47 results on '"Glenn E. Morris"'
Search Results
2. P.164Expression of alternative nebulin isoforms containing super repeat S21a or S21b in skeletal muscle
- Author
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Glenn E. Morris, Le Thanh Lam, Caroline Sewry, J. Laitila, Carina Wallgren-Pettersson, C. Pelin, and Ian Holt
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Gene isoform ,Nebulin ,medicine.anatomical_structure ,Neurology ,biology ,Chemistry ,Pediatrics, Perinatology and Child Health ,medicine ,biology.protein ,Skeletal muscle ,Neurology (clinical) ,Genetics (clinical) ,Cell biology - Published
- 2019
3. Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies
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Tatsushi Toda, Le Thanh Lam, Nguyen thi Man, Kazuhiro Kobayashi, Glenn E. Morris, and Tracy A. Lynch
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Glycosylation ,medicine.drug_class ,Molecular Sequence Data ,Biophysics ,Golgi Apparatus ,Monoclonal antibody ,medicine.disease_cause ,Biochemistry ,Mice ,chemistry.chemical_compound ,Peptide Library ,medicine ,Dystroglycan ,Animals ,Humans ,Amino Acid Sequence ,Muscular dystrophy ,Molecular Biology ,Mice, Inbred BALB C ,Mutation ,Hybridomas ,biology ,Immunodominant Epitopes ,Antibodies, Monoclonal ,Membrane Proteins ,Walker-Warburg Syndrome ,Cell Biology ,medicine.disease ,Fukutin ,Molecular biology ,Epitope mapping ,chemistry ,biology.protein ,Antibody ,Epitope Mapping ,HeLa Cells - Abstract
Mutations in the gene encoding fukutin protein cause Fukuyama muscular dystrophy, a severe congenital disorder that occurs mainly in Japan. A major consequence of the mutation is reduced glycosylation of alpha-dystroglycan, which is also a feature of other forms of congenital and limb-girdle muscular dystrophy. Immunodetection of endogenous fukutin in cells and tissues has been difficult and this has hampered progress in understanding fukutin function and disease pathogenesis. Using a new panel of monoclonal antibodies which bind to different defined sites on the fukutin molecule, we now show that fukutin has the predicted size for a protein without extensive glycosylation and is present at the Golgi apparatus at very low levels. These antibodies should enable more rapid future progress in understanding the molecular function of fukutin.
- Published
- 2012
4. Current research on SMN protein and treatment strategies for spinal muscular atrophy
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Glenn E. Morris, Heidi R. Fuller, and Emma L. Humphrey
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Neuromuscular disease ,Survival of motor neuron ,Spinal muscular atrophy ,SMN1 ,Biology ,Bioinformatics ,medicine.disease ,SMA ,nervous system diseases ,Exon ,nervous system ,Neurology ,Pediatrics, Perinatology and Child Health ,RNA splicing ,medicine ,snRNP ,Neurology (clinical) ,Genetics (clinical) - Abstract
The 7th UK SMA researchers meeting at Lake Vyrnwy Hotel, Oswestry UK in October 2010, was focussed on two main aspects of current SMA research: (1) to understand the normal functions of SMN protein (Fig. 1) and the mechanism of what goes seriously wrong when SMN levels are reduced to less than 10–15% of normal; and (2) to generate novel treatment options for current SMA patients and preventative options for future sufferers. Possible targets for treatment are shown in Fig. 1. Spinal Muscular Atrophy (SMA) is an inherited neuromuscular disease displaying a wide range of severity and characterised by loss of function and degeneration of lower motor neurons in the anterior horn of the spinal cord. It is caused by reduced levels of the small ubiquitous survival of motor neuron (SMN) protein, which is encoded by two genes, SMN1 and SMN2 [1]. Most of the mRNA transcribed from the SMN2 gene is alternatively spliced to omit exon 7 and any protein translated from such “delta7” mRNA is unstable and rapidly degraded [1–3]. In SMA patients, the SMN1 gene is deleted and only a small amount of functional SMN is produced from the SMN2 gene, with the more severe phenotypes having the least SMN [4,5]. In theory, therefore, only small increases in functional SMN are needed to produce a less severe SMA phenotype. This means that a concentrated effort on therapies that might increase the SMN protein levels in patients could help to protect the motor neurons [6]. Additional approaches without increasing SMN include modifying factors or stabilisation of neuromuscular junctions [7]. Shortly after the SMN1 gene was identified as the affected gene in SMA, the SMN protein was shown to have an essential role in the assembly of U snRNPs in the cytoplasm and their transport to the nucleus, where they are further modified for participation in RNA splicing [8]. It
- Published
- 2012
5. Muscleblind-Like Proteins
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Majid Fardaei, Ian Holt, Denis Furling, Caroline Sewry, J. David Brook, Virginie Jacquemin, Gillian Butler-Browne, and Glenn E. Morris
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Nucleoplasm ,Duchenne muscular dystrophy ,Alternative splicing ,Biology ,medicine.disease ,Myotonic dystrophy ,Molecular biology ,Pathology and Forensic Medicine ,chemistry.chemical_compound ,chemistry ,medicine ,MBNL1 ,Myocyte ,ITGA7 ,C2C12 - Abstract
In myotonic dystrophy, muscleblind-like protein 1 (MBNL1) protein binds specifically to expanded CUG or CCUG repeats, which accumulate as discrete nuclear foci, and this is thought to prevent its function in the regulation of alternative splicing of pre-mRNAs. There is strong evidence for the role of the MBNL1 gene in disease pathology, but the roles of two related genes, MBNL2 and MBNL3, are less clear. Using new monoclonal antibodies specific for each of the three gene products, we found that MBNL2 decreased during human fetal development and myoblast culture, while MBNL1 was unchanged. In Duchenne muscular dystrophy muscle, MBNL2 was elevated in immature, regenerating fibres compared with mature fibres, supporting some developmental role for MBNL2. MBNL3 was found only in C2C12 mouse myoblasts. Both MBNL1 and MBNL2 were partially sequestered by nuclear foci of expanded repeats in adult muscle and cultured cells from myotonic dystrophy patients. In adult muscle nucleoplasm, both proteins were reduced in myotonic dystrophy type 1 compared with an age-matched control. In normal human myoblast cultures, MBNL1 and MBNL2 always co-distributed but their distribution could change rapidly from nucleoplasmic to cytoplasmic. Functional differences between MBNL1 and MBNL2 have not yet been found and may prove quite subtle. The dominance of MBNL1 in mature, striated muscle would explain why ablation of the mouse mbnl1 gene alone is sufficient to cause a myotonic dystrophy.
- Published
- 2009
6. Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of α-dystroglycan
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Motoi Kanagawa, Tamao Endo, Hui Xiong, Nobuhiro Fujikake, Hiroshi Manya, Masaji Tachikawa, Akemi Nishimoto, Fan Wang, Tatsushi Toda, Kazuhiro Kobayashi, Satoshi Takeda, Tomohiro Chiyonobu, Glenn E. Morris, and Yoshitaka Nagai
- Subjects
Glycosylation ,Biophysics ,Biology ,N-Acetylglucosaminyltransferases ,medicine.disease_cause ,Biochemistry ,Gene product ,Dystroglycans ,Mice ,symbols.namesake ,chemistry.chemical_compound ,Transferases ,Chlorocebus aethiops ,Protein Interaction Mapping ,medicine ,Animals ,Humans ,Missense mutation ,Molecular Biology ,Genetics ,Mutation ,Proteins ,Cell Biology ,Golgi apparatus ,medicine.disease ,Fukutin ,Cell biology ,chemistry ,COS Cells ,symbols ,Congenital muscular dystrophy ,Protein Binding ,Signal Transduction - Abstract
The recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglycan (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that fukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity.
