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D M Shotton

Publications and source records attributed to D M Shotton.

At least 19 recordsLinked to original sources
PubMed1992

Expression of soluble isoforms of rat CD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45R monoclonal antibodies.

The CD45 or leucocyte-common antigens are encoded by a single gene but can be found in various forms due to alternative splicing of three exons near the 5' end of the gene. The CD45 antigens are major glycoproteins of all types of leucocytes. Monoclonal antibodies recognizing restricted epitopes of CD45 have been used to distinguish phenotypic and functional subsets of lymphocytes. To facilitate epitope mapping and biochemical studies, we have expressed the extracellular portions for four different isoforms of rat CD45 in Chinese hamster ovary cells. Constructs were prepared to give four soluble CD45 isoforms, with sequence incorporating either all three alternative exons (sCD45.ABC), the B exon (sCD45.B), the C exon (sCD45.C), or no alternative exons (sCD45.O). These were expressed at approximately 5 mg/l of spent tissue culture supernatant and were antigenically active with monoclonal antibodies (mAb) that recognize all CD45 isoforms. The MRC OX22 and OX32 mAb have been used to split rat CD4+ T cells into functionally distinct subpopulations and the epitopes for these were mapped to the product of exon C. The epitope for MRC OX33, a marker for B cells, requires expression of either the A exon or the A/B exon junction. Electron microscopy showed that the extra segments contributed to an extended structure as has been predicted from the sequence. The shape of the molecule is discussed with regard to other molecules at the leucocyte cell surface.

Animals↗
PubMed1991

Purification and properties of sialoadhesin, a sialic acid-binding receptor of murine tissue macrophages.

Macrophage subpopulations in the mouse express a lectin-like receptor, sialoadhesin (originally named sheep erythrocyte receptor, SER), which selectively recognizes sialoglycoconjugates and is likely to be involved in cellular interactions of stromal macrophages in haematopoietic and lymphoid tissues. In this report we describe the purification and ligand specificity of sialoadhesin isolated from mouse spleen. Purified sialoadhesin, a glycoprotein of 185 kd apparent Mr, agglutinated sheep or human erythrocytes at nanomolar concentrations in a sialic acid-dependent manner. Low angle shadowing and electron microscopy showed that sialoadhesin consisted of a globular head region of approximately 9 nm and an extended tail of approximately 35 nm. To investigate the specificity for sialic acid, we studied the interaction of sialoadhesin with derivatized human erythrocytes, glycoproteins, and glycolipids. In conclusion, sialoadhesin specifically recognizes the oligosaccharide sequence Neu5Ac alpha 2----3Gal beta 1----3GalNAc in either sialoglycoproteins or gangliosides. These findings imply that specific sialoglycoconjugates carrying this structure may be involved in cellular interactions between stromal macrophages and subpopulations of haematopoietic cells and lymphocytes.

Animals↗
PubMed1991

The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation.

Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions.

Amino Acid Sequence↗
PubMed1989

Imaging of immunogold labelled antigens on capping thymocytes by confocal reflection contrast scanning optical microscopy.

Confocal laser scanning optical microscopy (CLSM) in the reflection contrast mode has been used to image single 40 nm gold particles, and to study changes in the distribution of gold label associated with capping of the leukocyte sialoglycoprotein (LSGP) antigen on the surface of fixed rat thymocytes, labelled with the mouse monoclonal antibody W3/13 and a goat anti-mouse IgG immunogold conjugate. This imaging method has also been applied to live thymocytes labelled with gold-conjugated antibodies, to study the dynamics of the capping process.

Animals↗
PubMed1989

Effects of capping on the non-ionic detergent solubility of rat thymocyte glycoproteins.

