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G E Morris

Publications and source records attributed to G E Morris.

At least 55 records · Page 3Linked to original sources
PubMed1998

Colocalization of emerin and lamins in interphase nuclei and changes during mitosis.

Emerin is a nuclear membrane protein which is affected by mutation in X-linked Emery-Dreifuss muscular dystrophy. We have previously suggested that emerin is a member of a family of type II integral membrane proteins which associate with the nuclear lamina and which include lamina-associated proteins and the lamin B receptor. We now show that emerin in COS cells is not restricted to the nuclear rim but is also found at intranuclear sites, where it colocalizes with nuclear lamins B1, B2 and A/C. During mitosis, emerin is dispersed throughout the cell and then participates in the reconstitution of membranes around the daughter nuclei. Although emerin and lamins do not remain colocalized during mitosis, they all show some association with the midbody of the mitotic spindle.

Animals↗
PubMed1998

An epitope structure for the C-terminal domain of dystrophin and utrophin.

The muscular dystrophy protein, dystrophin, and the closely related protein, utrophin, are large cytoskeletal proteins which link actin microfilaments to the plasma membrane. A panel of 38 monoclonal antibodies (mAbs) has been produced against the C-terminal domains of dystrophin and utrophin. This domain interacts with both dystrobrevins, via their "leucine zipper" coiled-coil helices, and syntrophins, adaptor proteins which also interact with nitric oxide synthetase and transmembrane sodium channels. The amino acid sequences recognized by the mAbs have now been identified using a variety of epitope mapping techniques, including fragmentation by transposon mutagenesis, synthetic peptides, phage-displayed peptide libraries, and mutant dystrophins expressed in transgenic mice. In addition to defining antibody recognition sites, mapping was sufficiently precise to provide structural information, since individual amino acids accessible on the surface of the native protein were identified in many cases. In two regions of the domain, short linear epitopes were found in proline-rich sequences which may form surface loops, turns, or linkers, but these were separated by a third region which contained mainly conformational epitopes. The results are consistent with a loose and flexible structure for much of the C-terminal domain, especially around the highly conserved second leucine zipper or coiled-coil helix (CC-H2), but there is evidence for denaturation-resistant tertiary structure in the syntrophin-binding region and the first coiled-coil helix (CC-H1).

Amino Acid Sequence↗
PubMed1998

Identification of antigenic sites on three hepatitis C virus proteins using phage-displayed peptide libraries.

A novel approach to screening phage-displayed peptide libraries has been used to identify hepatitis C virus (HCV) core, NS4 and NS5 sequences, which are antigenic in humans. Two random peptide libraries were used for screening using a mixture of HCV-positive sera or individual antibodies to core, NS3, NS4, and NS5 HCV proteins affinity-purified from this mixture. Sequencing of 56 selected phage clones resulted in 28 different peptide sequences and identification of seven antigenic regions, three in the core protein (19-26, 34-49, and 73-83), three in the NS4 (1681-1693, 1712-1718, and 1726-1736) and one in the NS5 protein (2251-2260). No NS3-specific peptides were identified. The immune response to core, NS4 and NS5 proteins includes a variety of linear determinants whereas epitopes on the investigated part of NS3 protein appear to be conformation-dependent.

Amino Acid Sequence↗
PubMed1998

Epitope mapping.

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Amino Acid Sequence↗
PubMed1998

Early presentation of X-linked Emery-Dreifuss muscular dystrophy resembling limb-girdle muscular dystrophy.

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/I) 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 alpha-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.

Age of Onset↗
PubMed1998

Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies.

There is considerable confusion in the literature about the size of the myotonic dystrophy protein kinase (DMPK) and its localization within tissues. We have used a new panel of monoclonal antibodies (mAbs) to begin to resolve these issues, which are important for understanding the possible role of DMPK in myotonic dystrophy. Antisera raised against the catalytic and coil domains of DMPK recognized a major 55 kDa protein and a minor 72-80 kDa doublet on western blots of human skeletal muscle. Ten mAbs, five against the catalytic domain and five against the coil region, recognized only the 72-80 kDa doublet. The 72 kDa protein was present in all tissues tested, whereas the 80 kDa component was variably expressed, mainly in skeletal and cardiac muscles. The 72 kDa protein was absent in a DMPK knockout mouse and was greatly increased in a transgenic mouse overexpressing human DMPK, confirming its identity as authentic DMPK. Two mAbs against the catalytic domain recognized only the more abundant 55 kDa protein, which was found only in skeletal muscle. Nine out of 10 mAbs located DMPK to intercalated discs in human heart, an affected tissue in myotonic dystrophy. However, co-localization of DMPK with acetylcholine receptors at neuromuscular junctions was not observed with any of the mAbs. Subcellular fractionation and sedimentation analysis suggest that a major proportion of the DMPK in skeletal muscle and brain is cytosolic.

Animals↗
PubMed1998

Mutations in Emery-Dreifuss muscular dystrophy and their effects on emerin protein expression.

Seventeen families with Emery-Dreifuss muscular dystrophy (EDMD) have been studied both by DNA sequencing and by emerin protein expression. Fourteen had mutations in the X-linked emerin gene, while three showed evidence of autosomal inheritance. Twelve of the 14 emerin mutations caused early termination of translation. An in-frame deletion of six amino acids from the C-terminal transmembrane helix caused almost complete absence of emerin from muscle with no localization to the nuclear membrane, although mRNA levels were normal. This shows that mutant emerin proteins are unstable if they are unable to integrate into a membrane. A 22 bp deletion in the promoter region was expected to result in reduced emerin production, but normal amounts of emerin of normal size were found in leucocytes and lymphoblastoid cell lines. This shows that DNA analysis is necessary to exclude emerin mutations in suspected X-linked EDMD. Emerin levels in female carriers often deviated from the expected 50% and this was due, in at least two families, to skewed emerin mRNA expression from the normal and mutated alleles. In one family with a novel deletion of the last three exons of the emerin gene, a carrier had a cardiomyopathy and very low emerin levels (<5% of normal) due to skewed X-inactivation. In the three autosomal cases of EDMD, emerin was normal on western blots of blood cells, which suggests that autosomal EDMD is not caused by indirect reduction of emerin levels.