- Published
- 2006
7. Workshop on the nuclear envelope and Emery–Dreifuss muscular dystrophy 29th March 2006, Oswestry, UK
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Glenn E. Morris and K. Natalie Randles
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Plasma membrane protein ,medicine.medical_specialty ,Research groups ,Neuromuscular disease ,business.industry ,Emerin ,Laminopathy ,medicine.disease ,Neurology ,Pediatrics, Perinatology and Child Health ,medicine ,Physical therapy ,Neurology (clinical) ,Emery–Dreifuss muscular dystrophy ,Muscular dystrophy ,business ,Genetics (clinical) ,Muscle contracture - Abstract
A workshop on the nuclear envelope and Emery– Dreifuss muscular dystrophy (EDMD) was held at the Centre for Inherited Neuromuscular Disiease, Oswestry, UK, on 29th March 2006. Both structural defects and defects in signalling mechanisms that might link dysfunctional emerin and lamin A/C to muscle and heart disease were explored by researchers from eight different research groups within the UK. Quinlivan (Oswestry) welcomed visitors with an overview of the clinical services, pathology, locomotive assessment and molecular research undertaken by the Centre for Inherited Neuromuscular Disease (CIND) and described the new TORCH building that will house the Wolfson Neuromuscular Research Laboratory, the Keele University mass-spectrometry facility and a new gait laboratory from 2008. Emery (Oxford & Edinburgh) gave a historical account of ‘‘MD with contractures and heart block’’, as he modestly described the disease that bears his name, from the first family he studied in the USA in 1962 [1] to the identification of the genes responsible, emerin in 1994 [2] and lamin A/C in 1999 [3]. Morris (Oswestry) recalled that, when the emerin gene was first sequenced in 1994, people assumed it would encode a plasma membrane protein, like the
- Published
- 2006
8. Lamin A/C assembly defects in Emery–Dreifuss muscular dystrophy can be regulated by culture medium composition
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Nguyen thi Man, Glenn E. Morris, Ian Holt, and Manfred Wehnert
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Adult ,Male ,congenital, hereditary, and neonatal diseases and abnormalities ,animal structures ,Mutation, Missense ,Emerin ,Biology ,Transfection ,Mice ,medicine ,Animals ,Humans ,Muscular dystrophy ,Emery–Dreifuss muscular dystrophy ,Child ,Fibroblast ,Cells, Cultured ,Genetics (clinical) ,Skin ,Cell Nucleus ,Mice, Knockout ,Nucleoplasm ,integumentary system ,Fibroblasts ,Lamin Type A ,medicine.disease ,Immunohistochemistry ,Molecular biology ,Muscular Dystrophy, Emery-Dreifuss ,Culture Media ,Cell nucleus ,medicine.anatomical_structure ,Neurology ,embryonic structures ,Pediatrics, Perinatology and Child Health ,Nuclear lamina ,Neurology (clinical) ,Lamin - Abstract
Emery-Dreifuss muscular dystrophy results from mutations in either emerin or lamin A/C and is caused by loss of some unknown function of emerin-lamin A/C complexes. This function must be of special importance in the skeletal and cardiac muscles that are affected by the disease. Some lamin A/C mutant proteins form 'nuclear foci' in the nucleoplasm when overexpressed by transient transfection and similar aggregates have been seen in cultured skin fibroblasts from patients with Emery-Dreifuss muscular dystrophy, suggesting that mis-assembly of the A-type lamina may be involved in the pathogenesis. Whereas an earlier study of cultured skin fibroblasts compared several different missense mutations in lamin A/C, we have chosen to study one particular Emery-Dreifuss mutation (R249Q) in greater detail. We found that the proportion of fibroblast nuclei containing abnormal lamin A/C aggregates can vary from 0.5 to 23.6% depending on the culture conditions. In particular, switching from a 'slow growth' medium to 'rapid growth' media increased both the number and size of nuclear aggregates. Similar results were obtained with fibroblasts from a second unrelated patient with the same mutation. In contrast to these aggregates of endogenous lamin A/C, 'nuclear foci' formed after transfection of mouse embryo fibroblasts by mutant lamin A/C were not affected by culture conditions. Faulty assembly of the nuclear lamina by mutated lamin A/C molecules could be partly responsible for the disease phenotype, though this has not been proven. The present study suggests that inappropriate lamin A/C assembly may be preventable by manipulation of cell growth conditions.
- Published
- 2006
9. Indoprofen Upregulates the Survival Motor Neuron Protein through a Cyclooxygenase-Independent Mechanism
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Jianhua Zhou, Mitchell R. Lunn, Nguyen thi Man, Brent R. Stockwell, David E. Root, Glenn E. Morris, Elliot J. Androphy, Daniel D. Coovert, Charlotte J. Sumner, Arthur H.M. Burghes, Brian P. Kelley, Allison M. Martino, and Stephen P. Flaherty
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Clinical Biochemistry ,Drug Evaluation, Preclinical ,Nerve Tissue Proteins ,Endogeny ,SMN1 ,Biochemistry ,Article ,Mice ,Pregnancy ,Drug Discovery ,medicine ,Animals ,Humans ,Cyclooxygenase Inhibitors ,Cyclic AMP Response Element-Binding Protein ,Molecular Biology ,Gene ,Pharmacology ,biology ,Anti-Inflammatory Agents, Non-Steroidal ,RNA-Binding Proteins ,SMN Complex Proteins ,Indoprofen ,General Medicine ,Spinal muscular atrophy ,Fibroblasts ,Motor neuron ,medicine.disease ,SMA ,Survival of Motor Neuron 1 Protein ,Molecular biology ,Up-Regulation ,nervous system diseases ,Survival of Motor Neuron 2 Protein ,medicine.anatomical_structure ,Prostaglandin-Endoperoxide Synthases ,biology.protein ,Molecular Medicine ,Female ,Cyclooxygenase ,medicine.drug - Abstract
Most patients with the pediatric neurodegenerative disease spinal muscular atrophy have a homozygous deletion of the survival motor neuron 1 ( SMN1 ) gene, but retain one or more copies of the closely related SMN2 gene. The SMN2 gene encodes the same protein (SMN) but produces it at a low efficiency compared with the SMN1 gene. We performed a high-throughput screen of ∼47,000 compounds to identify those that increase production of an SMN2 -luciferase reporter protein, but not an SMN1 -luciferase reporter protein. Indoprofen, a nonsteroidal anti-inflammatory drug (NSAID) and cyclooxygenase (COX) inhibitor, selectively increased SMN2 -luciferase reporter protein and endogenous SMN protein and caused a 5-fold increase in the number of nuclear gems in fibroblasts from SMA patients. No other NSAIDs or COX inhibitors tested exhibited this activity.
- Published
- 2004
10. Autosomal dominant Emery–Dreifuss muscular dystrophy: a new family with late diagnosis
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Emilio Fernández-Alvarez, Gisèle Bonne, Jaume Colomer, C. Iturriaga, Ketty Schwartz, M. Puche, Glenn E. Morris, and S. Manilal
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musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,Weakness ,Contracture ,Adolescent ,Biopsy ,Cardiomyopathy ,Locus (genetics) ,Achilles Tendon ,LMNA ,Elbow Joint ,medicine ,Humans ,Emery–Dreifuss muscular dystrophy ,Muscular dystrophy ,Child ,Polymorphism, Single-Stranded Conformational ,Genetics (clinical) ,Genes, Dominant ,Muscle contracture ,Family Health ,Genetics ,business.industry ,Nuclear Proteins ,Middle Aged ,medicine.disease ,Lamins ,Muscular Dystrophy, Emery-Dreifuss ,Spine ,Pedigree ,Xq28 ,Neurology ,Pediatrics, Perinatology and Child Health ,Female ,Neurology (clinical) ,medicine.symptom ,Cardiomyopathies ,business - Abstract
Emery-Dreifuss muscular dystrophy is characterized by the clinical triad of early onset contractures of elbows, Achilles tendons and spine, wasting and weakness with a predominantly humero-peroneal distribution and life-threatening cardiac conduction defects and/or cardiomyopathy. Two main types of inheritance have been described: the X-linked form is caused by mutations in the STA gene on locus Xq28 and the gene for the autosomal dominant form (LMNA gene) has been localized on chromosome 1q11-q23. Recently, mutations in this LMNA gene have been also found to be responsible for the less frequent autosomal recessive form of the disease. Although all forms share a similar clinical presentation, some differences appear to exist between them as has been described recently in a large number of patients. We present the first documented Spanish family genetically confirmed to have autosomal dominant Emery-Dreifuss muscular dystrophy. Clinical, pathological and genetic data are described. We emphasize the difficulties in diagnosis, especially in sporadic cases or young patients in whom the clinical picture is not completely established.
- Published
- 2002
11. A Direct Interaction between the Survival Motor Neuron Protein and p53 and Its Relationship to Spinal Muscular Atrophy
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Philip J. Young, Patricia M. Day, Jianhua Zhou, Elliot J. Androphy, Glenn E. Morris, and Christian L. Lorson
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Time Factors ,animal diseases ,Mutation, Missense ,Apoptosis ,Nerve Tissue Proteins ,Biosensing Techniques ,SMN1 ,Biology ,Transfection ,medicine.disease_cause ,Biochemistry ,Cell Line ,Muscular Atrophy, Spinal ,Gene product ,SMN Complex Proteins ,Tumor Cells, Cultured ,medicine ,Humans ,Cyclic AMP Response Element-Binding Protein ,Molecular Biology ,Glutathione Transferase ,Mutation ,RNA-Binding Proteins ,Exons ,Cell Biology ,Spinal muscular atrophy ,Fibroblasts ,Motor neuron ,medicine.disease ,SMA ,Immunohistochemistry ,Precipitin Tests ,Survival of Motor Neuron 1 Protein ,Recombinant Proteins ,Protein Structure, Tertiary ,nervous system diseases ,Survival of Motor Neuron 2 Protein ,medicine.anatomical_structure ,Microscopy, Fluorescence ,nervous system ,Cajal body ,Cancer research ,Tumor Suppressor Protein p53 ,Dimerization ,Protein Binding - Abstract
Mutations in the SMN1 (survival motor neuron 1) gene cause spinal muscular atrophy (SMA). We now show that SMN protein, the SMN1 gene product, interacts directly with the tumor suppressor protein, p53. Pathogenic missense mutations in SMN reduce both self-association and p53 binding by SMN, and the extent of the reductions correlate with disease severity. The inactive, truncated form of SMN produced by the SMN2 gene in SMA patients fails to bind p53 efficiently. SMN and p53 co-localize in nuclear Cajal bodies, but p53 redistributes to the nucleolus in fibroblasts from SMA patients. These results suggest a functional interaction between SMN and p53, and the potential for apoptosis when this interaction is impaired may explain motor neuron death in SMA.
