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R C Hughes

Publications and source records attributed to R C Hughes.

At least 73 records · Page 4Linked to original sources
PubMed1991

Distribution of fibronectin and fibronectin-binding proteins, AGp110 and integrin alpha 5 beta 1, during chemically induced hepatocarcinogenesis in adult rats.

We have used single- and double-label immunocytochemistry to examine the distribution of AGp110, integrin alpha 5 beta 1 and fibronectin in adult rat liver during carcinogenesis induced by aflatoxin B1 or diethylnitrosamine. In normal liver fibronectin and the fibronectin integrin receptor alpha 5 beta 1 are localized on all three domains of the parenchymal cell surface: sinusoidal, lateral and canalicular. In contrast, AGp110, a non-integrin monomeric glycoprotein with fibronectin receptor properties, is confined to the bile canalicular (apical) plasma membrane of hepatocytes. Hepatocarcinogenesis induced by aflatoxin B1 causes altered cell foci to form in the parenchyma, followed by enlargement of these foci to form pre-neoplastic nodules and finally hepatocellular carcinomas of either poorly differentiated, trabecular or adenocarcinoma morphology. Expression of AGp110 decreased to a minimal level, at first selectively in altered cell foci, from the 9th week of treatment, and then indiscriminately in poorly differentiated carcinomas. The same lesions that were deficient in AGp110 also displayed a reduced level of fibronectin and alpha 5 beta 1, although the observed change in AGp110 demarcated altered foci and poorly differentiated tumour lesions more sharply, since expression of alpha 5 beta 1 and fibronectin, though substantially reduced, was still faintly apparent on the cell surface. Small acinar structures, observed in late hyperplastic nodules and in trabecular carcinomas, exhibited even, pericellular staining of fibronectin and alpha 5 beta 1, including prominent staining of the lumen area, whereas staining of AGp110 appeared to be confined to the lumen. In larger ducts of overt adenocarcinomas, fibronectin and alpha 5 beta 1 were distributed along the basal surface of the epithelium and AGp110 on the apical domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Experience of using rating scales for the assessment of vocational trainees in general practice.

For two years trainers in the northern half of the south western region have been asked to assess their trainees with a condensed version of the new Manchester rating scales for vocational training in general practice and to send copies to the regional adviser. The condensed version retains the 23 scales but does not include the subscales. Trainers' workshops and comments from individuals suggested that no radical improvements to the scales were required and that they were an adequate statement of what was required from trainees. The response rate of 89% for returning at least one assessment and 38% for returning all three suggested that use of this condensed version would be more feasible than using the full version of the scales. Analysis of 86 first assessments and 48 sets of three assessments showed a range of variation both in trainee ratings and use of individual scales that was consistent with expectations. A lack of significant rank correlations between the scales indicated that each of the scales measured a different characteristic of behaviour. We suggest that the most useful way to achieve systematic assessment of vocational trainees is by the use of the 23 main rating scales, with regional variations where appropriate.

Communication↗
PubMed1990

Identification of a novel glycoprotein (AGp110) involved in interactions of rat liver parenchymal cells with fibronectin.

We have identified an integral membrane glycoprotein in rat liver that mediates adhesion of cultured hepatocytes on fibronectin substrata. The protein was isolated by affinity chromatography of detergent extracts on wheat germ lectin-Agarose followed by chromatography of the WGA binding fraction on fibronectin-Sepharose. The glycoprotein (AGp110), eluted at high salt concentrations from the fibronectin column, has a molecular mass of 110 kD and a pI of 4.2. Binding of immobilized AGp110 to soluble rat plasma fibronectin required Ca2+ ions but was not inhibited by RGD peptides. Fab' fragments of immunoglobulins raised in rabbits against AGp110 reversed the spreading of primary hepatocytes attached onto fibronectin-coated substrata, but had no effect on cells spread on type IV collagen or laminin substrata. The effect of the antiserum on cell spreading was reversible. AGp110 was detected by immunofluorescence around the periphery of the ventral surface of substratum attached hepatocytes, and scattered on the dorsal surface. Immunohistochemical evidence and Western blotting of fractionated liver plasma membranes indicated a bile canalicular (apical) localization of AGp110 in the liver parenchyma. Expression of AGp110 is tissue specific: it was found mainly in liver, kidney, pancreas, and small intestine but was not detected in stomach, skeletal muscle, heart, and large intestine. AGp110 could be labeled by lactoperoxidase-catalyzed surface iodination of intact liver cells and, after phase partitioning of liver plasma membranes with the detergent Triton X-114, it was preferentially distributed in the hydrophobic phase. Treatment with glycosidases indicated extensive sialic acid substitution in at least 10 O-linked carbohydrate chains and 1-2 N-linked glycans. Immunological comparisons suggest that AGp110, the integrin fibronectin receptor and dipeptidyl peptidase IV, an enzyme involved in fibronectin-mediated adhesion of hepatocytes on collagen, are distinct proteins.

