Nebulin is a giant sarcomeric protein, binding to actin along the length of the thin filament. Mutations in NEB are the most common cause of autosomal recessive nemaline myopathy (NM). NEB has 183 exons, and undergoes extensive alternative splicing, giving rise to numerous different protein isoforms. The protein structure is modular, with 32-35 amino-acid actin-binding simple repeats forming 26 different super repeats spanning most of the protein. Two alternative isoforms of super repeat S21, S21a and S21b, are encoded by two mutually exclusive exons, exon 143 and exon 144, respectively. S21a and S21b differ in both charge and hydrophobicity. The amino-acid sequence encoded by exon 143 shows complete homology between mouse, rat and human, indicating a central, conserved role for this region of the nebulin protein. To study the expression and function of these developmentally and clinically intriguing alternatively spliced exons, we have developed specific monoclonal antibodies, NEB-143 and NEB-144, against the isoforms containing S21a or S21b, respectively. We have previously shown that nebulin containing exon 144 is the first isoform expressed in myogenesis in vitro, while regulated expression of nebulin containing exon 143 occurs at later stages of muscle development. In this study, we investigated the expression of the S21a (exon 143) nebulin isoform further, in several adult muscles and myofibre types. Our results revealed an interesting pattern of alternating correlation between the expression of S21a and different types of MyHC myofibres, both slow and fast, with preferential expression in fast, MyHC IIX fibres.

O-linked glycosylation of the central mucin-rich domain of alpha dystroglycan is key to mediating the binding to laminin, agrin and perlecan in the extracellular matrix. Mutations in up to 18 genes have now been associated with this group and are responsible for a spectrum of clinical phenotypes that range from Walker–Warburg syndrome and Muscle–Eye–Brain disease to milder forms of muscular dystrophy with onset in childhood or adult life. However, whilst the glycosylation status of alpha dystroglycan may be assessed using IIH6 which identifies a laminin binding epitope there are only a limited number of antibodies available to the core protein, an evaluation of which could shed new light on the pathogenesis of these disorders. Recently a new panel of monoclonal antibodies against the core epitope of alpha dystroglycan have been generated which have now been characterised using immunocytochemistry in both mouse and human tissue. All monoclonals react with unfixed frozen mouse and human muscle in addition to paraffin wax embedded tissue making them useful for both research and evaluation of the core protein in diagnostic biopsies. An analysis of the pattern of immunolabelling using these new antibodies in the skeletal muscle of various dystroglycanopathy mouse models and human skeletal muscle biopsies will be presented.

McArdle Disease (Glycogen Storage Disease V) is a rare inherited metabolic muscle disease caused by mutations in the Muscle Glycogen Phosphorylase (MGP) gene. MGP (myophosphorylase) is a muscle-specific enzyme needed for glycogen metabolism. The most common mutations in Caucasian patients are the early stop codon R49X (incidence around 80%) and missense G204S (incidence around 10%) mutations. Existing animal models do not have these mutations and there are no existing cell culture models of McArdle’s Disease, except from patient muscle biopsies. In order to study therapeutic approaches such as correction or read-through of the these mutations, we have made cell culture models of McArdle Disease using in vitro site-directed mutagenesis to introduce the R49X and G204S mutations into full length wildtype mouse MGP cDNA in the mammalian expression vector pCI-neo. Green fluorescent protein (GFP)-R49X and GFP-G204S mutant MGP constructs were also made to assess the stability of the RNA and production of protein. Stable cell lines were created by transfection into CHO-K1 cells, which does not express endogenous MGP. RT-PCR has shown that MGP mRNA is being transcribed by cells transfected with mutant R49X and G204S, and with GFP-R49X and GFP-G204S MGP. Work is in progress to quantify levels of mRNA expression. MGP was detected immunologically in cells with wildtype MGP but not in cells with R49X or G204S mutant MGP or GFP R49X or G204S mutant MGP fusion constructs. The cell models may be useful in identifying drugs or other treatments that might enable functional MGP production from the mutant form of the gene. This study was supported by Association for Glycogen Storage Disease and the RJAH Institute of Orthopaedics.