- Published
- 2002
12. The Relationship between SMN, the Spinal Muscular Atrophy Protein, and Nuclear Coiled Bodies in Differentiated Tissues and Cultured Cells
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thi Man N, Glenn E. Morris, Philip J. Young, Arthur H.M. Burghes, and Thanh Le
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endocrine system diseases ,Swine ,Blotting, Western ,Coiled Bodies ,Nerve Tissue Proteins ,RNA-binding protein ,Biology ,Muscular Atrophy, Spinal ,Mice ,SMN complex ,SMN Complex Proteins ,medicine ,Animals ,Humans ,snRNP ,Cyclic AMP Response Element-Binding Protein ,Skin ,Cell Nucleus ,Genetics ,Mice, Inbred BALB C ,Antibodies, Monoclonal ,Nuclear Proteins ,RNA-Binding Proteins ,Colocalization ,Cell Biology ,Spinal muscular atrophy ,Fibroblasts ,medicine.disease ,Cell biology ,Microscopy, Fluorescence ,Organ Specificity ,COS Cells ,RNA splicing ,Electrophoresis, Polyacrylamide Gel ,Coilin ,Rabbits ,HeLa Cells - Abstract
The spinal muscular atrophy protein, SMN, is a cytoplasmic protein that is also found in distinct nuclear structures called "gems." Gems are closely associated with nuclear coiled bodies and both may have a direct role in snRNP maturation and pre-RNA splicing. There has been some controversy over whether gems and coiled bodies colocalize or form adjacent/independent structures in HeLa and other cultured cells. Using a new panel of antibodies against SMN and antibodies against coilin-p80, a systematic and quantitative study of adult differentiated tissues has shown that gems always colocalize with coiled bodies. In some tissues, a small proportion of coiled bodies (10%) had no SMN, but independent or adjacent gems were not found. The most striking observation, however, was that many cell types appear to have neither gems nor coiled bodies (e.g., cardiac and smooth muscle, blood vessels, stomach, and spleen) and this expression pattern is conserved across human, rabbit, and pig species. This shows that assembly of distinct nuclear bodies is not essential for RNA splicing and supports the view that they may be storage sites for reserves of essential proteins and snRNPs. Overexpression of SMN in COS-7 cells produced supernumerary nuclear bodies, most of which also contained coilin-p80, confirming the close relationship between gems and coiled bodies. However, when SMN is reduced to very low levels in type I SMA fibroblasts, coiled bodies are still formed. Overall, the data suggest that gem/coiled body formation is not determined by high cytoplasmic SMN concentrations or high metabolic activity alone and that a differentiation-specific factor may control their formation.
- Published
- 2000
13. Expression and synthesis of alternatively spliced variants of Dp71 in adult human brain
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Glenn E. Morris, Peter N. Ray, Perry L. Howard, Henry J. Klamut, and Richard C. Austin
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Transcription, Genetic ,medicine.drug_class ,Duchenne muscular dystrophy ,Immunoblotting ,Biology ,Monoclonal antibody ,Polymerase Chain Reaction ,Epitope ,Dystrophin ,Exon ,medicine ,Humans ,RNA, Messenger ,Cloning, Molecular ,Genetics (clinical) ,Brain Chemistry ,Regulation of gene expression ,Messenger RNA ,Alternative splicing ,medicine.disease ,Molecular biology ,Muscular Dystrophy, Duchenne ,Alternative Splicing ,Gene Expression Regulation ,Neurology ,Pediatrics, Perinatology and Child Health ,biology.protein ,Neurology (clinical) - Abstract
Transcripts encoding the 70-75 kDa C-terminal protein product of the dystrophin gene (Dp71) are alternatively spliced to generate multiple protein products in a number of adult human tissues. In this report, reverse transcriptase-polymerase chain reaction was used to clone and characterize a subpopulation of truncated Dp71 transcripts in adult human brain tissue which did not contain exons 71-74, resulting in an in-frame deletion of 330 bp encoding the syntrophin-binding domain. These truncated Dp71 transcripts are also alternatively spliced for exon 78. Immunoblot analysis, using dystrophin-specific C-terminal antibodies directed against epitopes in either exon 77 (MANDRA1), or 78 (1461), identified full-length dystrophin, Dp140 and Dp71, in total protein lysates from adult human brain tissue. In addition, a minor immunoreactive protein of approximately 58 kDa was also identified (designated Dp71 big up tri, open(110)). The observation that a monoclonal antibody directed against epitopes within exons 73-74 (MANEX7374A) failed to detect this 58 kDa protein provides definitive evidence that Dp71 big up tri, open(110) is derived from Dp71 transcripts deleted for the syntrophin-binding domain. These results, as well as previous findings, demonstrate that alternative splicing of Dp71 in the human brain generates a variety of mRNA transcripts encoding distinct protein variants of Dp71, and further supports the use of exon-specific antibodies in characterizing these variants. The presence of these Dp71 protein variants in brain tissue points to their interaction with various cellular proteins and their involvement in different cellular functions.
- Published
- 2000
14. Direct Interaction between Emerin and Lamin A
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Donald R. Love, Glenn E. Morris, Lisa Clements, and S. Manilal
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congenital, hereditary, and neonatal diseases and abnormalities ,animal structures ,Barrier-to-autointegration factor ,Biophysics ,Emerin ,Thymopoietins ,Biology ,medicine.disease_cause ,Biochemistry ,medicine ,Humans ,Inner membrane ,Cloning, Molecular ,Muscular dystrophy ,Emery–Dreifuss muscular dystrophy ,Molecular Biology ,Mutation ,Binding Sites ,integumentary system ,Antibodies, Monoclonal ,Membrane Proteins ,Nuclear Proteins ,Cell Biology ,Lamin Type A ,medicine.disease ,Molecular biology ,Lamins ,Muscular Dystrophy, Emery-Dreifuss ,Peptide Fragments ,Recombinant Proteins ,embryonic structures ,Nuclear lamina ,Lamin - Abstract
Emerin is the protein of the inner nuclear membrane that is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. The autosomal dominant form of the disease is caused by mutations in the lamin A/C gene. Several lines of circumstantial evidence have suggested an interaction of emerin with lamins in the nuclear lamina but direct interaction between the two proteins has not yet been demonstrated. We now demonstrate direct interaction between recombinant emerin and lamin A molecules using biomolecular interaction analysis (BIA) and monoclonal antibodies. An emerin–lamin A interaction system may be related in function to the LAP2-lamin B system at the inner nuclear rim.
- Published
- 2000
15. Molecular analysis of a spontaneous dystrophin 'knockout' dog
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Matthew M. Binns, Matthew Breen, Natasha J. Olby, Helen F Dickens, Scott J. Schatzberg, Joe N. Kornegay, Glenn E. Morris, Louise V.B. Anderson, Cordelia Langford, Stephen D. Wilton, Caroline J. Zeiss, and Nicholas J.H. Sharp
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Male ,X Chromosome ,Biopsy ,Duchenne muscular dystrophy ,Blotting, Western ,Biology ,Polymerase Chain Reaction ,Dystrophin ,Dogs ,Utrophin ,medicine ,Animals ,Dog Diseases ,Muscular dystrophy ,Muscle, Skeletal ,Creatine Kinase ,In Situ Hybridization, Fluorescence ,Genetics (clinical) ,X chromosome ,medicine.diagnostic_test ,Skeletal muscle ,DNA ,Muscular Dystrophy, Animal ,medicine.disease ,Molecular biology ,Disease Models, Animal ,medicine.anatomical_structure ,Neurology ,Mutation ,Pediatrics, Perinatology and Child Health ,biology.protein ,Neurology (clinical) ,Chromosome Deletion ,ITGA7 ,Fluorescence in situ hybridization - Abstract
We have determined the molecular basis for skeletal myopathy and dilated cardiomyopathy in two male German short-haired pointer (GSHP) littermates. Analysis of skeletal muscle demonstrated a complete absence of dystrophin on Western blot analysis. PCR analysis of genomic DNA revealed a deletion encompassing the entire dystrophin gene. Molecular cytogenetic analysis of lymphocytes from the dam and both dystrophic pups confirmed a visible deletion in the p21 region of the affected canine X chromosome. Utrophin is up-regulated in the skeletal muscle, but does not appear to ameliorate the dystrophic canine phenotype. This new canine model should further our understanding of the physiological and biochemical processes in Duchenne muscular dystrophy.
- Published
- 1999
16. Localization of rabbit huntingtin using a new panel of monoclonal antibodies
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Glenn E. Morris, James Neal, Nguyen thi Man, S. Manilal, Philip Thomas, Fiona L. Wilkinson, Peter S. Harper, and A L Jones
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congenital, hereditary, and neonatal diseases and abnormalities ,Huntingtin ,medicine.drug_class ,Recombinant Fusion Proteins ,Blotting, Western ,Nerve Tissue Proteins ,Cross Reactions ,Biology ,Monoclonal antibody ,Epitope ,Epitopes ,Mice ,Cellular and Molecular Neuroscience ,Trinucleotide Repeats ,Huntington's disease ,Antibody Specificity ,SETD2 ,mental disorders ,medicine ,Animals ,Bacteriophages ,Molecular Biology ,Gene Library ,Brain Chemistry ,Huntingtin Protein ,Mice, Inbred BALB C ,Pyramidal Cells ,Antibodies, Monoclonal ,Nuclear Proteins ,Polyglutamine tract ,medicine.disease ,Molecular biology ,nervous system diseases ,Huntington Disease ,nervous system ,Cytoplasm ,Langerhans Cells ,Immunology ,Rabbits ,Cell fractionation - Abstract
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat which is expressed as a polyglutamine tract near the N-terminus of the gene product, huntingtin. N-terminal huntingtin fragments form intranuclear aggregates in HD patients and these may be involved in the pathogenesis. Monoclonal antibodies (mAbs) against three different regions of huntingtin (amino acids 997-1276, 1844-2131 and 2703-2911) have been produced and two of the epitopes have been identified using phage displayed peptide libraries. All mAbs reacted with 350 kDa huntingtin on Western blots and one mAb from each region was selected for further study by strong immunoreactivity with neurons in different regions of rabbit brain and by ability to immunoprecipitate native huntingtin. Subcellular fractionation and sucrose density centrifugation of rabbit brain extract showed that most of the huntingtin exists as a high molecular weight complex in the cytoplasm. Two outstanding problems have been addressed; the location of huntingtin in tissues outside the central nervous system and whether huntingtin is present in the nucleus of normal cells. We conclude that huntingtin is present at low levels in most non-neuronal cells though we have identified an interstitial cell type in skin with very high immunoreactivity. Using both immunolocalization and nuclear purification methods, we were unable to exclude the possibility that a small proportion of full-length huntingtin is present in the nucleus.