Amino Acid Sequence↗
PubMed1990

Stereotactic implantation of fetal mesencephalon.

Twelve patients with advanced Parkinson's disease have been improved by transplantation of fetal mesencephalon into the caudate nucleus. No immunosuppression had been used. The human allogeneic transplantation window is wide.

Adult↗
PubMed1990

An endogenous carbohydrate-binding protein of baby hamster kidney (BHK21 C13) cells. Temporal changes in cellular expression in the developing kidney.

Asialofetuin (ASF) coupled to Sepharose has been used to isolate a Mr 30,000 protein from Triton X-100 extracts of the baby hamster kidney cell line BHK21 C13. Binding to ASF-Sepharose was specific for terminal beta-galactosyl residues. The lectin requires detergent for optimal solubilization and binding is independent of Ca2+ or reducing reagents. The lectin was labelled in a lactoperoxidase-catalysed iodination of intact BHK21 C13 cells, suggesting that it is associated with the cell surface. Antibodies to the lectin identify in Western blotting cross-reactive components in established cell lines of kidney (MDCK, NRK) and non-kidney (L, CHO, 3T3) origin. In young adult hamsters, the lectin is expressed in colon and duodenum and in lesser amounts in ileum, stomach, lung, liver and testes but is absent in kidney. The lectin is expressed in late embryonic and newborn hamster kidney but expression declines during 14 days after birth. Immunofluorescent staining of cryostat sections of newborn hamster kidney and intestine show that the lectin is expressed at apical epithelial surfaces. The presence of the lectin at the luminal surface of kidney tubules suggests a tubular origin for the BHK21 C13 cell line. Possible functions of the Mr 30,000 lectin in kidney development are discussed.

Animals↗
PubMed1990

Localization of two fibronectin-binding glycoproteins in rat liver and primary hepatocytes. Co-distribution in vitro of integrin (alpha 5 beta 1) and non-integrin (AGp110) receptors in cell-substratum adhesion sites.

We have compared the localization of integrin alpha 5 beta 1 and AGp110 (apical glycoprotein of Mr 110 x 10(3]] in rat liver parenchyma and in primary hepatocyte cultures. Integrin alpha 5 beta 1 is a heterodimeric fibronectin receptor. AGp110 is a newly described monomeric glycoprotein of the apical (bile canalicular) membrane domain of liver parenchyma that binds in an RGD-independent manner to fibronectin and mediates spreading of hepatocytes onto fibronectin-coated substrata. Using Western blotting of fractionated liver membranes and immunocytochemistry of liver sections at light- and electron-microscope levels, we have confirmed that AGp110 is a canalicular glycoprotein and have established that integrin is located in approximately equal proportions in the sinusoidal, lateral and canalicular membrane domains. In the canalicular surface domain both glycoproteins are associated with microvilli. Examination of immunolabelled primary hepatocytes spread on fibronectin-coated substrata by light and laser scanning confocal microscopy revealed colocalization of AGp110, integrin, actin and vinculin in substratum-attached microextensions at the periphery of the basal cell surface. Actin filaments that terminated at these cell processes originated from circular sub-cortical actin fibres. Interference reflection microscopy revealed focal adhesive contacts at the edge of the basal cell periphery at the same location where AGp110 and integrin were observed by immunofluorescence. In vitro, a proportion of the primary hepatocytes seeded onto fibronectin-coated substrata aggregated into colonies of several cells with intercellular contacts between neighbouring cells. Cell-substratum contacts containing integrin, AGp110, actin and vinculin followed the contours of these colonies in the same manner as they delineated the basal periphery of single, substratum-attached cells. We conclude that both integrin and AGp110 contribute to hepatocyte-fibronectin adhesive interactions and that intercellular adhesion and cooperation among hepatocytes in their response to fibronectin matrices leads to colony formation and morphological differentiation of parenchymal cell monolayers in vitro.

