Genes in the diagnosis of malignant disease.
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Humans↗
Biomedical subjects
C Wagener
Publications and source records attributed to C Wagener.
Purification and N-terminal amino acid sequence of a chondroitin sulphate/dermatan sulphate proteoglycan isolated from intima/media preparations of human aorta.
A proteoglycan (PG) was purified to homogeneity from intima/media preparations of human aorta specimens by the following chromatographic steps: Sepharose Q anion exchange, Sepharose CL-4B size exclusion, hydroxyapatite, MonoQ anion exchange and TSK G 4000 SW size exclusion. The purity of the preparation was established by SDS/PAGE using direct staining by silver or Dimethylmethylene Blue, as well as by Western blots of biotin-labelled samples. The electrophoretic mobility of the native PG was less than that of a 200,000-Mr standard protein. After treatment with chondroitin sulphate lyase ABC, a core protein of Mr 15,000 was revealed. The Mr of the glycosaminoglycan (GAG) peptides was less than 24,000, by comparison with a keratan sulphate peptide. The composition of the GAG chains was determined by differential digestion of the PG by chondroitin sulphate lyases AC/ABC or chondroitin sulphate lyase AC alone followed by anion-exchange chromatography of the resulting disaccharides. The GAG chains are composed of approximately one-third of dermatan sulphate and two-thirds chondroitin sulphate disaccharide units. The sequence of the 20 N-terminal amino acids is identical with the sequence previously reported for PG I isolated from human developing bone [Fisher, Termine & Young (1989) J. Biol. Chem. 264, 4571-4576]. The assignment of glycosylation sites to the serine residues in positions 5 and 10 was confirmed. The findings indicate that the chondroitin sulphate/dermatan sulphate PG is a major PG in intima/media preparations of human aorta and represents a biglycan-type PG.
Amino Acid Sequence↗
Identification of membrane antigens in granulocytes and colonic carcinoma cells by a monoclonal antibody specific for biliary glycoprotein, a member of the carcinoembryonic antigen family.
The gene coding for 'biliary glycoprotein (BGP)' is a member of the carcinoembryonic antigen (CEA) gene family. A monoclonal antibody (MAb) was induced against a BGP-preparation isolated from human bile. The antibody did not crossreact with the carcinoembryonic antigen (CEA) and different non-specific crossreacting antigens. The anti-BGP MAb was used to identify BGP-related antigens in membrane extracts from granulocytes and the colonic carcinoma cell line HT-29. In granulocyte membranes, a single antigen of Mr 160,000 was bound. In membranes from HT-29 cells, a main antigen of Mr 85,000 was present. At high antigen concentration, an additional antigen of Mr 115,000 was identified. Since several transcripts of the BGP gene have been identified, the different BGP related antigens are probably products of alternatively spliced mRNAs.
Antibodies, Monoclonal↗
The putative role of members of the CEA-gene family (CEA, NCA an BGP) as ligands for the bacterial colonization of different human epithelial tissues.
Immobilized purified CEA (carcinoembryonic antigen), NCA (non-specific crossreacting antigen) and BGP I (biliary glycoprotein I) bind strains of E. coli (including EPEC) and some Salmonella species (including S. typhi, S. paratyphi A + B and S. java) while Shigella-, Yersinia- and Bacteroides- strains showed no adhesion. The binding was of high avidity, heat sensitive, dose dependent, saturable and nearly completely abolished in the presence of 10 mM alpha-methylmannoside. From inhibition studies with aromatic mannose compounds, it was suggested that in contrast to Salmonella strains E. coli strains exhibit a higher hydrophobicity in the binding region adjacent to the CEA-, NCA- and BGP-binding site. By further inhibition experiments it could be demonstrated that E. coli and Salmonella strains bind to high-mannose type oligosaccharides of these molecules via lectins on bacterial type I fimbriae. We conclude that the expression of products of this gene family on different human epithelial cells (colon-, bile canaliculi, uroepithel etc.) may function as ligands for bacterial colonization of epithelial tissues.
Antigens, CD↗
Binding of Escherichia coli and Salmonella strains to members of the carcinoembryonic antigen family: differential binding inhibition by aromatic alpha-glycosides of mannose.