- Published
- 1999
17. Early presentation of X-linked Emery–Dreifuss muscular dystrophy resembling limb-girdle muscular dystrophy
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Glenn E. Morris, Jacqueline Taylor, D. Recan, Stéphane Llense, Ewa J Lichtarowicz-Krynska, S. Manilal, Victor Dubowitz, Francesco Muntoni, and Caroline Sewry
- Subjects
Male ,musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,Pathology ,medicine.medical_specialty ,X Chromosome ,Genetic Linkage ,Biopsy ,Duchenne muscular dystrophy ,Emerin ,Thymopoietins ,Muscular Dystrophies ,Diagnosis, Differential ,Dysferlin ,Leukocytes ,medicine ,Humans ,Age of Onset ,Muscular dystrophy ,Emery–Dreifuss muscular dystrophy ,Genetics (clinical) ,Skin ,Muscle contracture ,biology ,business.industry ,Muscles ,Membrane Proteins ,Nuclear Proteins ,Anatomy ,medicine.disease ,Muscular Dystrophy, Emery-Dreifuss ,Pedigree ,Neurology ,Child, Preschool ,Mutation ,Pediatrics, Perinatology and Child Health ,biology.protein ,Neurology (clinical) ,business ,ITGA7 ,Limb-girdle muscular dystrophy - Abstract
Emery–Dreifuss muscular dystrophy is an X-linked neuromuscular disorder caused by defects in the STA gene on Xq28, which codes for a nuclear protein named emerin. Affected patients usually present in early adolescence with scapulo-peroneal muscle weakness and wasting, and contractures of the tendo Achilles, elbows and paraspinal muscles, resulting in spine rigidity. We present here a case of Emery–Dreifuss muscular dystrophy with an unusually severe, early presentation. He presented at 2.5 years with predominantly proximal weakness and mild equinovarus deformity of the right foot. Serum creatine kinase activity was elevated (1994 IU/l) and a muscle biopsy at the age of 4 years showed marked dystrophic abnormalities. Normal expression of dystrophin, and no detectable deletion in the corresponding gene, excluded a diagnosis of Duchenne muscular dystrophy. Similarly, normal expression of α-sarcoglycan made a limb-girdle muscular dystrophy caused by a defect in a sarcoglycan unlikely. Several years later, examination of the proband's maternal cousin, aged 14 years, suggested Emery–Dreifuss muscular dystrophy. This was confirmed in both affected boys by the absence of emerin in muscle and leucocytes, and identification of a mutation in exon 4 of the STA gene. Carrier status in both mothers was also confirmed by mutational and protein analysis. Emery–Dreifuss muscular dystrophy should therefore be considered in the differential diagnosis of cases of early onset muscular dystrophy, even in the absence of the typical clinical features.
- Published
- 1998
18. Dystrophin is replaced by utrophin in frog heart; implications for muscular dystrophy
- Author
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Glenn E. Morris
- Subjects
musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,Utrophin ,animal diseases ,Xenopus ,Muscular Dystrophies ,Dystrophin ,Xenopus laevis ,medicine ,Dystroglycan ,Animals ,Muscular dystrophy ,Genetics (clinical) ,Membranes ,Sarcolemma ,biology ,Myocardium ,Cardiac muscle ,Membrane Proteins ,Skeletal muscle ,Muscular Dystrophy, Animal ,musculoskeletal system ,medicine.disease ,biology.organism_classification ,Molecular biology ,Cell biology ,Cytoskeletal Proteins ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Neurology ,Pediatrics, Perinatology and Child Health ,biology.protein ,Neurology (clinical) - Abstract
The possibility of using utrophin upregulation as a treatment for dystrophin-deficient muscular dystrophies has focused attention on the question of how many of dystrophin's various functions can be performed by the closely-related protein, utrophin. In Xenopus heart, little or no dystrophin was found on Western blots but the dystrophin-related protein, utrophin, was abundant. This utrophin was shown by immunofluorescence microscopy to be associated with cardiac muscle membranes and its distribution was similar to that of dystrophin in rabbit heart. The utrophin distribution pattern in the frog heart was shared by beta-dystroglycan, a transmembrane protein responsible for localizing both dystrophin and utrophin at cell membranes. The results suggest that utrophin in Xenopus heart can perform similar functions to dystrophin in mammalian heart, lending further support to the possibility of utrophin upregulation therapy in muscular dystrophy. In skeletal muscle, however, Xenopus resembles mammals in expressing dystrophin at the sarcolemma and very little utrophin.
- Published
- 1997
19. The molecular basis for cross-reaction of an anti-dystrophin antibody with alpha-actinin
- Author
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Yvonne H. Edwards, Glenn E. Morris, Marian James, and Nguyen thi Man
- Subjects
Phage display ,medicine.drug_class ,Molecular Sequence Data ,Autoimmunity ,Cross Reactions ,Succinyl CoA synthetase ,Monoclonal antibody ,Epitope ,Dystrophin ,Complementary DNA ,medicine ,Animals ,Humans ,Phage display peptide library ,Actinin ,Amino Acid Sequence ,Peptide sequence ,Molecular Biology ,biology ,Sequence Homology, Amino Acid ,cDNA library ,Antibodies, Monoclonal ,λgt11 library ,Molecular biology ,Rats ,Epitope mapping ,Enzyme ,biology.protein ,Molecular Medicine ,Epitope Mapping - Abstract
The epitope recognised by the anti-dystrophin monoclonal antibodies MANDYS141 and MANDYS142 has been characterised using a phage display peptide library and a bacteriophage lambda cDNA library. Using a phage display library of random 15-mer peptides, the epitope recognised by the two antibodies was identified as EEXF. A lambda gt11 clone obtained by screening a human muscle cDNA library was shown to contain part of the out-of-frame human mitochondrial succinyl CoA synthetase (alpha-subunit) cDNA sequence which contains the sequence EEPL, suggesting a minimum requirement of EEXF/L for antibody binding. The sequence EEDF is located in the helical rod region of dystrophin and the N-terminal domain of alpha-actinin; this may explain why native dystrophin is not detected, since the alpha-helical, coiled-coil folding of the rod region of dystrophin may obscure the epitope in the native protein. The antibody cross-reaction between dystrophin and alpha-actinin is likely to be fortuitous and not due to any structural homology that exists between these two members of the spectrin superfamily.
- Published
- 1997
- Full Text
- View/download PDF
20. Evaluation of a panel of new monoclonal antibodies to α913-DG
- Author
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Glenn E. Morris, Susan C. Brown, V. Pagalday, and Caroline Sewry
- Subjects
Neurology ,medicine.drug_class ,Pediatrics, Perinatology and Child Health ,medicine ,Neurology (clinical) ,Biology ,Monoclonal antibody ,Virology ,Genetics (clinical) - Published
- 2016
21. Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy
- Author
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Christer Holmberg, Glenn E. Morris, S. K. Singhrao, Juhani Rapola, Carina Wallgren-Pettersson, Bharat Jasani, Angus John Clarke, Ismo Virtanen, Sally Jones, and Geoffrey R. Newman
- Subjects
Adult ,Pathology ,medicine.medical_specialty ,Biopsy ,Blotting, Western ,Muscle Fibers, Skeletal ,macromolecular substances ,Gömöri trichrome stain ,Myopathies, Nemaline ,Desmin ,law.invention ,Nemaline myopathy ,law ,medicine ,Humans ,Actinin ,Intermediate filament ,Nemaline bodies ,Genetics (clinical) ,Inclusion Bodies ,biology ,Antibodies, Monoclonal ,musculoskeletal system ,medicine.disease ,Immunohistochemistry ,Molecular Weight ,Microscopy, Electron ,Actinin, alpha 1 ,Neurology ,Pediatrics, Perinatology and Child Health ,biology.protein ,Female ,Neurology (clinical) ,Electron microscope ,Dystrophin - Abstract
To elucidate the protein composition of the nemaline bodies present in the muscle fibres of patients with congenital nemaline myopathy (CNM), we studied muscle biopsies with monoclonal antibodies against alpha-actinin and desmin in combination with a modified Gomori trichrome method. Electron microscopy of immunolabelled resin embedded sections was used for cytochemical localisation of alpha-actinin and desmin. Light microscopy of sections immunolabelled for alpha-actinin showed a cross-striation of the muscle fibres corresponding to the Z band pattern, focal thickening of the Z bands and additional reactivity with a granular pattern corresponding to the presence of nemaline bodies. Labelling of desmin did not show a similar pattern. Electron microscopy confirmed the presence of alpha-actinin in the nemaline bodies and Z bands, whereas desmin was only found in intermediate filaments around the Z bands. Western blots showed single, sharp alpha-actinin bands indistinguishable from normal. Our results provide direct evidence for the presence of alpha-actinin in nemaline bodies and a lack of quantitative or qualitative differences between the alpha-actinin of normal and CNM muscle.