The non-ionic detergent solubility of surface antigens during capping, following their cross-linking by specific antibodies, was investigated for three rat thymocyte glycoproteins, the leucocyte-common antigen (L-CA), the leucocyte sialoglycoprotein (LSGP) and Thy-1, using a combination of immunofluorescence microscopy, covalent surface radiolabeling and quantitative analysis using a radiolabeled antibody. Prior to the addition of cross-linking antibody, both L-CA and LSGP were soluble in the non-ionic detergent Triton X-100, while Thy-1 was largely insoluble. Addition of sufficient antibody to induce capping led to a significant reduction in the solubility of L-CA and LSGP, even prior to warming to induce capping of the antigen under investigation. Subsequent capping did not increase the amount of insoluble antigen, suggesting that ligand binding, rather than the process of capping itself, is sufficient to cause this partial Triton insolubility. These results indicate the formation of an association between these liganded glycoproteins and the cell's detergent-insoluble cytoskeleton. In contrast, the Thy-1 antigen became progressively more extractable by Triton X-100 as the antigen was capped, suggesting that the insertion of the Thy-1 lipid tail into the plasma membrane was progressively more easily perturbed by the non-ionic detergent during the capping process. The reciprocal maintenance of solubility of either LSGP or L-CA during the capping of the other shows that the majority of the 180 kDa thymocyte L-CA molecules play no role in the mechanism of capping of LSGP. Furthermore, immunoprecipitates of solubilized L-CA contained no detectable amounts of the cytoskeletal protein fodrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Ultrastructural identification of type 1 fibres in human skeletal muscle. Immunogold labelling of thin cryosections with a monoclonal antibody against slow myosin.

Existing methods for the ultrastructural identification of fibre types in human skeletal muscle are fallible. This has prompted us to develop a reliable immunoelectron microscopic approach for the identification of human skeletal muscle fibre types. Here we report the unambiguous electron microscopic identification of human type 1 muscle fibres, achieved by combining cryoultramicrotomy with colloidal gold immunocytochemical labelling, using a monoclonal antibody (N0Q7.5.4D) which is specific for the heavy chain of the slow myosin isoform of human skeletal muscle. This method for the identification of muscle fibre types and determination of myosin isoform distributions may have important applications in the ultrastructural study of pathological muscle and in the analysis of myofibrillar assembly during myogenesis.

Antibodies, Monoclonal↗
PubMed1988

Video-enhanced light microscopy and its applications in cell biology.

The combination of novel optical microscopic techniques with advanced video and digital image-processing technology now permits dramatic improvements in the quality of light-microscope images. Such video-enhanced light microscopy has lead to a renaissance in the applications of the light microscope for the study of living cells in two important areas: the intensification of faint fluorescence images, permitting observation of fluorescently labelled cells under conditions of very low illuminating intensity; and the enhancement of extremely low contrast images generated by minute cellular structures, so that these may be clearly seen and their normal intracellular movements recorded. Application of both these aspects of video-enhanced light microscopy have recently led to major discoveries concerning the functioning of the living cell. In this review I discuss the equipment, procedures and image-processing principles employed in these applications, and describe and illustrate some of the spectacular results that have recently been obtained.

Animals↗
PubMed1988

Cytoskeletal involvement in the sequential capping of rat thymocyte surface glycoproteins.

The independent capping of the three major rat thymocyte glycoproteins, the leucocyte-common (L-C) antigen, the leucocyte sialoglycoprotein (LSGP) and Thy-1, was investigated using specific monoclonal antibodies. The capping of each antigen did not require redistribution of the other major surface glycoproteins, and was accompanied by a partial co-capping of the cytoskeletal proteins fodrin and actin, but not of tubulin. A study of the ability of a cell that already possesses one glycoprotein cap to cap a second different glycoprotein showed that this was possible in all cases to varying degrees, the second cap always forming at the same position on the cell surface as the first. Colchicine failed to perturb this observed sequential capping polarity, indicating that microtubules did not direct this second capping event.

Actins↗
PubMed1987

Isolation and initial biochemical characterisation of caps of two major rat thymocyte glycoproteins: evidence for the involvement of a 205 K Con A binding protein and cytoskeletal components in capping.

