Monoclonal antibodies for clinical trials of Duchenne muscular dystrophy therapy.

Most pathogenic mutations in Duchenne and Becker muscular dystrophies involve deletion of single or multiple exons from the dystrophin gene, so exon-specific monoclonal antibodies (mAbs) can be used to distinguish normal and mutant dystrophin proteins. In Duchenne therapy trials, mAbs can be used to identify or rule out dystrophin-positive "revertant" fibres, which have an internally-deleted dystrophin protein and which occur naturally in some Duchenne patients. Using phage-displayed peptide libraries, we now describe the new mapping of the binding sites of five dystrophin mAbs to a few amino-acids within single exons.

Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies.

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.

Monitoring duchenne muscular dystrophy gene therapy with epitope-specific monoclonal antibodies.

Several molecular approaches to Duchenne muscular dystrophy (DMD) therapy are at or near the point of clinical trial and usually involve attempts to replace the missing dystrophin protein. Although improved muscle function is the ultimate measure of success, assessment of dystrophin levels after therapy is essential to determine whether any improved function is a direct consequence of the treatment or, in the absence of improved function, to determine whether new dystrophin is present, though ineffective. The choice of a monoclonal antibody (mAb) to distinguish successful therapy from naturally occurring "revertant" fibres depends on which dystrophin exons are deleted in the DMD patient.

Valproate and bone loss: iTRAQ proteomics show that valproate reduces collagens and osteonectin in SMA cells.

Valproate is commonly used as an anticonvulsant and mood stabilizer, but its long-term side-effects can include bone loss. As a histone deacetylase (HDAC) inhibitor, valproate has also been considered for treatment of spinal muscular atrophy (SMA). Using iTRAQ labeling technology, followed by two-dimensional liquid chromatography and mass spectrometry analysis, a quantitative comparison of the proteome of an SMA cell line, with and without valproate treatment, was performed.

The SMN interactome includes Myb-binding protein 1a.

Understanding networks of interacting proteins is a major goal in cell biology. The survival of motor neurons protein (SMN) interacts, directly or indirectly, with a large number of other proteins and reduced levels of SMN cause the inherited disorder spinal muscular atrophy (SMA). Some SMN interactions are stable and stoichiometric, such as those with gemins, while others are expected to be transient and substoichiometric, such as the functional interaction of SMN with coilin in Cajal bodies.

A two-site ELISA can quantify upregulation of SMN protein by drugs for spinal muscular atrophy.

Objectives: Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons during early or postnatal development. Severity is variable and is inversely related to the levels of survival of motor neurons (SMN) protein. The aim of this study was to produce a two-site ELISA capable of measuring both the low, basal levels of SMN protein in cell cultures from patients with severe SMA and small increases in these levels after treatment of cells with drugs.

Endosomal location of dopamine receptors in neuronal cell cytoplasm.

Five subtypes of dopamine receptor exist in two subfamilies: two D(1)-like (D(1) and D(5)) and three D(2)-like (D(2), D(3) and D(4)). We produced novel monoclonal antibodies against all three D(2)-like receptors and used them to localize receptors in Ntera-2 (NT-2) cells, the human neuronal precursor cell line. Most of the immunostaining for all three receptors colocalized with mannose-6-phosphate receptor, a marker for late endosomes formed by internalization of the plasma membrane.

Lamin A/C assembly defects in Emery-Dreifuss muscular dystrophy can be regulated by culture medium composition.

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.

Characterisation of the transcription factor, SIX5, using a new panel of monoclonal antibodies.

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.

Indoprofen upregulates the survival motor neuron protein through a cyclooxygenase-independent mechanism.

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 approximately 47,000 compounds to identify those that increase production of an SMN2-luciferase reporter protein, but not an SMN1-luciferase reporter protein.

Effect of pathogenic mis-sense mutations in lamin A on its interaction with emerin in vivo.

Mutations in lamin A/C can cause Emery-Dreifuss muscular dystrophy (EDMD) or a related cardiomyopathy (CMD1A). Using transfection of lamin-A/C-deficient fibroblasts, we have studied the effects of nine pathogenic mutations on the ability of lamin A to assemble normally and to localize emerin normally at the nuclear rim. Five mutations in the rod domain (L85R, N195K, E358K, M371K and R386K) affected the assembly of the lamina.

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.

Characterization of genetic deletions in Becker muscular dystrophy using monoclonal antibodies against a deletion-prone region of dystrophin.

We have produced a new panel of 20 monoclonal antibodies (mAbs) against a region of the dystrophin protein corresponding to a deletion-prone region of the Duchenne muscular dystrophy gene (exons 45-50). We show that immuno-histochemistry or Western blotting with these "exon-specific" mAbs can provide a valuable addition to Southern blotting or PCR methods for the accurate identification of genetic deletions in Becker muscular dystrophy patients. The antibodies were mapped to the following exons: exon 45 (2 mAbs), exon 46 (6), exon 47 (1), exons 47/48 (4), exons 48-50 (6), and exon 50 (1).

Epitope mapping of recombinant antigens by transposon mutagenesis.

We describe a method for generating a plasmid library expressing random truncations of a recombinant protein and for epitope mapping by screening the library with monoclonal antibodies. The key step is the random introduction of the transposon, Tn1000, which carries stop codons in all three reading frames, into a bacterial expression plasmid by using a simple bacterial mating procedure. Antibody-positive clones are then selected and the point of protein truncation is determined by sequencing the plasmid DNA at the point of transposon insertion.

Specificity and VH sequence of two monoclonal antibodies against the N-terminus of dystrophin.

We have used a random library of 15-mer peptides expressed on phage to show that two monoclonal antibodies (mAbs) require only the first three amino acids of dystrophin (Leu-Trp-Trp) for binding. Since the mAbs recognize dystrophin in frozen muscle sections, the results suggest that this hydrophobic N-terminus of dystrophin is accessible to antibody in situ. Quantitative binding studies suggested minor differences in specificity between the two mAbs, so the Ig heavy-chain variable region (VH) sequences of the two hybridomas were determined by RT-PCR and cDNA sequencing.

Immunoreactivity of skate electrocytes towards monoclonal antibodies against human dystrophin and dystrophin-related (DMDL) protein.

Monoclonal antibodies against human dystrophin have been used to demonstrate the existence of a dystrophin-like protein in the electrocytes of skate electric organ. This protein is also present in skate muscle and resembles that found in Torpedo electric organ. Monoclonal antibodies against a human autosomal homologue of dystrophin (DMDL protein) did not detect a similar protein in skate or Torpedo.

Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies.

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.

Monoclonal antibodies against defined regions of the muscular dystrophy protein, dystrophin.

Nineteen monoclonal antibodies which bind to native dystrophin in the plasma membrane of frozen muscle sections were obtained using a recombinant fusion protein as immunogen. On Western blots of normal mouse muscle extracts, the antibodies bind specifically to a 400,000 Mr protein which is absent from dystrophic mouse (mdx) muscle. At least four distinct epitopes have been identified by cleavage mapping methods.

Monoclonal antibodies against a nucleolar protein from differentiating chick muscle cells.

Two monoclonal antibodies, NORM and NORA, bind specifically to a 37 000 molecular weight protein in total protein extracts of chick skeletal muscle cell cultures. They have been used to follow changes in the amount and distribution of this protein during terminal differentiation and the associated cessation of cell division. Immunofluorescence microscopy shows that the antigen is found almost exclusively in the nucleolus in interphase cells and is dispersed in the cytoplasm during mitosis.