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Biomedical subjects

D S Bailey

Publications and source records attributed to D S Bailey.

15 recordsLinked to original sources
PubMed1989

Immunohistochemical analysis of antiserum from rhesus monkeys immunized with human colon carcinoma.

In an attempt to generate antibodies which recognize novel tumor-associated antigens we have immunized Rhesus monkeys (Macaca mulatta) with human colon carcinoma cells prepared from freshly excised tumors. Immunohistochemical characterization of polyclonal antisera from one monkey (DF6) revealed preferential reactivity with primary and metastatic colon carcinoma tissue, and a general lack of recognition of nonneoplastic mucosa. Immunoreactivity was localized to the luminal contents of glandular structures and to the apical surfaces of cells lining these glands. Immunoreactivity was not observed with any normal tissue examined. Examination of neoplastic tissues revealed reactivity with two gastric carcinoma specimens (n = 2) and one breast carcinoma (n = 7). In reactive colon carcinoma tissues, the pattern of staining with DF6 was similar to that of several other antibodies including anti-carcinoembryonic antigen, B72.3, anti-Le(x) and anti-Le(y). However, the panel of tissues recognized by these antibodies and DF6 differed significantly, suggesting that the DF6-reactive epitopes are unique. Human colon carcinoma cell lines maintained in vitro also expressed antigens recognized by DF6 in a pattern similar to that of surgically excised tissue. This preliminary characterization of DF6 antiserum suggests that immunization of Rhesus monkeys is a potentially useful protocol for identifying antigens preferentially expressed by human colon carcinoma.

Animals↗
PubMed1989

Early biochemical responses of the small intestine of coeliac patients to wheat gluten.

The pathogenesis of coeliac disease has been investigated by studying the response of small intestinal hydrolases in patients with coeliac disease subject to gluten challenge. Small intestinal biopsies taken before and two and a half hours after a gluten challenge in five patients with coeliac disease who had been maintained on a gluten free diet were examined by a combination of electron and light microscopy, organ culture, pulse chase biosynthetic labelling, SDS-PAGE and autoradiography. Before the challenge, the small intestinal biopsies showed nearly normal morphology. Two and a half hours after the challenge there was deterioration in villus architecture, distortion of microvillus structure, disorganisation of the intermicrovillus pit region, an increase in lysosome like bodies in the apical cytoplasm of the luminal enterocytes and pronounced hypertrophy of the rough endoplasmic reticulum of these cells. SDS-PAGE of small intestinal biopsies from four treated coeliac patients before gluten challenge revealed normal microvillus membrane and hydrolase composition. There was a generalised reduction but no specific alteration in the pattern of polypeptide synthesis in the mucosa of the small intestine in these subjects two and a half hours after the gluten challenge. These results suggest that the generalised reduction in small intestinal brush border enzymes in coeliac patients is not the primary pathogenetic mechanism and represents a secondary effect.

Adult↗
PubMed1987

Alterations in lipid-linked oligosaccharide metabolism in human melanoma cells concomitant with induction of stress proteins.

Challenge of human A375 melanoma cells with sodium arsenite induced the synthesis of stress proteins and stimulated [3H]mannose incorporation into a novel component migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 14 kDa (designated M14). Enhanced M14 expression was elicited by heavy metals (zinc, copper, cadmium, and nickel), thiol-reactive agents (iodoacetamide and auranofin), and hyperthermia. The kinetics of M14 induction and recovery from stress were similar to those of the stress proteins, but M14 half-life was only 15 min. Incorporation of [3H]mannose into M14 was inhibited by tunicamycin but not by cycloheximide or actinomycin D. M14 was metabolically labeled with [32P]orthophosphate but not by [35S] methionine or [3H]asparagine. Further studies revealed that M14 was selectively soluble in chloroform/methanol/water (10:10:3) and sensitive to both endo-beta-N-acetylglucosaminidase H digestion and mild acid hydrolysis. The latter released a water-soluble mannose-labeled moiety which eluted from Bio-Gel P-6 in a manner similar to Glc3Man9GlcNAc2. Together, these data suggest that M14 is a lipid-oligosaccharide intermediate of N-linked protein glycosylation and that enhanced expression of this class of molecule in response to chemical insults and hyperthermia is a newly described cellular reaction to stress.

Arsenic↗
PubMed1986

Cell-free synthesis of high-molecular-weight small intestinal polypeptides.

An intrinsic membrane glycoprotein, Mr 131 000, is a major developmentally specific component of the neonatal guinea-pig small intestinal microvillar membrane. Such high-molecular-weight proteins are often difficult to translate in vitro. In this study we report a successful strategy for the identification of the primary translation product of this glycoprotein, a high-molecular-weight precursor polypeptide of approximate Mr 225 000.

Animals↗
PubMed1986

Cloning of mRNA sequences from the human colon: preliminary characterisation of defined mRNAs in normal and neoplastic tissues.

RNA has been extracted from the normal human colon, converted into cDNA and cloned in the bacterial plasmid pBR322. About 4,000 sequences from this library were screened with probes derived from normal mucosa, familial polyposis mucosa, colonic adenocarcinomas and the colon tumour cell line HT29. Some mucosal sequences showed greatly reduced levels of transcription in neoplastic conditions, while a few showed elevated transcription. These have been further characterised by Northern and RNA dot-blot analysis.