Two major rat thymocyte surface glycoproteins, the leucocyte-common (L-C) antigen and the leucocyte sialoglycoprotein (LSGP), were induced to cap independently, using the specific monoclonal antibodies OX-1 and W3/13, respectively, and an appropriate fluorescently labeled second antibody layer. The caps were subsequently isolated from detergent extracted cells by a procedure involving gentle shearing. TRITC-phalloidin staining of the isolated caps demonstrated the presence of F-actin within these structures, and lectin-affinity staining after fractionation on SDS polyacrylamide gels revealed the presence of a concanavalin A (Con A) binding protein of relative molecular weight (Mr) 205,000, gp205, in both the L-C antigen and LSGP caps, but absent from the detergent-insoluble residue isolated from unchallenged cells. These results suggest that gp205 may be involved in the association of cross-linked glycoproteins with the cytoskeleton during capping.

Animals↗
PubMed1987

Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles.

We have used light microscopic histomorphometry to quantify the developmental histopathological changes induced by muscular dystrophy in the soleus and extensor digitorum longus (EDL) muscles of the mdx mouse. We find that this X-linked disease exhibits early fibre necrosis with foci of invasive cells, clustering of affected fibres, hyaline fibres, and, in the mixed soleus muscle, a progressive increase in the proportion of type 1 fibres, the mdx soleus containing 58 +/- 5% type 1 fibres by 26 weeks, compared with 27 +/- 4% in control C57BL/10 ScSn mice. This increase is not due to atrophy or slow axon reinnervation of type 2 fibres. Although only 5% of all original fibres survive by 26 weeks in the EDL, the diseased mdx fibres are continuously and successfully replaced by new fibres with internal nuclei, the affected mice thus avoiding the end-stage histopathology and physical disability characteristic of the X-linked human Duchenne and Emery-Dreifuss muscular dystrophies. Homozygous mdx mice share the life expectancy of normal C57BL/10 mice and appear behaviourly normal. The mdx mouse is therefore an excellent mammalian model in which to study the processes of muscle fibre degeneration and regeneration.

Animals↗
PubMed1986

Electron-microscopic studies of the CA antigen, epitectin.

Epitectin, the mucin-like glycoprotein defined by the monoclonal antibodies CA1, CA2 and CA3, has been examined by electron microscopy to determine its shape and size. It appears to be a single extended strand with a mean length of about 270 nm. The antibodies CA1 and CA2 appear to bind preferentially to a terminal site on the epitectin molecule.

Antigens, Neoplasm↗
PubMed1985

Visualisation by low-angle shadowing of the leucocyte-common antigen. A major cell surface glycoprotein of lymphocytes.

The leucocyte-common antigen (L-CA) from rat thymocytes is a cell surface glycoprotein of 180 000 apparent mol. wt. with an 80-kd cytoplasmic domain. This paper reports the molecular dimensions of the molecule visualised by electron microscopy after low-angle shadowing. The L-CA monomer consists of a globular head region of approximately 12 nm diameter and a short tail approximately 18 nm long. In deoxycholate both monomers and multimers are seen with aggregation occurring at the head groups. When the detergent is removed, larger clusters are formed with tails extending from a central aggregate. A 100-kd tryptic fragment of L-CA that is known to include the extracellular parts of the molecule also exists in monomer and multimer forms and is seen to have a rod-like structure of length 28 nm without evidence of the head group. Altogether the data indicate that the rod-like structure is found outside the cell and that the extra sequence that forms the head is inside. The tryptic fragment is likely to be derived by cleavage after the transmembrane sequence.

Animals↗
PubMed1985

A novel procedure for pattern analysis of features present on freeze-fractured plasma membranes.

We have used statistical methods for the analysis of two-dimensional point patterns to derive quantitative descriptions of the distributions of caveolae on freeze-fractured muscle fibre membranes. One method was based on a quadrat analysis while the second was a new procedure that we have called the interpoint distance analysis. We show that the latter analysis can unambiguously distinguish random, clustered and dispersed patterns and that a single parameter can be derived that can be used to compare different distributions. It is readily applicable to patterns containing several hundred points. Practical details of the method are given and a simple algorithm that can be implemented on a microcomputer is provided. The interpoint distance analysis should prove generally useful in situations where the two-dimensional distribution of objects has to be quantified.

