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Nuclear speckles are storage sites for small nuclear RNPs (snRNPs) and other splicing factors. Current ideas about the role of speckles suggest that some pre-mRNAs are processed at the speckle periphery before being exported as mRNA. In myotonic dystrophy type 1 (DM1), the export of mutant DMPK mRNA is prevented by the presence of expanded CUG repeats that accumulate in nuclear foci. We now show that these foci accumulate at the periphery of nuclear speckles. In myotonic dystrophy type 2 (DM2), mRNA from the mutant ZNF9 gene is exported normally because the expanded CCUG repeats are removed during splicing. We now show that the nuclear foci formed by DM2 intronic repeats are widely dispersed in the nucleoplasm and not associated with either nuclear speckles or exosomes. We hypothesize that the expanded CUG repeats in DMPK mRNA are blocking a stage in its export pathway that would normally occur at the speckle periphery. Localization of the expanded repeats at the speckle periphery is not essential for their pathogenic effects because DM1 and DM2 are quite similar clinically.

SIX5 is a member of the human SIX family of transcription factors, many of which are involved in eye development. However, SIX5 transcripts are known to be present at very low levels in cells and no study has yet convincingly demonstrated detection of endogenous SIX5 protein by Western blotting or immunolocalisation. We have produced a new panel of 18 monoclonal antibodies (mAbs) that recognise at least four different epitopes in order to identify authentic human SIX5 protein in cells and tissues. Phage-displayed peptide libraries were used to identify individual amino-acids important for antibody binding within each epitope. Endogenous SIX5 migrated in SDS-PAGE with an apparent M(r) of 100 kDa and was present at similar levels in all foetal tissues and cell lines tested. In HeLa cells, it was located in the nucleoplasm with a granular distribution. An mRNA for a shorter splicing isoform of SIX5, with an altered carboxy-terminus, has been described, but further mAbs specific for this isoform did not detect any endogenous protein. We conclude that the full-length isoform is the major functional protein in vivo while the putative shorter protein is undetectable and may not be expressed at all.

We present our observations on the skeletal muscle pathology of nine cases from seven families of autosomal dominant Emery-Dreifuss muscular dystrophy (ADEDMD) with identified mutations in the lamin A/C gene, aged 2-35 years at the time of biopsy. The severity of pathological change was moderate and the most common features were variation in fibre size (hypertrophy and atrophy), an increase in internal nuclei and smaller diameter fibres with high oxidative enzyme activity. Only one case showed necrosis, which was present in two separate samples taken from the quadriceps and tibialis anterior, at different ages. Immunocytochemistry detected an age-related reduction of laminin beta1 on the muscle fibres in adolescent and adult cases. Antibodies to lamins A and A/C, and emerin did not reveal any detectable differences from controls. Electron microscopy of two out of three cases showed an abnormal distribution of heterochromatin in many fibre nuclei. Our results show that dystrophic changes in skeletal muscle are not a major feature of ADEDMD, and that nuclear abnormalities may be detected with electron microscopy. Immunodetection of reduced laminin beta1 may be a useful secondary marker in adults with this disorder, as immunocytochemistry of lamins is not yet of diagnostic use.

A novel method for cloning DNase I fragments into bacteriophage display vector fUSE2 was used to create libraries expressing hepatitis C virus (HCV) protein fragments on the phage surface. Selection by panning with a mixture of sera from five HCV-seropositive individuals enabled identification of antigenic determinants in NS3 (amino acids 1,383-1,415), NS4 (amino acids 1, 930-1,938), and NS5 (amino acids 2,088-2,104). The NS3 result is the most accurate location to date of a major conformational determinant that cannot be mimicked by short peptides. Any expressed sequence from the phage library can be excised with Bgl II and cloned directly into the Bgl II site of an appropriate plasmid for bacterial expression. This enables production of chimeric proteins containing multiple antigenic determinants, illustrated by co-expression of the NS4P (amino acids 1,930-1,938) epitope with an NS4N fragment (amino acids 1,644-1,812) containing at least three linear HCV epitopes. When used to screen 35 individual HCV-positive sera by enzyme-linked immunosorbent assay (ELISA), the chimeric antigen detected eight more positives than NS4N alone and gave increased immunoreactivity with others. This approach of identifying antigenic regions by phage display and then co-expressing them as chimeric proteins may be generally applicable to the production of improved diagnostic antigens and recombinant vaccines.

The homodimeric muscle isoform of creatine kinase (MM-CK) unfolds on exposure to low levels of guanidinium chloride (GdmCl) to yield a partly folded monomeric intermediate. Those regions of MM-CK that experience local unfolding were previously identified through an extensive study of antibody accessibility and protease sensitivity. Since these studies were completed, the coordinates of the rabbit isoform (MM-CK) were released. In light of this, we have determined the minimum changes to this structure required to explain our data on protease and epitope accessibility in the intermediate. We propose that the observed changes occur through (a) disruption of the monomer-monomer interface during dissociation, (b) separation and/ or unfolding of domains or subdomains, and (c) the partial unfolding of solvent-exposed helices. The proposed structure for the intermediate is consistent both with current models of unfolding intermediates and the results of independent studies pertaining to the unfolding of creatine kinase. Proteins 2001;42:269–278. © 2000 Wiley-Liss, Inc.