Various Escherichia coli and Salmonella strains bound to glycoproteins of the family of carcinoembryonic antigens (CEA). As judged from plateau regions of the binding curves, CEA, nonspecific cross-reacting antigen of Mr 55,000 (NCA-55), and biliary glycoprotein of Mr 85,000 (BGP-85) showed similar binding activities. The binding to ovalbumin was significantly lower and the binding to fetuin was insignificant under identical experimental conditions. The binding of E. coli and S. typhi to the different glycoproteins was similar as judged from the binding curves. In comparison with alpha-methyl-D-mannopyranoside, aromatic alpha-glycosides of mannose were more potent binding inhibitors of E. coli but not of salmonellae to CEA and NCA-55. These results are similar to those previously obtained with intestinal epithelial cells and yeast cells (N. Firon, S. Ashkenazi, D. Mirelman, I. Ofek, and N. Sharon, Infect. Immun. 55:472-476, 1987). The binding of E. coli to CEA was inhibited by purified type 1 fimbriae. On the basis of the distribution of CEA-like glycoproteins in tissues and body fluids, the results indicate that glycoproteins of the CEA family may be involved in the recognition of bacteria and the regulation of bacterial colonization.
Animals↗
Binding of nonspecific cross-reacting antigen, a granulocyte membrane glycoprotein, to Escherichia coli expressing type 1 fimbriae.
Nonspecific cross-reacting antigen (NCA) is a well-characterized membrane glycoprotein on granulocytes, macrophages, and lung epithelium. Structural studies at the protein and genomic levels have revealed that NCA is a member of the immunoglobulin supergene family, and hybridization studies showed that the transcript level of NCA is induced by treatment with gamma interferon. These studies, as well as the expression of NCA on granulocytes, suggest a role for NCA in immune response. For a first step in studying this possible role, we have examined the binding of two glycoforms of NCA designated NCA-50 (Mr, 50,000) and TEX-75 (Mr, 75,000). Here we report the results from binding assays which demonstrate carbohydrate-mediated binding of Escherichia coli expressing type 1 fimbriae and of isolated type 1 fimbriae to NCA-50. TEX-75 did not bind to the purified fimbriae but bound slightly to the bacterial strain. Inhibition studies showed that the binding to NCA-50 involved interaction of mannose moieties on NCA-50 and lectins on the fimbriae. The binding of NCA-50 to bacterial fimbriae was confirmed by electron microscopy studies, using immunolabeling techniques. In addition, we show that the surface expression of NCA-50 (and presumably of other NCA species) on isolated polymorphonuclear leukocytes is increased following activation with the bacterial peptide formylmethionyl-leucyl-phenylalanine, consistent with a role for NCA in immune response.
Antigens, Neoplasm↗
Escherichia coli of human origin binds to carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA).
Immobilized carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA) bound 3 strains of E. coli of human origin. The binding was dose dependent, saturable, and of high avidity. Binding of the bacteria to CEA and NCA was completely abolished in the presence of 10 mM alpha-methyl D-mannopyranoside. Bacteria did not bind to concanavalin A. In addition, binding to deglycosylated CEA was either absent or significantly reduced. These findings indicate that the E. coli strains bind to D-mannosyl residues in CEA and NCA. Considering the tissue distribution of CEA (brush border of colonic epithelium) and NCA (granulocytes), these glycoproteins may be involved in the recognition of bacteria.
Antigens, Neoplasm↗
Quantification of carcinoembryonic antigen in serum and analysis of epitope specificities of monoclonal antibodies.
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Antibodies, Monoclonal↗
Selective precipitation of biotin-labeled antigens or monoclonal antibodies by avidin for determining epitope specificities and affinities in solution-phase assays.
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Antibodies, Monoclonal↗
Determination of epitope specificities and affinities of monoclonal antibodies in solution phase using biotin-labeled carcinoembryonic antigen and avidin as precipitating agent.
A generally applicable method for the selective precipitation of antigens from solution phase is presented. The method is based on the rapid and quantitative binding of avidin to biotin-labeled antigen in the presence of biotinylated carrier protein. Using carcinoembryonic antigen (CEA) as a model antigen biotin-labeled CEA was precipitated to greater than 97% at amounts of up to 5000 ng/tube. The method was applied to the determination of epitope specificities and affinities of monoclonal anti-CEA antibodies in solution phase. Non-specific precipitation did not exceed 2% for radiolabeled Fab fragments and 6% for radiolabeled IgG. Because antigen-antibody binding takes place in solution, artifacts introduced by the immobilization of reactants to solid supports are omitted.