Actins↗
PubMed1989

Fetal brain tissue.

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Abortion, Induced↗
PubMed1989

Bowringia milbraedii agglutinin. Specificity of binding to early processing intermediates of asparagine-linked oligosaccharide and use as a marker of endoplasmic reticulum glycoproteins.

We have purified a protein with hemagglutinating activity from the seeds of a West African legume, Bowringia milbraedii. The purified protein, designated BMA, has a native Mr = 38,000 on gel filtration and a subunit size of Mr = 16,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions. Hemagglutination was inhibited most effectively by Man alpha 1----2 linked sugars. Affinity chromatography of oligosaccharides on BMA-Sepharose showed that Man alpha 1----2Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol (where GlcNAcol is N-acetylglucosaminitol) and Man alpha 1----2Man alpha 1----3Man beta 1----4GlcNAcol were retarded on the column, whereas Man alpha 1----3Man beta 1----4GlcNAcol did not bind. Oligomannosidic-type glycans obtained by treatment of [3H] mannose-labeled baby hamster kidney cells with endo-beta-N-acetylglucosaminidase H bound more strongly to BMA-Sepharose and required 10 or 200 mM methyl-alpha-mannoside for elution. Oligosaccharides bearing the sequence Man alpha 1----2Man alpha 1----6Man alpha 1----6Man, i.e. Man9GlcNAc and certain isomers of Man8GlcNAc and Man7GlcNAc, bound more tightly than other Man8 GlcNAc and Man7GlcNAc isomers lacking this sequence. Man6GlcNAc and Man5GlcNAc were weakly bound. These results suggest that BMA binds preferentially to glycoproteins that are subjected to early steps of oligosaccharide processing in the endoplasmic reticulum but not to glycoproteins that are exposed to more extensive processing by Golgi mannosidases. Staining of permeabilized cells with BMA-chromophore conjugates revealed a reticular cytoplasmic pattern consistent with a preferential visualization of the endoplasmic reticulum. BMA staining was less evident in the juxtanuclear regions that were stained brightly with wheat germ agglutinin, a lectin that binds preferentially to sialylated glycoproteins located in Golgi compartments.

Animals↗
PubMed1989

Transplantation in Parkinson's disease: stereotactic implantation of adrenal medulla and foetal mesencephalon.

The two possible dopamine donor sites for transplantation are autologous adrenal medulla and human foetal substantia nigra or adrenal medulla. There is increasing experience with the use of adrenal medulla transplantation for Parkinson's disease and much less experience in foetal substantia nigra transplantation. The particular problems of each technique are discussed with examples and postoperative progress of cases with the special emphasis on management problems.

Adrenal Medulla↗
PubMed1988

Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases.

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.

Alkaloids↗
PubMed1988

Substrate recognition by UDP-N-acetyl-alpha-D-galactosamine: polypeptide n-acetyl-alpha-D-galactosaminyltransferase. Effects of chain length and disulphide bonding of synthetic peptide substrates.

A synthetic peptide AcTPPP, based on a threonine-containing sequence present in bovine myelin basic protein, is a potent acceptor of glycosyl transfer from UDP-N-acetylgalactosamine catalyzed by extracts of baby hamster kidney (BHK) cells or rabbit lymph node tissue. In contrast, the disulphide-linked peptide (AcTCPPP)2, based on a glycosylated sequence present in the hinge region of rabbit immunoglobulin G, is not an acceptor and inhibits glycosylation of AcTPPP. Extension of the cystine-containing peptide at the N-terminus produced weak acceptors but strong acceptors resulted when the cystine residue was reduced to form monomeric peptides. The acceptor specificity of the N-acetylgalactosaminyl-transferase activity of BHK cells is very similar to that of rabbit lymph node tissue. The results indicated that tissues actively secreting immunoglobulin do not contain a transferase activity adapted specifically for glycosylation of sequences containing cystine residues, and suggested that addition of an N-acetylgalactosamine to a threonine residue in the hinge region of rabbit immunoglobulin takes place during biosynthesis prior to the formation of the inter-chain disulphide bridge of fully assembled immunoglobulins.

Amino Acid Sequence↗
PubMed1988

Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties.

In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from the known alteration in assembly of N-linked glycans affecting the carbohydrate chains on the proteoglycan or some other combination of factors is discussed.

