A prospective survey of patients undergoing day surgery was performed with the main aim of identifying the need for post-operative follow-up after minor or intermediate surgery. The degree of patient satisfaction and the increased workload that this group of patients caused general practitioners was also analysed. Patients underwent either varicose vein surgery, inguinal hernia repair or vasectomy and were asked to return a prepaid reply questionnaire two weeks after surgery. Ninety-eight patients were recruited into the survey, 89 (90.8 per cent) correctly completed forms were returned. Of these patients 58 (69 per cent) believed that they would not benefit from an outpatient appointment, 19 (21.3 per cent) made one or more visits to their general practitioner but had no continuing problem at two weeks postoperation. Six (6.7 per cent) continued to have a problem and thought they would benefit from a surgical outpatient appointment. Of the five patients given routine follow-up appointments before discharge four expected no benefit from an outpatient appointment and only one wished to be seen. This survey suggests that patients should be given the option of an outpatient appointment after minor or intermediate surgery via a postal questionnaire. Unnecessary appointments would thereby be reduced.

Adult↗

PubMed1995

Apo-dystrophins (Dp140 and Dp71) and dystrophin splicing isoforms in developing brain.

PCR studies have shown that exons 71-74 are spliced out in most dystrophin mRNA transcripts in the brain. We have prepared new monoclonal antibodies against the syntrophin-binding region of dystrophin encoded by exons 73-74 and examined three protein products of the dystrophin gene in brain; the widely distributed Dp71, the recently discovered, brain-specific Dp140 and dystrophin itself. Exon 73-74 mAbs bound to all three proteins in brain and the extent of binding suggests that alternatively spliced dystrophins are less prominent at the protein level than predicted by PCR data. Dp140, unlike Dp71, was found to be present at much higher levels in foetal brain than in adult brain. If lack of functional Dp140 is the cause of the cognitive impairment in some Duchenne muscular dystrophy patients, this result suggests that the effects may occur early in development, which would reduce the options for therapeutic intervention.

Adult↗

PubMed1995

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). PCR amplification of single exons or groups of exons was used both to produce specific dystrophin immunogens and to map the mAbs obtained. PCR-mediated mutagenesis was also used to identify regions of dystrophin important for mAb binding. Because the mAbs can be used to characterize the dystrophin produced by individual muscle fibres, they will also be useful for studying "revertant" fibres in Duchenne muscle and for monitoring the results of myoblast therapy trials in MD patients with deletions in this region of the dystrophin gene.

Amino Acid Sequence↗

PubMed1995

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. After elimination of PCR errors, the two cDNA sequences were found to be identical except for five somatic mutations which resulted in three amino acid changes in the second hypervariable region (CDR2). The results suggest that the two hybridomas originated from the same lymphocyte clone in a germinal centre of the spleen, but underwent different point mutations and subtype switches during clonal expansion to form blast cells.

Amino Acid Sequence↗

PubMed1995

The N-terminal half of dystrophin is protected from proteolysis in situ.

Using a panel of "exon-specific" monoclonal antibodies, we have examined the products of degradation of dystrophin by endogenous proteases in post-mortem human muscle. Four main sites of dystrophin digestion were identified, all of them in the C-terminal half of the molecule. Two of them correspond to "hinges" in the central rod region and a third in the C-terminal domain follows the dystroglycan binding site. The results support the Koenig and Kunkel model for the tertiary structure of dystrophin (J. Biol. Chem. 265 (1990) 4560-4566), but suggest that much of the N-terminal half of dystrophin is protected from proteolysis, possibly by interaction with the sub-sarcolemmal cytoskeleton. Although the results seem inconsistent with an anti-parallel dimer model of dystrophin in which hinge 2 and hinge 3 are close together, possible ways of reconciling them with such a model are also considered.

Antibodies, Monoclonal↗

PubMed1995

Full-length and short forms of utrophin, the dystrophin-related protein.