Base Sequence↗
PubMed1985

Two-dimensional isoelectric focussing/sodium dodecyl sulphate polyacrylamide gel electrophoretic mapping and some molecular characteristics of the proteins of the adult guinea-pig small intestinal microvillus membrane.

The adult guinea-pig small intestinal microvillus membrane was purified approximately 25-fold by both cation-precipitation and differential centrifugation methods. Comparison by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed no substantial differences in polypeptide composition between the two preparations. One-dimensional SDS-PAGE and two-dimensional isoelectric focussing (IEF)/SDS-PAGE, together with Coomassie-blue, silver and lectin-staining, showed three major high molecular weight polypeptides, Mr 108 000, 116 000 and 127 000, as well as a 47 kDa protein (actin), as major constituents of the membrane. The proteins of Mr 108 000 and 116 000 were strongly concanavalin A reactive. A detailed two-dimensional IEF/SDS-PAGE map of the membrane was constructed. Sodium carbonate treatment showed the two concanavalin A-reactive glycoproteins, Mr 108 000 and 116 000, comprising the sucrase-isomaltase complex, to be loosely-associated 'extrinsic' microvillus membrane proteins. Two proteins, Mr 127 000 and 135 000, were tightly-associated 'intrinsic' microvillus proteins. Despite regional differences in specific activity of some small intestinal microvillar enzymes, most noticeably enterokinase (EC 3.4.21.9) and dipeptidyl peptidase IV (EC 3.4.14.x), no substantial regional differences were seen in microvillus membrane polypeptide composition. In contrast, a substantial increase in the major high molecular weight proteins of Mr 108 000 and 116 000 accompanied a 10-fold rise in sucrase-isomaltase activity, and loss of a major protein of Mr 131 000 accompanied the complete loss of lactase activity from the membrane during postnatal development.

Age Factors↗
PubMed1984

Structural and biochemical differentiation of the mammalian small intestine during foetal development.

Microscopical studies showed that initial differentiation of the guinea-pig small intestine occurs between days 35 and 55 of foetal development. Changes observed at this time include formation of villi (by day 42), elaboration of submucosal duodenal Brunner's glands (by day 49) and the appearance of a well-developed microvillus membrane (by day 56). Different microvillus membrane-associated hydrolases appear at different stages of foetal and postnatal development. The 'early' enzymes such as aminopeptidase, alkaline phosphatase and sucrase show a sharp increase and reach their maximal levels between days 35 and 50, whereas the late enzymes such as dipeptidyl peptidase IV and lactase increase gradually between days 35 and 50, and reach maximal activity between days 50 and 60. A combination of techniques involving precipitation with Mg2+ followed by fractionation on sucrose density gradients has enabled us to prepare, for the first time, a 21-fold enriched microvillus membrane fraction from the foetal intestine. Polypeptide analysis of this membrane fraction by sodium dodecyl sulphate/polyacrylamide gel electrophoresis showed the presence of developmentally specific polypeptides at different stages of foetal and postnatal development. Three polypeptides of molecular weights 205 000, 80 000 and 47 000 are major microvillus membrane components at the 40-day foetal stage. Two other polypeptides of molecular weights 60 000 and 131 000 are major microvillar components at 56-day and older foetal stages as well as at the 3-day neonatal stage. The adult microvillus membrane contained 112 000 and 122 000 Mr polypeptides as major components. The above results were confirmed using two-dimensional isoelectric focussing-sodium dodecyl sulphate/polyacrylamide gel electrophoretic techniques.

Animals↗
PubMed1981

Glycoprotein biosynthesis in animal cells grown in suspension culture. Assembly of lipid-linked saccharides and formation of protein-bound 'high-mannose' oligosaccharides.

Glycoprotein biosynthesis was studied with mouse L-cells grown in suspension culture. Glucose-deprived cells incorporated [3H]mannose into 'high-mannose' protein-bound oligosaccharides and a few relatively high-molecular-weight lipid-linked oligosaccharides. The latter were retained by DEAE-cellulose and turned over quite slowly during pulse--chase experiments. Increased heterogeneity in size of lipid-linked oligosaccharides developed during prolonged glucose deprivation. Sequential elongation of lipid-linked oligosaccharides was also observed, and conditions that prevented the assembly of the higher lipid-linked oligosaccharides also prevented the formation of the larger protein-bound 'high-mannose' oligosaccharides. In parallel experiments, [3H]mannose was incorporated into a total polyribosome fraction, suggesting that mannose residues were transferred co-translationally to nascent protein. Membrane preparations from these cells catalysed the assembly from UDP-N-acetyl-D-[6-3H]glucosamine and GDP-D-[U-14C]mannose of polyisoprenyl diphosphate derivatives whose oligosaccharide moieties were heterogeneous in size. Elongation of the N-acetyl-D-[6-3H]glucosamine-initiated glycolipids with mannose residues produced several higher lipid-linked oligosaccharides similar to those seen during glucose deprivation in vivo. Glucosylation of these mannose-containing oligosaccharides from UDP-D-[6-3H]glucose was restricted to those of a relatively high molecular weight. Protein-bound saccharides formed in vitro were mainly smaller in size than those assembled on the lipid acceptors. These results support the involvement of lipid-linked saccharides in the synthesis of asparagine-linked glycoproteins, but show both in vivo and in vitro that protein-bound 'high-mannose' oligosaccharide formation can occur independently of higher lipid-linked oligosaccharide synthesis.