Animals↗
PubMed1988

Miniature radiation dosimeter for in vivo radiation measurements.

We have made initial characterization measurements on a miniature radiation dosimeter which can be used for in vivo radiation measurements. It consists of a radiation sensing field effect transistor (RADFET) mounted in a 0.8 mm OD plastic catheter. The RADFET acts as a dosimeter by storing trapped charge proportional to absorbed dose. The stored charge signal can be differentiated to give dose rate. We report on the techniques for mounting, a circuit for dose readout, drift of the readings, linearity of response, temperature and angular dependence, and unpowered operation of the device.

Radiometry↗
PubMed1987

Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing.

Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.

1-Deoxynojirimycin↗
PubMed1987

Characterization of a mannosidase acting on alpha 1----3- and alpha 1----6-linked mannose residues of oligomannosidic intermediates of glycoprotein processing.

Baby hamster kidney (BHK) cell extracts catalyze the conversion of [3H]mannose-labelled (Man)5GlcNAc and (Man)6GlcNAc oligosaccharides to a (Man)3GlcNAc species that retains affinity for concanavalin-A-Sepharose and appears to be Man alpha 1----3[Man alpha 1----6]Man beta 1----4GlcNAc. The properties of the (Man)5GlcNAc-hydrolase activity differ from lysosomal alpha-mannosidases as well as previously described processing mannosidases acting on oligosaccharide intermediates of N-glycan assembly. Mosquito cell extracts catalyze hydrolysis of (Man)6GlcNAc but lack the (Man)5GlcNAc hydrolase activity detected in BHK cell extracts. Glycopeptide analysis has been carried out on a ricin-resistant BHK mutant RicR14 that lacks N-acetylglucosaminyl transferase I and fails to convert oligomannosidic N-glycans to complex-type chains, and mosquito cells that constitutively lack N-acetylglucosaminyl transferase I. In both cell lines, the cellular glycoproteins contain (Man)5GlcNAc oligosaccharide as the major stable component equivalent to a 15-20-fold increase compared with normal BHK cells. Although containing very high amounts of asparagine-linked (Man)5(GlcNAc)2, RicR14 cells exhibit (Man)5GlcNAc hydrolase activity at levels similar to wild-type BHK cells. This result, together with previous work [Foddy, L., Feeney, J. & Hughes, R.C. (1986) Biochem. J. 233, 697-706] showing the complete inhibition of conversion of oligomannosidic intermediates to complex-type N-glycans in BHK cells treated with swainsonine, an inhibitor of mannosidase II but not the (Man)5GlcNAc hydrolase activity, argues against a major role for the (Man)5GlcNAc hydrolase activity in N-glycan assembly and suggesting other functions for the mannosidase activity in BHK cells.

Aedes↗
PubMed1987

Distinct patterns of glycosylation of colligin, a collagen-binding glycoprotein, and SPARC (osteonectin), a secreted Ca2+-binding glycoprotein. Evidence for the localisation of colligin in the endoplasmic reticulum.

Mouse parietal endoderm PYS cells were labelled with [2-3H]mannose for 16-24 h. Colligin, an Mr-47000 collagen-binding protein, and SPARC, a Mr-43000 protein, highly homologous to the Ca2+-binding protein osteonectin, were isolated from labelled cell extracts and culture medium respectively. Glycopeptides obtained by exhaustive digestion with pronase were analysed by lectin-affinity, ion-exchange, and gel-filtration chromatography and by paper chromatography of high-mannose oligosaccharides after endo H release. The results show that the N-linked carbohydrate chains of colligin are exclusively the high-mannose type, of which (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2 make up 77%. This carbohydrate structure provides strong evidence that colligin is a component of the endoplasmic reticulum, and argues against a role in cell-surface interactions. By contrast to colligin, SPARC secreted by PYS cells contains predominantly a diantennary complex type of chain containing a variable number of sialic acid and core-substituted fucose residues. Similar glycosylation patterns to those discussed above were seen in colligin isolated from primary mouse embryonic parietal endoderm cells and the murine 3T3 cell line, and in SPARC secreted by bovine corneal endothelial cells. Unlike the type-IV-collagen-binding glycoprotein studied by Dennis, J., Waller, C. and Schirrmacher, V. [J. Cell Biol. 99, 1416-1423 (1984)], removal of N-linked oligosaccharides from colligin had no effect on its binding to native type IV collagen.

Animals↗