All previous studies of the localization of utrophin (the dystrophin-related protein) in muscle and other tissues have been performed only with antibodies against the C-terminal region of the protein. Since several short forms of dystrophin, the apo-dystrophins, are produced from the 3' end of the dystrophin gene, there is a possibility that similar short forms of utrophin exist and that these could be responsible for some of the many different localizations of 'utrophin' in muscle. We have produced a new panel of 15 mAbs against the N-terminal region of utrophin and we have used it together with mAbs against the C-terminal region to show that full-length utrophin is present at neuromuscular junctions, in nerves, blood vessels and capillaries in normal muscle and in the sarcolemma of patients with muscular dystrophy and dermatomyositis. However, two of the 15 mAbs also recognised rat/mouse utrophin and both of these detected an additional 62 kDa protein on Western blots of rat C6 glioma cells. This potential 62 kDa 'apo-utrophin' was not detected in human cerebral cortex, in rat Schwannoma cells nor in any of the non-nerve cells and tissues tested.

Animals↗

PubMed1995

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. One advantage of the method is that no subcloning or in vitro manipulation of DNA is necessary.

Antibodies, Monoclonal↗

PubMed1995

Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy.

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.

Actinin↗

PubMed1995

Endovascular femoropopliteal bypass: a cadaveric study.

OBJECTIVE: Patency rates of standard femoropopliteal bypass in infra-inguinal occlusive disease have yet to be matched by minimally invasive percutaneous procedures. We report a feasibility study of a less invasive endovascular femoropopliteal bypass technique. METHODS: (1) groin exposure of femoral artery, (2) guidewire passage and mechanical dilatation of superficial femoral artery (SFA), (3) expandable helical cutter endarterectomy of SFA, (4) transluminal placement of PTFE graft, (5) graft balloon dilatation to shape and set distal interface and (6) end-to-end anastomosis of proximal graft to femoral artery. Development and testing was undertaken in 48 limbs of 26 fresh human cadavers. Limbs with no demonstrable SFA disease were excluded. Seventeen limbs had mild, diffuse disease. Three limbs had a single, short, tight stenosis. Seventeen limbs had multiple, high grade stenotic lesions 12-40 cm long (mean 24 cm). Eleven limbs had occlusive lesions, 8-38 cm long (mean 24 cm). RESULTS: We successfully completed the procedure in 39 (81%) limbs. We failed to complete the procedure in nine limbs; four from failed guidewire passage, four from vessel avulsion, and one from graft deployment failure. Histology confirmed endarterectomy cleavage in the standard plane. Angiography and explants demonstrated a patent graft and popliteal artery, and smooth distal graft/arterial interface with no obvious defects in 24 (62%) cases. Defects included combinations of: contrast extravasation/reflux, graft malpositioned/incorrectly sized, distal graft fold, and distal intimal flap. CONCLUSION: Endovascular femoropopliteal bypass is feasible and warrants further studies for possible clinical application.

Aged↗

PubMed1995

Evidence for a utrophin-glycoprotein complex in cultured cell lines and a possible role in cell adhesion.

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Actins↗

PubMed1995

Probing protein structure with proteases: studies of an equilibrium intermediate in protein unfolding.

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Amino Acid Sequence↗

PubMed1995

A novel dystrophin isoform is required for normal retinal electrophysiology.

Dystrophin is present in the outer plexiform layer of the retina and is required for normal retinal function as measured by electroretinography. We describe the identification of a novel isoform of dystrophin (Dp260) present in the mouse retina. The unique 5' terminus of the mRNA originates from a newly identified exon and is spliced in frame to exon 30 of the Duchenne muscular dystrophy (DMD) gene. The retinal isoform of dystrophin has 13 novel amino acids as its N-terminus followed by most of the dystrophin rod domain and the cysteine-rich C-terminal domains. Analysis of mouse tissues indicated this isoform of dystrophin is expressed in retina, brain and cardiac tissue. Comparison of retinal electrophysiology in mdx and mdxCv3 mouse suggests that Dp260 is required for normal retinal function.

Amino Acid Sequence↗