- Published
- 1995
22. The Economics of Household Solid Waste Generation and Disposal
- Author
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Duncan M. Holthausen and Glenn E. Morris
- Subjects
Economics and Econometrics ,Municipal solid waste ,Economy ,Natural resource economics ,Unit price ,media_common.quotation_subject ,Production model ,Economics ,Production (economics) ,Policy initiatives ,Management, Monitoring, Policy and Law ,Welfare ,media_common - Abstract
We develop a household production model of waste management that explicitly incorporates many of the technical and behavioral elements germane to current regulatory and non-regulatory solid waste policy initiatives. Examination of first-order conditions shows the interaction among household preferences, these production options, and external prices and fees. A simplified simulation of our model illustrates these relationships, showing that household response elasticities can vary widely over common price ranges and that relatively large household welfare gains may be obtained by adopting curbside recycling and unit pricing programs.
- Published
- 1994
23. Expression of the 43 kDa dystrophin-associated glycoprotein in human neuromuscular disease
- Author
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Nguyen thi Man, Timothy R. Helliwell, and Glenn E. Morris
- Subjects
musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,Neuromuscular disease ,Vascular smooth muscle ,Utrophin ,Duchenne muscular dystrophy ,Biology ,Muscular Dystrophies ,Dystrophin ,Reference Values ,medicine ,Humans ,Dystroglycans ,Genetics (clinical) ,Membrane Glycoproteins ,Sarcolemma ,Muscles ,Antibodies, Monoclonal ,Membrane Proteins ,musculoskeletal system ,medicine.disease ,Immunohistochemistry ,Molecular biology ,Cytoskeletal Proteins ,Neurology ,Pediatrics, Perinatology and Child Health ,Congenital muscular dystrophy ,biology.protein ,Neurology (clinical) ,ITGA7 - Abstract
The expression of the 43 kDa dystrophin-associated glycoprotein (43DAG) has been studied using immunohistochemical labelling with a monoclonal antibody, MANDAG-1, and compared with immunolabelling for dystrophin and the dystrophin-related protein, utrophin, in normal muscle and in muscle from 50 patients with neuromuscular disease. 43DAG and dystrophin were expressed in vascular smooth muscle and at the sarcolemma of normal muscle fibres, with increased labelling at neuromuscular and myotendinous junctions. 43DAG expression was reduced in Duchenne and Becker dystrophies with patchy labelling, more intense around presumptive satellite cells. In Duchenne dystrophy, there was increased 43DAG expression in “revertant” fibres. In Becker dystrophy, 43DAG expression was more extensive around individual fibres, showed more interfibre variation and was more closely related to the intensity of immunolabelling for both dystrophin and utrophin than in Duchenne dystrophy. In other neuromuscular diseases, including congenital muscular dystrophy, no abnormalities of 43DAG expression were identified. The results suggest that in the absence of dystrophin, 43DAG is synthesized but is not stabilized in the sarcolemma. Stability is greater in Becker dystrophy but a normal dystrophin molecule appears to be required for the complete and stable membrane integration of 43DAG. Utrophin may confer some additional stability to the membrane integration of 43DAG but this is incomplete where dystrophin is absent or abnormal.
- Published
- 1994
24. A quantitative ELISA for dystrophin
- Author
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Nguyen thi Man, J.M. Ellis, and Glenn E. Morris
- Subjects
musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,Anticorps monoclonal ,medicine.drug_class ,Immunology ,Enzyme-Linked Immunosorbent Assay ,In Vitro Techniques ,Monoclonal antibody ,Muscular Dystrophies ,Epitope ,Dystrophin ,Mice ,medicine ,Animals ,Humans ,Immunology and Allergy ,Muscular dystrophy ,biology ,medicine.diagnostic_test ,Muscles ,Antibodies, Monoclonal ,Elisa assay ,Muscular Dystrophy, Animal ,musculoskeletal system ,medicine.disease ,Molecular biology ,Immunoassay ,biology.protein - Abstract
A novel approach to the quantitation of the muscular dystrophy protein, dystrophin, in muscle extracts is described. The two-site ELISA uses two monoclonal antibodies against dystrophin epitopes which lie close together in the rod domain of the dystrophin molecule in order to minimize the effects of dystrophin degradation. Dystrophin is assayed in its native form by extracting with non-ionic detergents and avoiding the use of SDS.
- Published
- 1993
25. Expression of the transcripts initiated in the 62nd intron of the dystrophin gene
- Author
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Glenn E. Morris, Annette Koulakoff, Axel Kahn, Jean-Philippe Hugnot, Yoheved Berwald-Netter, Philippe Chafey, Martine Lambert, Hélène Gilgenkrantz, J.C. Kaplan, and C. Billard
- Subjects
Brain development ,Transcription, Genetic ,Molecular Sequence Data ,Gene Expression ,Biology ,Polymerase Chain Reaction ,Muscular Dystrophies ,Dystrophin ,Animals ,Humans ,Lymphocytes ,Child ,Promoter Regions, Genetic ,Genetics (clinical) ,DNA Primers ,Base Sequence ,Lymphoblast ,Intron ,A protein ,Promoter ,Molecular biology ,Dystrophin gene ,Introns ,Rats ,Real-time polymerase chain reaction ,Liver ,Neurology ,Pediatrics, Perinatology and Child Health ,Neurology (clinical) ,Gene Deletion - Abstract
The pattern of expression of two distal transcripts initiated in the 62nd intron of the dystrophin gene was investigated under different circumstances: (i) during the development of different rat tissues these transcripts and Dp 71, a protein encoded by one of them, increased with brain development and decreased with muscle development; (ii) in cultured glial and neuronal cells, the distal promoter was coactivated with tissue-specific upstream promoters, the muscle-type promoter in glial cells and the brain-type promoter in neuronal cells, which suggests that activity of the upstream promoter does not interfere with activity of the distal promoter; (iii) in lymphoblasts of DMD patients with various deletions of the dystrophin gene, the most distal of which included the 56th intron, the production of the distal transcript was not perturbed.
- Published
- 1993
26. The dystrophin-related protein, utrophin, is expressed on the sarcolemma of regenerating human skeletal muscle fibres in dystrophies and inflammatory myopathies
- Author
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Kay E. Davies, Man Nguyen thi, Glenn E. Morris, and Timothy R. Helliwell
- Subjects
Adult ,Vascular smooth muscle ,Adolescent ,Utrophin ,animal diseases ,Duchenne muscular dystrophy ,In Vitro Techniques ,Muscular Dystrophies ,Neuromuscular junction ,Dystrophin ,Sarcolemma ,Muscular Diseases ,medicine ,Humans ,Child ,Genetics (clinical) ,Inflammation ,biology ,Chemistry ,Muscles ,Antibodies, Monoclonal ,Infant ,Membrane Proteins ,Skeletal muscle ,Anatomy ,Middle Aged ,musculoskeletal system ,medicine.disease ,Immunohistochemistry ,Nerve Regeneration ,Cell biology ,Cytoskeletal Proteins ,medicine.anatomical_structure ,Neurology ,Child, Preschool ,Pediatrics, Perinatology and Child Health ,biology.protein ,Neurology (clinical) ,ITGA7 - Abstract
Utrophin is the 400 kDa protein product of an autosomal homologue (DMDL) of the dystrophin gene. In normal skeletal muscle, utrophin is expressed in vascular smooth muscle, endothelium and nerves but not in mature muscle fibres except at the neuromuscular junction. We have examined the expression of utrophin in a wide range of human skeletal muscle diseases using monoclonal antibodies against three C-terminal epitopes. Utrophin is consistently expressed in all basophilic, regenerating fibres irrespective of the underlying disease or expression of dystrophin. It is also found in regenerating fibres from a normal volunteer. In Duchenne and Becker dystrophies, as well as in dermatomyositis, sarcolemmal staining for utrophin is also seen in larger fibres which are not obviously regenerating. These studies do not support the idea that utrophin occupies membrane attachment sites only when dystrophin is absent or reduced, but would be consistent with utrophin expression as part of an activated foetal programme during regeneration.
- Published
- 1992
27. Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies
- Author
-
Alison J. Cartwright, Mark Osborne, Glenn E. Morris, and Nguyen thi Man
- Subjects
medicine.drug_class ,Molecular Sequence Data ,Biophysics ,Biology ,Monoclonal antibody ,Biochemistry ,Epitope ,Epitopes ,chemistry.chemical_compound ,Structural Biology ,Sequence Homology, Nucleic Acid ,medicine ,Peptide synthesis ,Animals ,Humans ,Amino Acid Sequence ,Creatine Kinase ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Antibodies, Monoclonal ,Brain ,Molecular biology ,Amino acid ,Isoenzymes ,Kinetics ,Epitope mapping ,chemistry ,biology.protein ,Protein folding ,Antibody ,Protein Binding - Abstract
Epitopes on human brain creatine kinase (B-CK) recognized by three monoclonal antibodies have been located by chemical cleavage methods, followed by peptide synthesis or analysis of specificity for natural variants (isoforms). One antibody, CK-HTB, recognizes a conformational, or assembled, surface epitope on native CK which is also present on partially unfolded forms. It requires an Asn residue at position 300 in the amino acid sequence and will not recognize variants with Lys or His in this position. This results in a striking specificity of the antibody, which binds to B-CK only in chicken and man, but to muscle-form (M-CK) only in the rat. The results suggest that Asn-300 is exposed on the enzyme surface as part of a relatively denaturation-resistant region. Two monoclonal antibodies, CK-END1 and CK-END2, recognise epitopes within 53 amino acids of the C-terminus and bind to a synthetic hexapeptide representing the last six amino acids of human B-CK (Leu-375-Lys-380). The two antibodies show overlapping, but distinct, specificities in their binding to CK variants. CK-END1 requires Met-376 and will not tolerate Ile in this position, whereas CK-END2 requires Leu-375 and will not tolerate Met. Neither antibody binds to native CK, though both will bind to a folding intermediate and to partially unfolded states. This shows that the C-terminus of CK becomes inaccessible to the antibodies during those later stages of protein folding associated with recovery of enzyme activity and suggests that the protein may ‘tuck in its tail’ during one of the final steps.