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-dependant. J. Med. Virol. 56:105–111, 1998. © 1998 Wiley-Liss, Inc.

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, β-dystroglycan (43DAG) in cultured cell lines which contain little if any dystrophin. We have shown also that utrophin and β-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 β-dystroglycan staining co-localised with α-actinin in a punctate distribution outside classical vinculin-rich focal adhesions. β-dystroglycan, utrophin, syntrophin (59DAP), and α-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. © 1996 Wiley-Liss, Inc.

Emery–Dreifuss muscular dystrophy (EDMD) is a rare neuromuscular disorder typically characterised by early contractures, slowly progressive muscular wasting, and life-threatening heart conduction disturbances, which can develop into a cardiomyopathy. Some severe neonatal cases have also been identified. There is a wide intrafamilial and interfamilial clinical variability. Genetically, X-linked recessive (EMD1 and EDMD6), autosomal dominant (EMD2, EDMD4 and EDMD5) and autosomal recessive (EMD3) forms can be distinguished. By molecular genetic methods, EDMD can be associated with mutations in the STA (emerin gene symbol), LMNA (lamin A/C gene symbol), SYNE (nesprin gene symbol)1, SYNE2 and Four and a Half LIM Domain 1 (FHL1) genes. Female carriers of the X-linked forms may manifest with cardiac symptoms but heterozygous carriers of the autosomal forms do not show symptoms. Only approximately 46% of unrelated EDMD patients have a mutation in known genes pointing to further genetic heterogeneity in EDMD. The molecular pathogenesis is unresolved, but there is evidence for gene expression changes via altered nuclear signalling and for reduced resistance of muscles to physical damage during contraction. Key Concepts: Emery–Dreifuss muscular dystrophy (EDMD) is a neuromuscular disorder recognised clinically by three features: early contractures, a characteristic pattern of muscle wasting and cardiac conduction defects. EDMD was originally described as an X-linked disorder, later found to be caused by mutations in the STA gene encoding a nuclear membrane protein, emerin. Molecular genetic analysis allows precise subtyping of EDMD into X-linked forms (STA and FHL1 associated), autosomal dominant forms (LMNA, SYNE1/SYNE2 associated) and an autosomal recessive form (LMNA associated). Wide clinical variability of EDMD and clinical overlap with other clinical entities frequently require consideration of patients not completely fulfilling the EDMD diagnostic criteria for molecular genetic differentiation in STA, LMNA, SYNE1/SYNE2 and FHL1. Digenic pathogenesis has been observed including LMNA/STA, LMNA/DES and SYNE1/SYNE2. In most cases, EDMD mutations affect proteins located in the nuclear membrane where they interact with each other. Many patients do not have mutations in genes identified so far, so further genes are expected to be involved in the pathogenesis of EDMD. Keywords: Emery–Dreifuss muscular dystrophy; contractures; cardiomyopathy; STA; SYNE; FHL1; emerin; lamin A/C; nesprin; nuclear membrane

Using monoclonal antibodies against the M and B subunit isoforms of creatine kinase (CK) we have investigated their distribution in developing human skeletal and cardiac muscle immunohistochemically. It is demonstrated that in skeletal muscle, a switch from CK-B to CK-M takes place around the week 8 of development, whereas in the developing heart, CK-M is the predominant isoform from the earliest stage examined onward (i.e., 4 1/2 weeks of development). In all hearts examined, local differences in concentration of the CK isoforms are observed. The CK-M expression in the developing outflow tract (OFT) and conduction system is described in detail. Between the weeks 5 and 7 of development, the distal portion of the OFT is characterized by low CK-M expression, whereas around the week 8-10 of development the myocardium around the developing semilunar valves in the OFT expresses a very high level of CK-M. At all stages examined, a relatively low CK-M level is observed in those regions in which the "slow" components of the conduction system do develop (e.g., the sinoatrial junction and atrioventricular junction), whereas a relatively high concentration of CK-M is observed in those areas that are destined to become the "fast" components, i.e., the subendocardial myocardium of the ventricles. The high expression of CK-M in the developing "fast components" of the conduction system contrasts with the relatively low expression of CK-M in the force-producing myocardium of the interventricular septum and free ventricular wall.

A cell preparation method by which large numbers of embryonic chick skeletal muscle cells may be obtained is described. The procedure requires fewer manipulations and much less time than standard trypsinization. By the criteria used, both methods are comparable with respect to percent viable cells and survival of plated cells. However, in addition to the ease of preparation, the mechanical dissociation method offers the significant advantage that the cell suspension is greatly enriched for myoblasts without the necessity of an additional preplating step.