Since the MDA Monoclonal Antibody Resource was set up over two years ago, it has distributed over 600 units of antibody to 136 laboratories in 15 countries. The available antibodies are listed on a new website at (www.rjah.nhs.uk/cind/MonoclonalAntibodyDatabase/tabid/303/Default.aspx) and include mAbs for research into Duchenne/Becker muscular dystrophy, Emery–Dreifuss MD, spinal muscular atrophy and myotonic dystrophy. Datasheets are now available on-line for nearly every antibody; they are continually up-dated and provide information on validated applications, epitope mapping, species-specificity and any known cross-reaction problems. They also show examples of western blot and immunolocalization applications and give references to published uses. Further recent developments include (a) trials of Zenon technology, which enables double-labelling with two or more mouse mAbs and avoids secondary antibody background staining when mouse mAbs are used on mouse tissue sections, and (b) further expansion of the dystrophin mAb library with mapped antibodies against exons 57–60. This project is supported by the Muscular Dystrophy Association (USA).

Alzheimer's disease is classified as a degenerative dementia while vascular dementia is known to be associated with atherothrombosis and classical vascular risk factors. However, over the last decade, there is increasing evidence of the role of haemostatic factors and inflammatory mechanisms in the pathogenesis of Alzheimer's disease. Serum markers of hypercoagulability and markers of inflammation could lead to thrombosis, accelerated atherogenesis and resulting dementia of both Alzheimer's and vascular dementia. In this case control study, we studied these serum markers of coagulation and inflammation in patients suffering from dementia.

In 1994, residents of Marietta, Georgia, participated in a pay-as-you-throw solid waste demonstration project. Rather than pay a fixed monthly fee for collection, half of the residents pain a fee per reusable trash can, and half paid for each nonreusable trash bag collected. Data from both a sample of households and city-wide totals indicate that the programs significantly reduced waste set-outs, even after accounting for increases in (unpriced) recycling. The bag program caused larger reductions (36%) than the subscription can program (14%). Rough estimates of the program indicate both savings for residents and social welfare increases. (JEL Q20).

This paper describes the Fiscal Environmental Integration Model (FEIM) and its use to examine the merits of introducing a set of air pollutant emission taxes and stringent abatement requirements based on best commonly available control technology. These environmental protection strategies are examined both independently and in combination. In addition, Hungary has very high VAT, employment, and income tax rates and therefore might receive more than the usual advantage from using environmental tax revenues to reduce other taxes. We therefore also examine the economic and environmental implications of different uses of the revenues generated by the air pollutant emission taxes. FEIM is a CGE model of the Hungarian economy that includes sectoral air pollution control costs functions and execution options that allow examination of the key policy choices involved. We developed and ran a baseline and seven scenarios with FEIM. The scenarios centered on introduction of environmental load fees (ELF) on emissions of SO2, NOx, and particulates and emission abatement requirements (EAR) for these pollutants.

Epitope mapping of proteins defines the structural elements of a molecule needed for recognition by individual antibodies. X-ray crystallography of anti- body-antigen complexes (1) is perhaps the most precise means of defining an epitope. However, several quicker but less detailed methods are also available. These alternative approaches include fragmentation of the antigen by chemical or proteolytic cleavage (2), chemical synthesis of peptide fragments (3), competition between antibodies (4), protection by antibody against proteolytic cleavage (5) or against chemical modification (6), and natural variants in different tissues or species (7). With antigens expressed from cDNA in bacterial plasmids, antigenic fragments can be produced by deletion mutagenesis with restriction enzymes (8) or exonucleases (9), and by construction of epitope libraries of random cDNA fragments in bacteriophage (10) or plasmid (11) vectors. Site-directed mutagenesis can be used to define important amino acid residues in epitopes (12). These methods, however, involve extensive in vitro manipulations or are limited by available sites within the cloned sequence. A simple and inexpensive in vivo alternative (13), which is not constrained by restriction site availability, uses the bacterial transposon, Tn1000 (14), to interrupt a coding sequence. Tn1000 introduces early termination codons and, hence, generates a library of clones expressing N-terminal protein fragments with progressively fewer epitopes as they get shorter (Fig. 1).

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K 'nicks' native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to 'nicked' CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.

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.