- Published
- 1991
28. P.10.20 Recent and future additions to the MDA Monoclonal Antibody Resource for Neuromuscular Disorders
- Author
-
L. Le Thanh Lam, M. Nguyen thi Man, Glenn E. Morris, Caroline Sewry, Emma L. Humphrey, and Ian Holt
- Subjects
Neuromuscular disease ,medicine.drug_class ,Spinal muscular atrophy ,Biology ,medicine.disease ,Monoclonal antibody ,Myotonic dystrophy ,Fukutin ,Nemaline myopathy ,Neurology ,Pediatrics, Perinatology and Child Health ,Immunology ,Utrophin ,medicine ,biology.protein ,Neurology (clinical) ,Dystrophin ,Genetics (clinical) - Abstract
Specific antibodies are vital tools in the fight against neuromuscular disease, but no single antibody can perform all the necessary functions. Over a period of 20 years, we have produced large numbers of well-characterized antibodies for studies of the most common neuromuscular diseases (Duchenne/Becker and Emery–Dreifuss muscular dystrophies, spinal muscular atrophy and myotonic dystrophy), often in close collaboration with other research groups worldwide. This MDA Monoclonal Antibody (mAb) Resource currently contains nearly 400 different mAbs, including 150 exon-specific dystrophin mAbs. The antibody panels are currently used by the research community (a) for assessing the outcome of gene therapy, cell therapy, drug therapy or oligonucleotide therapy trials (e.g. dystrophin switch-on or alternative splicing) (b) for animal or cellular model systems to look for novel drug treatments (e.g. SMN or utrophin up-regulation) or novel approaches to gene/protein replacement, and (c) for basic research into understanding disease pathogenesis with the eventual aim of discovering novel treatment targets (e.g. muscleblind and msh3 in myotonic dystrophy). All mAbs are freely available for academic research, subject to an agreed form of acknowledgement in any publications. In most cases, the binding site for each mAb on the target proteins has been identified using epitope mapping techniques. The website: www.glennmorris.org.uk/mabs.htm has also been improved to enable automated antibody requests on-line. We now describe the most recent additions to the Resource, including new panels of mAbs against dystrophin, msh3, fukutin and nesprins 1 and 2. We will also describe collaborative projects to produce mAbs relevant to dystroglycanopathies, cardiomyopathies and nemaline myopathy for addition to the MDA Resource in future.
- Published
- 2013
29. P.1.17 Immunostaining of the sarcolemma with a new monoclonal antibody against alpha-dystroglycan core and its relevance to diagnosis
- Author
-
Tatsushi Toda, Francesca Sciandra, Susan C. Brown, Glenn E. Morris, E. Wilson, Ian Holt, Jane E. Hewitt, Kazuhiro Kobayashi, Caroline Sewry, E. Lacey, and Andrea Brancaccio
- Subjects
Sarcolemma ,Glycosylation ,medicine.drug_class ,Biology ,Monoclonal antibody ,Molecular biology ,Transmembrane protein ,Epitope ,Dystroglycans ,chemistry.chemical_compound ,Neurology ,Biochemistry ,chemistry ,Pediatrics, Perinatology and Child Health ,medicine ,biology.protein ,Neurology (clinical) ,Antibody ,Genetics (clinical) ,Immunostaining - Abstract
Alpha-dystroglycan ( α DG) is an extracellular, glycosylated protein attached to the outer plasma membrane by its interaction with the transmembrane protein, beta-dystroglycan. Genetic mutations that alter the glycosylation state of α DG cause a number of inherited disorders, including Fukuyama MD and certain congenital and limb-girdle MDs. Many of the pathogenic mutations are in proteins that carry out glycosylation of α DG and not in the DG gene itself. These enzymes are usually located in the Golgi where proteins are modified before export to the extracellular space. Amino-acid mutations in α DG itself can also cause congenital MD when sugar attachment to the α DG backbone is affected. Antibodies against dystroglycans immunostain the sarcolemma in a similar manner to dystrophin antibodies. It would be useful to the muscle pathologist to have monoclonal antibodies (mAbs) that recognize specific glycosylated forms of α DG associated with different mutations or disease states, since such mAbs would be useful for diagnosis using muscle biopsy sections. There are problems of reproducibility with existing mAbs against α DG, but it is still unclear whether those problems are attributable to the antibodies themselves or to the nature of the glycosylated epitopes on α DG. To determine whether it is possible to obtain improved mAbs for diagnostic use, we have begun a collaborative programme, supported by LGMD2I and CureCMD, to produce new mAbs against glycosylated and non-glycosylated forms of α DG. As a first step, we now describe a new mAb raised against bacterial recombinant α DG core sequence, which gives good staining of the sarcolemma in human muscle biopsy sections. We have mapped the epitope recognized by this mAb to short sequence that appears to be accessible to antibody in BOTH glycosylated and non-glycosylated forms of α DG.
- Published
- 2013
30. G.P.147
- Author
-
Catherine M. Shanahan, Caroline Sewry, L.T. Le, Qiuping Zhang, T.D. Nguyen, Glenn E. Morris, and Ian Holt
- Subjects
Nesprin ,biology ,Calponin ,Alternative splicing ,Skeletal muscle ,medicine.disease ,Actin cytoskeleton ,Molecular biology ,medicine.anatomical_structure ,Neurology ,KASH domains ,Pediatrics, Perinatology and Child Health ,medicine ,biology.protein ,Spectrin ,Neurology (clinical) ,Muscular dystrophy ,Genetics (clinical) - Abstract
Nesprin proteins have a central rod domain of spectrin repeats and are intracellular linkers and scaffolds. Full length or “giant” nesprin-1 and nesprin-2 have N-terminal calponin homology domains that bind the actin cytoskeleton and C-terminal transmembrane KASH domains which anchor the nesprins to the outer nuclear membrane. A number of short isoforms of nesprin-1 and nesprin-2 are produced by internal promotion and by alternative splicing. Mutations in nesprin-1 and nesprin-2 can cause Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. Nesprin-1-alpha-2 is found almost exclusively in skeletal muscle and heart and all the known mutations in nesprins that are associated with EDMD or DCM lie within the alpha isoform sequence. Two novel “epsilon” isoforms of nesprin-2 were predicted by bioinformatics and we have demonstrated the existence of corresponding proteins for the first time. Because they are similar to nesprin-1 alpha in size and structure, we evaluated the significance of the epsilon isoforms in 20 human tissues and 7 human cell lines using qPCR. N2-epsilon-1 was expressed only in ovary and early embryonic cells (Ntera-2 and ESC), while N2-epsilon-2 was expressed in several mature tissues, including cardiac, but not skeletal, muscle. Western blotting confirmed the presence of epsilon-1 protein in Ntera-2 and ESC and epsilon-2 protein in heart and brain. Total nesprin transcript in ESC was mainly nesprin-2-giant (77%) and nesprin-2-epsilon-1 (21%). PCR indicated that most of the nesprin-2 mRNA in ESC lacked the KASH domain, whereas heart and skeletal muscle appeared to contain high levels of nesprin-2 KASH. Immunofluorescence microscopy with a monoclonal antibody against nesprin-2 showed nuclear rim staining in heart and skeletal muscle sections, but in KASH-less ESC there was an intense nucleoplasmic speckle-like distribution. Supported by British Heart Foundation (PG/11/71/29091).
- Published
- 2014
31. G.P.321
- Author
-
Christopher Morris, Rita Barresi, Emma L. Humphrey, Caroline Sewry, Ian Holt, Jane E. Hewitt, Francesca Sciandra, Andrea Brancaccio, Susan C. Brown, Glenn E. Morris, E. Lacey, and L.T. Le
- Subjects
musculoskeletal diseases ,animal structures ,Glycosylation ,biology ,medicine.drug_class ,Duchenne muscular dystrophy ,Mucin ,musculoskeletal system ,Monoclonal antibody ,medicine.disease ,Molecular biology ,Epitope ,Dystroglycan complex ,chemistry.chemical_compound ,Neurology ,chemistry ,Antibody Repertoire ,Pediatrics, Perinatology and Child Health ,medicine ,biology.protein ,lipids (amino acids, peptides, and proteins) ,Neurology (clinical) ,Antibody ,Genetics (clinical) - Abstract
We have generated novel monoclonal antibodies (mAbs) against alpha-dystroglycan to immunolabel the sarcolemma in human muscle biopsies. For one of these, DAG-6F4, a seven amino-acid epitope, PNQRPEL, was identified using phage-displayed peptides and is located immediately after the highly-glycosylated mucin domain of alpha-dystroglycan. On western blots of recombinant alpha-dystroglycan, epitope accessibility was reduced, but not entirely prevented, by glycosylation. DAG-6F4 immunolabelling was markedly reduced in muscle biopsies from Duchenne muscular dystrophy patients consistent with a disruption in the dystroglycan complex. In a range of dystroglycanopathy patients with reduced/altered glycosylation, staining by DAG-6F4 was generally less reduced than staining by IIH6 (an antibody commonly used to identify glycosylated alpha-dystroglycan), though the extent of the differences between the two antibodies varied between different patients, some biopsies showing reductions in core protein as well as its glycosylation. A second mAb, DAG-3H9, gave similar results, but recognised a different epitope, GDRAP, closer to the C-terminus. There are currently few antibodies available against core alpha-dystroglycan, so DAG-6F4 represents a useful addition to the antibody repertoire for evaluating the dystroglycan complex in neuromuscular disorders.