Cell Membrane↗
PubMed1984

Monoclonal antibodies detect a spectrin-like protein in normal and dystrophic human skeletal muscle.

Spectrin is the major protein of the erythrocyte membrane skeleton, which is bound to the cytoplasmic surface of the membrane's lipid bilayer and is responsible for cell shape and membrane elasticity. Inability to identify spectrin in other cell types led to the assumption that this protein was unique to erythrocytes. However, spectrin-like proteins have been demonstrated recently in a variety of cell types, including skeletal and cardiac muscle, in several species. We used monoclonal antibodies against human erythrocyte spectrin subunits in an immunocytochemical study to detect related proteins in normal and diseased human skeletal muscle. Six of seven monoclonal antibodies against beta-spectrin determinants were bound at the cytoplasmic surface of muscle fiber plasma membranes, whereas none of six monoclonal antibodies against alpha-spectrin determinants was bound. Muscle fibers of patients with neuromuscular diseases showed similar distribution and specificity of antibody binding to those of normal subjects, but the intensity of binding was increased. In contrast, probable regenerating fibers in muscle of patients with muscular dystrophies showed reduced binding of antibodies, but reduced binding was not seen in fetal muscle fibers nor in those of a patient with a myotubular myopathy. We conclude that human skeletal muscle fibers possess a spectrin-related protein associated with their plasma membrane that shows extensive beta-chain similarities to erythrocyte spectrin but differs significantly with respect to the alpha-subunit. Its function may be associated with the maintenance of membrane and myofibril integrity during contraction, and the increased antibody binding in diseased muscle may reflect a structural rearrangement of spectrin or a compensatory increase in spectrin abundance in response to increased stress on these systems.

Antibodies, Monoclonal↗
PubMed1983

Erythrocyte membrane skeleton abnormalities in hereditary spherocytosis.

Erythrocyte ghosts from eight individuals with hereditary spherocytosis have been compared with respect to their protein compositions as judged by SDS gel electrophoresis, their ease of spectrin extractability, and their freeze-etch electron microscopic appearance after incubation in condition designed to promote aggregation of the intramembrane particles. Four of these HS cases were unrelated, while the other four represented two generations from a single family, including a pair of identical twins, one of whom had not undergone splenectomy when this investigation was initiated. Of the four unrelated cases, one showed no departures from normal under the conditions of this investigation, whereas the other three exhibited features which suggested a membrane skeleton lesion. In one of these there was a reduced proportion of spectrin tetramers relative to dimers in 4 degrees C extracts, while the two remaining cases exhibited abnormal intramembrane particle aggregation. The four related cases had almost identical variations from normal. Spectrin was not extractable from their ghost membranes during a mild extraction incubation which removed spectrin from normal control ghosts. However, the intramembrane particle aggregation subsequently induced in these ghosts was of a degree unobtainable in normal ghosts without such spectrin extraction. In addition the ghosts from one twin, the only one of these patients who had not undergone splenectomy at the start of this investigation, showed a reduced amount of band 4.2. However, when this patient's blood was re-tested after splenectomy, this protein was found to be at normal levels. Our results support the view that hereditary spherocytosis is not a single disease, but is rather a term used to describe a variety of different molecular lesions of the erythrocyte membrane skeleton with similar clinical manifestations.

Actins↗
PubMed1982

Dissection of the human erythrocyte spectrin molecule using monoclonal antibodies.

One IgM and three IgG monoclonal antibodies specific to band 1 of human erythrocyte spectrin have been characterised. The antigenic sites of the IgG antibodies have been identified and mapped by radioimmune labelling of tryptic fragments of spectrin fractionated by SDS slab gel electrophoresis and blotted onto nitrocellulose filters. The binding site of one of these antibodies has also been directly visualised in the electron microscope after low-angle shadowing of the antibody-spectrin dimer complex, and lies at that end of the dimer which is responsible for tetramer formation.

Animals↗