Animals↗
PubMed1979

The assembly of lipid-linked oligosaccharides in plant and animal membranes.

Membrane preparations from growing regions of pea stems and actively-dividing mouse L-cells form lipid-linked saccharides from GDP-mannose and UDP-N-acetylglucosamine. These lipids have properties which are consistent with those of mono- and di-phosphoryl polyisoprenyl derivatives. In experiments using plant membranes, the monophosphoryl derivative labeled with GDP-(14C) mannose contains mannose only, while the diphosphoryl derivative labeled with the same nucleotide sugar is heterogeneous, containing oligosaccharides corresponding to mannosaccharides of 5, 7, and 9--12 residues. Only the diphosphoryl polyisoprenyl derivatives are labeled with UDP-(14C)glucosamine and these contain predominantly chitobiose and N-acetylglucosamine itself. Unlabeled GDP-mannose added after UDP-N-acetyl-(14C)glucosamine results in the formation of higher lipid-linked oligosaccharides which are apparently the same as those which are labeled with GDP-(14C)mannose alone. Incubation of the membranes with GDP-(14C)mannose in the presence of Mn2+, unlabeled UDP-glucose or unlabeled UDP-N-acetylglucosamine results in marked changes in the accumulation of both the polyisoprenyl monophosphoryl mannose and polyisoprenyl diphosphoryl oligosaccharides. Animal cell membranes synthesise lipid-linked oligosaccharides when incubated with UDP-N-acetylglucosamine and GDP-mannose. These oligosaccharides are similar in size to those synthesised by the plant membranes but their formation is more efficient. The potential roles of these compounds in glycoprotein biosynthesis in both plant and animal tissues is discussed.

Glycoproteins↗
PubMed1977

An ultrastructural study of the relationship between the plasma membrane and the cell wall of the coenocytic alga hydrodictyon africanum.

A wall/plasma membrane unit was prepared from Hydrodictyon africanum by microdissection. A replica of the inner surface of the membrane was made and by freeze-fracture of the whole cell, 2 corresponding internal fracture faces were obtained. The large coenocytes of the alga were plasmolysed and the wall separated by cutting it away. Its inner surface was directly viewed in the electron microscope after shadowing with Pt/C. Particles were found on the outer half of the internal fracture face of the membrane which were oriented in the same 2 directions as the microfibrils laid down at the inner surface of the wall. No structures were found at the inner surface of the membrane. Some evidence was obtained for a structural connexion between the innermost layers of the wall and the plasma membrane.

Cell Membrane↗
PubMed1976

Phospholipid composition of the plasma membrane of the green alga, Hydrodictyon africanum.

A plasma-membrane fraction was isolated from the alga Hydrodictyon africanum by micro-dissection and its phospholipid components were analysed. Phosphatidylcholine was the major phospholipid of the preparation. Both phosphatidylserine and diphosphatidylglycerol were enriched in the fraction compared with the whole cell, but the relative amount of phosphatidylglycerol present was less than that in the whole cell. Phosphatidylinositol was absent from the plasma-membrane preparation.

Cell Membrane↗
PubMedDate not supplied

Effect of the lectin concanavalin A on the neonatal guinea pig gastrointestinal mucosa in vivo.

The effect of intrapharyngeal infusion and subsequent passage of the lectin concanavalin A through the gastrointestinal tract of neonatal guinea pigs was examined. The lectin, administered at a concentration of 1 mg/ml, caused considerable mucosal damage in the stomach. Subsequent passage of the lectin through the small intestine caused little cellular damage to the mucosa, as judged by light and electron microscopy. Parallel morphometric analyses of the jejunum showed a decrease in mean villus height and an increase in mean crypt depth of 8% after 5 h of lectin administration. A 4.7-fold increase in jejunal crypt cell production rate was observed, accompanied by a statistically significant increase in the number of intraepithelial lymphocytes. These morphologic and kinetic changes were accompanied by a three-fold increase in the urinary lactulose:mannitol excretion ratio, indicative of a loss of mucosal integrity. No systemic antibody response to concanavalin A was demonstrated within 5 h of intragastric infusion of the lectin. Concanavalin A, conjugated with 10 nm colloidal gold particles, was also directly infused into the lumen of the jejunum in vivo. Within 60 min, both villous and crypt epithelial cells contained gold particles, demonstrating the rapid accessibility of crypt cells to the lectin. Intercellular spaces appeared within the villous epithelium, although both junctional complexes and spot desmosomes were retained. The brush border was abnormal with signs of incipient vesicularization. These findings have implications for our understanding of the pathogenesis of childhood coeliac disease and for the vulnerability of the neonatal gut to food proteins.

Animals↗