- Published
- 2014
32. G.P.27
- Author
-
Glenn E. Morris, C.A. Sewry, L.T. Le, and T.M. Nguyen
- Subjects
Neuromuscular disease ,biology ,business.industry ,medicine.drug_class ,Genetic enhancement ,Spinal muscular atrophy ,medicine.disease ,Monoclonal antibody ,Bioinformatics ,Myotonic dystrophy ,Cell therapy ,Neurology ,Pediatrics, Perinatology and Child Health ,Immunology ,Utrophin ,biology.protein ,Medicine ,Neurology (clinical) ,business ,Dystrophin ,Genetics (clinical) - Abstract
Specific antibodies are vital research tools in the fight against neuromuscular disease, but no single antibody can perform all the necessary functions. Over a period of 20 years, we have produced large numbers of well-characterized antibodies for studies of the most common neuromuscular diseases (Duchenne/Becker and Emery-Dreifuss muscular dystrophies, spinal muscular atrophy and myotonic dystrophy). This MDA Monoclonal Antibody (mAb) resource currently contains over 350 different mAbs, including 150 exon-specific dystrophin mAbs. The antibody panels are currently used by the research community (a) for assessing the outcome of gene therapy, cell therapy, drug therapy or oligonucleotide therapy trials (e.g. dystrophin switch-on or alternative splicing) (b) for animal or cellular model systems to look for novel drug treatments (e.g. SMN or utrophin up-regulation) or novel approaches to gene/protein replacement, and (c) for basic research into understanding disease pathogenesis with the eventual aim of discovering novel treatment targets (e.g. muscleblind and msh3 in myotonic dystrophy). All mAbs are freely available for academic research, subject to an agreed form of acknowledgement in any publications. We now describe the most recent additions to the resource, including new mAbs against nebulin and nesprins 1 and 2. Technical advice freely available from the resource, including datasheets and protocols for producing and applying mAbs, will also be presented. The resource website can be found at www.glennmorris.org.uk/mabs.htm .
- Published
- 2014
33. P22 High content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC independent pathway in myotonic dystrophy cell lines
- Author
-
Ian Holt, J.D. Brook, Christopher J. Hayes, C.Z. Chen, S. Arya, Soraya Chaouch, B. Haworth, P. Shinn, I. Udosen, W. Zheng, T.E. Robinson, Denis Furling, Glenn E. Morris, N. Southall, C. Austin, X. Li, Ami Ketley, and J. Granados-Riveron
- Subjects
Biology ,medicine.disease ,Myotonic dystrophy ,Molecular biology ,Small molecule ,CELF1 Protein ,Neurology ,Cell culture ,High-content screening ,Pediatrics, Perinatology and Child Health ,medicine ,Distribution (pharmacology) ,Neurology (clinical) ,Genetics (clinical) ,Protein kinase C - Published
- 2014
34. D.P.4.09 Defective mRNA in myotonic dystrophy accumulates at the periphery of nuclear splicing speckles
- Author
-
Glenn E. Morris, Ian Holt, Gillian Butler-Browne, Denis Furling, J.D. Brook, and Saloni Mittal
- Subjects
Messenger RNA ,Neurology ,Chemistry ,Pediatrics, Perinatology and Child Health ,RNA splicing ,medicine ,Neurology (clinical) ,medicine.disease ,Myotonic dystrophy ,Genetics (clinical) ,Cell biology - Published
- 2008
35. P.5.18 Tissue-specific expression of nesprin isoforms and its relevance to muscular dystrophy and dilated cardiomyopathy
- Author
-
Caroline Sewry, Glenn E. Morris, Ian Holt, D. Nguyen Thuy, Catherine M. Shanahan, L. Le Thanh Lam, and Qiuping Zhang
- Subjects
Gene isoform ,Nesprin ,Cardiac muscle ,Emerin ,Skeletal muscle ,Biology ,medicine.disease ,Embryonic stem cell ,Molecular biology ,medicine.anatomical_structure ,Neurology ,Pediatrics, Perinatology and Child Health ,medicine ,Neurology (clinical) ,Muscular dystrophy ,Genetics (clinical) ,Lamin - Abstract
A combination of isoform-specific, quantitative RT-PCR and site-specific monoclonal antibodies has been used to produce consistent evidence at both RNA and protein levels for the principal isoform products of SYNE1 and SYNE2, the genes encoding nesprin-1 and nesprin-2 proteins. Western blots show that both genes produce three groups of isoforms that are homologous in size and structure: giant, or full-length, isoforms of about 1000 kDa, medium isoforms of about 380 kDa (nesprin-1-beta and nesprin-2-gamma) and short isoforms of about 110 kDa (nesprin-1-alpha and nesprin-2-epsilon). SYNE2 alone produces a very short isoform at 60 kDa (nesprin-2-alpha) in skeletal muscle. Although the giant forms were the dominant isoforms in all tissues, major differences between tissues in expression of shorter isoforms were observed. From SYNE1, nesprin-1-alpha was found in skeletal and cardiac muscle only. Several cell lines, including embryonic stem cells, did not produce any detectable nesprin-1 at all. From SYNE2, we observed a developmental transition from nesprin-2-epsilon-1 at very early stages (embryonic stem cells, embryonal teratocarcinoma and ovary tissue) to nesprin-2-epsilon-2 in adult and fetal tissues. Both skeletal muscle and heart have a high proportion of short isoforms compared with other tissues, but heart expresses the nesprin-2-epsilon-2 isoform where skeletal muscle produces nesprin-2-alpha-1. This study has clarified the major nesprin isoforms expressed in cells and tissues, especially in cardiac and skeletal muscle, which are the major affected tissues in neuromuscular disease. Mutations in nesprin-1 and/or nesprin-2 can cause both Emery-Dreifuss muscular dystrophy and dilated cardiomyopathy. The operative functional unit appears to be the “LINC” complex of emerin, lamin A/C, nesprins and SUN proteins, since mutations in any single component can result in a similar pathology.
- Published
- 2013
36. D.P.4.10 Muscleblind-like proteins: Similarities and differences in normal and myotonic dystrophy muscle
- Author
-
V. Jacquemin, Majid Fardaei, Ian Holt, Gillian Butler-Browne, Glenn E. Morris, J.D. Brook, Caroline Sewry, and Denis Furling
- Subjects
medicine.medical_specialty ,Endocrinology ,Neurology ,Internal medicine ,Pediatrics, Perinatology and Child Health ,medicine ,Neurology (clinical) ,Biology ,medicine.disease ,Myotonic dystrophy ,Genetics (clinical) - Published
- 2008
37. M.P.4.06 Model systems for developing therapies for McArdle disease
- Author
-
Caroline Sewry, K. Wright, Glenn E. Morris, and Ros Quinlivan
- Subjects
Oncology ,medicine.medical_specialty ,MCARDLE DISEASE ,Neurology ,business.industry ,Internal medicine ,Pediatrics, Perinatology and Child Health ,medicine ,Neurology (clinical) ,business ,Genetics (clinical) - Published
- 2007
38. Early onset X-linked Emery-Dreifuss muscular dystrophy resembling limb-girdle muscular dystrophy
- Author
-
E.J. Lichtarowicz-Krynska, Francesco Muntoni, Victor Dubowitz, D. Recan, Stéphane Llense, Caroline Sewry, J.-C. Kaplan, Glenn E. Morris, Jeremy M. G. Taylor, and S. Manilal
- Subjects
Neurology ,business.industry ,Pediatrics, Perinatology and Child Health ,X-Linked Emery-Dreifuss Muscular Dystrophy ,medicine ,Neurology (clinical) ,Anatomy ,medicine.disease ,business ,Genetics (clinical) ,Early onset ,Limb-girdle muscular dystrophy - Published
- 1997
39. A family with severe pseudo-dominant Emery-Dreifuss muscular dystrophy due to emerin deficiency
- Author
-
F. Leturcq, J. Bensaid, Jon Andoni Urtizberea, D. Recan, Glenn E. Morris, F. Fraisse, P. Warrot, Stéphane Llense, J.-C. Kaplan, N. Deburgrave, J.-M. Dupont, D. Amsallem, C. Giraudet, and J.-C. Barbot
- Subjects
Pathology ,medicine.medical_specialty ,Neurology ,business.industry ,Pediatrics, Perinatology and Child Health ,Emerin ,Medicine ,Neurology (clinical) ,Emery–Dreifuss muscular dystrophy ,business ,medicine.disease ,Genetics (clinical) - Published
- 1997
40. X-linked Emery-Dreifuss muscular dystrophy: molecular diagnosis by protein analysis and use of the skin biopsy in female carriers
- Author
-
Glenn E. Morris, J. Pradas, S. Manilal, M. Guitet, Jaume Colomer, J. Corbera, and J. Vila
- Subjects
Pathology ,medicine.medical_specialty ,Neurology ,medicine.diagnostic_test ,business.industry ,Pediatrics, Perinatology and Child Health ,Skin biopsy ,X-Linked Emery-Dreifuss Muscular Dystrophy ,medicine ,Neurology (clinical) ,business ,Genetics (clinical) - Published
- 1997
41. Exon-specific monoclonal antibodies against dystrophn for diagnosis and genetic deletion analysis in muscular dystrophy
- Author
-
Le Thiet Thanh, Glenn E. Morris, and Nguyen thi Man
- Subjects
Exon ,Neurology ,medicine.drug_class ,business.industry ,Pediatrics, Perinatology and Child Health ,medicine ,Neurology (clinical) ,Muscular dystrophy ,Monoclonal antibody ,business ,medicine.disease ,Molecular biology ,Genetics (clinical) - Published
- 1996
42. Monoclonal antibodies against emerin, for the diagnosis of emery-dreifuss muscular dystrophy
- Author
-
Glenn E. Morris, Sushila Maniw, Nguyen thi Man, and Caroline Sewry
- Subjects
Pathology ,medicine.medical_specialty ,business.industry ,medicine.drug_class ,Emerin ,medicine.disease ,Monoclonal antibody ,Neurology ,Pediatrics, Perinatology and Child Health ,medicine ,Neurology (clinical) ,Emery–Dreifuss muscular dystrophy ,business ,Genetics (clinical) - Published
- 1996
43. Asbestos fibre supply and the regulation of asbestos exposure
- Author
-
Glenn E. Morris and Stephen E. Margolis
- Subjects
Economics and Econometrics ,Sociology and Political Science ,Asbestos fibre ,Natural resource economics ,medicine ,Business ,Management, Monitoring, Policy and Law ,medicine.disease_cause ,Law ,Domestic market ,Asbestos - Abstract
The USA and Canada are each considering measures to reduce general exposure to asbestos. Product bans and workplace exposure rules may each reduce general exposure by reducing domestic asbestos demand. The effectiveness of demand-reducing measures will depend upon the elasticity of asbestos supply to the domestic market. This paper provides calculations of this elasticity for three fibre grade groupings, using a framework that recognizes international trade in asbestos and the joint product nature of most asbestos fibre production. The results suggest that reductions in North American demand result in relatively substantial changes in asbestos use but very small changes in price.