Adolescent↗
PubMed1997

The molecular basis for cross-reaction of an anti-dystrophin antibody with alpha-actinin.

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.

Actinin↗
PubMed1997

Laminin-induced clustering of dystroglycan on embryonic muscle cells: comparison with agrin-induced clustering.

The effect of laminin on the distribution of dystroglycan (DG) and other surface proteins was examined by fluorescent staining in cultures of muscle cells derived from Xenopus embryos. Western blotting confirmed that previously characterized antibodies are reactive in Xenopus. In control cultures, alphaDG, betaDG, and laminin binding sites were distributed as microclusters (<1 microm2 in area) over the entire dorsal surface of the muscle cells. Treatment with laminin induced the formation of macroclusters (1-20 microm2), accompanied by a corresponding decline in the density of the microclusters. With 6 nM laminin, clustering was apparent within 150 min and near maximal within 1 d. Laminin was effective at 30 pM, the lowest concentration tested. The laminin fragment E3, which competes with laminin for binding to alphaDG, inhibited laminin-induced clustering but did not itself cluster DG, thereby indicating that other portions of the laminin molecule in addition to its alphaDG binding domain are required for its clustering activity. Laminin-induced clusters also contained dystrophin, but unlike agrin-induced clusters, they did not contain acetylcholine receptors, utrophin, or phosphotyrosine, and their formation was not inhibited by a tyrosine kinase inhibitor. The results reinforce the notion that unclustered DG is mobile on the surface of embryonic muscle cells and suggest that this mobile DG can be trapped by at least two different sets of molecular interactions. Laminin self binding may be the basis for the laminin-induced clustering.

Agrin↗
PubMed1997

Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase.

Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 degrees C, but not at 12 degrees C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.

Amino Acid Sequence↗
PubMed1997

Diagnosis of X-linked Emery-Dreifuss muscular dystrophy by protein analysis of leucocytes and skin with monoclonal antibodies.

The X-linked form of Emery-Dreifuss muscular dystrophy (EDMD) was recently shown to be due to mutations in the STA gene on chromosome Xq28. We have demonstrated a simple test for the diagnosis of this condition, looking for altered expression of the protein, emerin, in leucocytes and skin with a monoclonal antibody. Full-length emerin is completely absent in affected boys from the EDMD families studied. The method has also enabled identification of a female carrier of the disease by reduced levels of the protein on the leucocyte Western blot and a mosaic pattern of expression by immunofluorescence microscopy of the skin biopsy.

Adolescent↗
PubMed1997

Dystrophin is replaced by utrophin in frog heart; implications for muscular dystrophy.

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.

Animals↗
PubMed1996

Utrophin-dystroglycan complex in membranes of adherent cultured cells.

In skeletal muscle, dystrophin binds to an oligomeric, transmembrane complex (DAGc; dystrophin-associated glycoprotein complex) which interacts with laminin in the extracellular matrix. We now present biochemical evidence for an association between utrophin (dystrophin-related protein, DRP) and a major DAGc component, beta-dystroglycan (43DAG) in cultured cell lines which contain little if any dystrophin. We have shown also that utrophin and beta-dystroglycan co-localise at or near the plasma membrane and that they co-sediment in large complexes on sucrose density gradients. On the lower plasma membrane, in contact with the substratum, part of the utrophin and beta-dystroglycan staining co-localised with alpha-actinin in a punctate distribution outside classical vinculin-rich focal adhesions. beta-dystroglycan, utrophin, syntrophin (59DAP), and alpha-actinin were found in all adhesion-competent cell lines studied, but levels of the last three proteins were greatly reduced in myeloma cells, which cannot readily attach to substrata. Possible roles for utrophin in cultured cells are considered in the light of recent evidence for involvement of utrophin-glycoprotein complexes in muscle in signal transduction and recruitment of acetylcholine receptors to neuromuscular junctions.

Actinin↗
PubMed1996

The Emery-Dreifuss muscular dystrophy protein, emerin, is a nuclear membrane protein.

A large fragment of emerin cDNA was prepared by PCR and expressed as a recombinant protein in Escherichia coli. Using this as immunogen, we prepared a panel of 12 monoclonal antibodies which recognise at least four different epitopes on emerin in order to ensure that emerin can be distinguished from non-specific cross-reacting proteins. All the mAbs recognised a 34 kDa protein in all tissues tested, though minor emerin-related bands were also detected in some tissues. Immunofluorescence microscopy showed that emerin is located at the nuclear rim in all tissues examined. A muscle biopsy from an Emery-Dreifuss muscular dystrophy (EMDM) patient showed complete absence of emerin by both Western blotting and immunohistochemistry, suggesting a simple diagnostic antibody test for EDMD families. Biochemical fractionation of brain and liver tissues showed that emerin was present in nuclei purified by centrifugation through 65% sucrose and was absent from soluble fractions (post-100,000 g). From these results, together with sequence and structural homologies between emerin, thymopoietins and the nuclear lamina-associated protein, LAP2, we suggest that emerin will prove to be one member of a family of inner nuclear membrane proteins.

Adult↗