- Published
- 1986
44. Two-dimensional gel analysis of nuclear proteins during muscle differentiation in vitro
- Author
-
Nguyen thi Man, R.J. Cole, and Glenn E. Morris
- Subjects
Gel electrophoresis ,Lysis ,Cellular differentiation ,Phosphatase ,Cell Biology ,Biology ,Molecular biology ,Tropomyosin ,In vitro ,humanities ,chemistry.chemical_compound ,Tubulin ,chemistry ,Biochemistry ,Cytoplasm ,biology.protein ,Myocyte ,Phosphorylation ,Protein phosphorylation ,Nuclear protein ,Protein kinase A ,Actin ,DNA - Abstract
Isolated nuclei from chick skeletal muscle cell cultures at different stages of differentiation were labelled in vitro with γ-[ 32 P]ATP. Nuclear proteins were separated into saline-soluble and non-histone (NHP) fractions and analysed by two-dimensional gel electrophoresis and autoradiography. Relatively few proteins (10–12) were phosphorylated to any extent and most of these corresponded to minor, or even undetectable, components on stained gels. 32 P incorporation into most of these proteins increased between 24 and 40 h and decreased sharply between 40 and 66 h, the changes being more marked in some phosphoproteins than in others. Pulse-chase data suggest that these overall changes in phosphorylation reflect changes in protein kinase, rather than protein phosphatase activities. One highly phosphorylated acidic protein (mol. wt 10 000 D; pI 4.2) which was undetectable on stained gels, showed a continuous increase in 32 P incorporation between 24 and 66 h in both saline-soluble and NHP fractions. This protein may be the muscle counterpart of a phosphorylated nuclear protein partially characterized in other cell types. Both nuclear protein phosphorylation and low molecular weight non-histone proteins have been particularly implicated in activation of gene transcription in other systems, although we have no direct evidence, as yet, to connect them with control of myoblast growth and differentiation.
- Published
- 1980
45. Treatment of human muscle creatine kinase with glutaraldehyde preferentially increases the immunogenicity of the native conformation and permits production of high-affinity monoclonal antibodies which recognize two distinct surface epitopes
- Author
-
Alison J. Cartwright, Glenn E. Morris, Nguyen thi Man, and Karen M. Andrews
- Subjects
biology ,Protein Conformation ,medicine.drug_class ,Immunogenicity ,Immunology ,Antibody Affinity ,Antibodies, Monoclonal ,Monoclonal antibody ,Molecular biology ,Epitope ,Epitopes ,Epitope mapping ,Antigen ,Biochemistry ,Antibody Specificity ,Glutaral ,biology.protein ,medicine ,Native state ,Humans ,Immunology and Allergy ,Creatine kinase ,Antibody ,Creatine Kinase - Abstract
Treatment of human muscle creatine kinase (MM-CK) with glutaraldehyde produced highly aggregated forms which retained the native antigenic structure. Immunization of BALB/c mice with CK aggregates instead of untreated CK produced over ten-fold higher titres of antibody against native CK without increasing the tires of antibody against denatured enzyme. Production of high-affinity monoclonal antibodies specific for both the muscle isoenzyme and the native conformation became possible where the use of untreated CK had failed. Four monoclonal antibodies have been characterized by an epitope mapping technique and compared with a commercially available monoclonal antibody. One antibody has a much higher affinity for MM-CK than the other three and the commercial antibody. Competition studies show that it also recognizes a different epitope on the CK surface from the other three monoclonal antibodies which bind to the same surface region as the commercial antibody. Immunoassays based on the high affinity antibody can easily measure less than 1 ng of CK, a sensitivity comparable to, or better than, standard enzymatic assays.
- Published
- 1989
46. Monoclonal antibodies to intermediate filaments in chick muscle cell cultures
- Author
-
Linda P. Head and Glenn E. Morris
- Subjects
Hot Temperature ,Fluorescent Antibody Technique ,Muscle Proteins ,Vimentin ,Chick Embryo ,macromolecular substances ,chemistry.chemical_compound ,Intermediate Filament Proteins ,Antibody Specificity ,Animals ,Myocyte ,Isoelectric Point ,Intermediate filament ,Cells, Cultured ,Cytoskeleton ,Immunosorbent Techniques ,Actin ,biology ,Colcemid ,Myogenesis ,Muscles ,Demecolcine ,Antibodies, Monoclonal ,Cell Biology ,Molecular biology ,Molecular Weight ,chemistry ,biology.protein ,Desmin ,Myofibril - Abstract
Two monoclonal antibodies, FIFI and PHIL, have been prepared using detergent-washed myogenic cells as immunogen. On Western blots of total protein extracts of muscle cells, both antibodies bind to vimentin (52 kD) and its degradation products (major band at 42 kD), but do not bind to mouse proteins or to actin (42 kD). Specificity for a determinant common to vimentin and desmin was confirmed by 2-D gel electrophoresis of muscle cell extracts and purified desmin. Western blots with FIFI reveal particularly well the extreme sensitivity of intermediate filaments (IFs) to proteolysis, which was preventable in brain tissue only by boiling in 1% SDS, although it could be reduced in both brain and muscle by less extreme methods. Western blots suggest a large increase in IF content of differentiating myoblast cell cultures at the time of cell fusion and an increase of at least 4-fold is confirmed by a quantitative immunoassay using a direct ELISA method. Immunofluorescence microscopy shows that this increase is due to the appearance of high concentrations of the intermediate filament antigen at the ends of early myotubes, preceding the appearance of cross-striations in myofibrils. Furthermore, whereas the polar filaments detected by FIFI run right to the ends of the early myotubes and only sparingly penetrate the central area, cross-striated myofibrils (as detected by the monoclonal antibody, SAM) run the length of the myotube but do not reach the ends. Colcemid and colchicine cause the vimentin filaments in fibroblasts to collapse into perinuclear rings or caps, but do not have this effect on the polar fluorescence in early myotubes. Heat shock (2 h at 45 °C) has a similar differential effect. The results suggest that early in muscle differentiation intermediate filament proteins accumulate rapidly at myotube ends, where they are organized differently from those in fibroblasts.
- Published
- 1985
47. Protein kinase and cyclic AMP levels in differentiating myoblasts are altered by extracellular calcium
- Author
-
Nguyen Thi Mân and Glenn E. Morris
- Subjects
Biophysics ,Chick Embryo ,Biochemistry ,Ca2+/calmodulin-dependent protein kinase ,Cyclic AMP ,medicine ,Extracellular ,Animals ,Myocyte ,Protein kinase A ,Molecular Biology ,Cell Nucleus ,biology ,Akt/PKB signaling pathway ,Chemistry ,Muscles ,Skeletal muscle ,Cell Differentiation ,Cell Biology ,Kinetics ,medicine.anatomical_structure ,biology.protein ,Calcium ,Creatine kinase ,Protein Kinases ,cGMP-dependent protein kinase - Abstract
Protein kinase specific activities and cyclic AMP levels show a similar pattern of response, when the Ca2+ concentration is altered in the culture medium of differentiating chick skeletal muscle cells; an increase at intermediate Ca2+ concentrations (0.05–0.2mM), followed by a decrease at higher concentrations (2mM). Effects of Ca2+ on protein kinase appear to be on cyclic AMP-independent enzymes in both nucleus and cytoplasm, and are quite the reverse of Ca2+ effects on the muscle-specific enzyme, creatine kinase.
- Published
- 1980
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