Animals↗
Characterization of biotin-labeled proteoglycans by electrophoretic separation on minigels and blotting onto nylon membranes prior and after enzymatic digestion.
Biotinylated proteoglycans were separated by sodium dodecyl sulfate electrophoresis prior and after enzymatic digestion by glycan-specific enzymes using polyacrylamide minigels. The biotin-labeled compounds were blotted onto nylon membranes either by electrophoresis or by diffusion and detected by avidin-enzyme conjugates. The method allows the nonisotopic detection of native proteoglycans and core proteins. Proteoglycans can be visualized at protein amounts as low as 0.7 ng per lane. In comparison with sensitive protein stains, compounds of enzyme preparations do not interfere with bands corresponding to core proteins. Electrophoresis, blotting, and staining of up to 12 samples per gel are accomplished in less than 3 h.
Amino Acids↗
Activation of the phosphatidylinositol cycle in spreading cells.
Metabolites of the phosphatidylinositol cycle were analyzed in BHK-21 (C13) cells spreading on fibronectin-coated culture plates in comparison with attached nonspreading cells 45 min after plating. Among the water-soluble metabolites (glycerophosphoinositol, inositol, inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate), significant elevations were found for inositol monophosphate, inositol bisphosphate, and inositol tetrakisphosphate. In the lipid fraction, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate were significantly elevated. The activation of the phosphatidylinositol cycle in spreading versus nonspreading attached BHK-21 (C13) cells may be involved in the permissive effect of the extracellular matrix on cell proliferation.
Animals↗
Limited improvement of tumour diagnosis by the simultaneous determination of carcinoembryonic antigen (CEA) and of a tumour-associated CEA-related antigen of Mr 128,000 in serum.
Two biotin-avidin based enzyme immunoassays were developed using three monoclonal anti-CEA antibodies with distinct epitope- and antigen-specificities. A broadly cross-reactive monoclonal anti-CEA antibody (T84.1) was immobilized on a solid support. Either monoclonal antibody T84.66 or CEA.11 was used as the second, biotin-labeled monoclonal antibody. Both antibodies do not cross-react with normal granulocytes or bile canaliculi. Monoclonal antibody CEA.11 binds to CEA and to an antigen with a relative molecular mass of 128,000 present in extracts from solid carcinomas. Monoclonal antibody T84.66 does not cross-react with the antigen of Mr 128,000. After adsorption of tumour extracts to a CEA-specific immunosorbent, residual activity was measurable by the CEA.11 assay, but not by the T84.66 assay. Serum samples (n = 726) from patients with malignant and non-malignant disease, as well as from healthy volunteers, were analysed by both immunoassays and by two commercial CEA immunoassays. In comparison with the T84.66 assay and the commercial assays, the CEA.11 assay did not significantly increase the sensitivity or specificity of tumour diagnosis.
Antibodies, Monoclonal↗
In vitro influence of Mycoplasma species on the stimulation of human polymorphonuclear granulocytes.
The influence of Mycoplasma species (sp.) on the stimulation of human polymorphonuclear neutrophil granulocytes (PMNG) was determined by means of the luminol-dependent chemiluminescence (CL) method. When opsonized Mycoplasma sp. were used the CL response of PMNG was greater than in the presence of nonopsonized strains. Nonopsonized and nonspecifically opsonized Mycoplasma sp. showed a different CL response pattern. The stimulation of PMNG was with M. pneumoniae significantly weaker than with the other Mycoplasma sp. Using isolated M. hominis strains always the same CL-reaction of PMNG was observed. On the other hand, with 12 isolated U. urealyticum strains different results were obtained; 9 strains isolated from the upper urogenital tract lead to a slight PMNG stimulation comparable to that of M. pneumoniae. No correlation was found between CL response and bacterial killing. The weak stimulation of PMNG by M. pneumoniae and most of the U. urealyticum isolates suggest that this behaviour could be a factor of pathogenicity.
Humans↗
Molecular cloning of a cDNA coding biliary glycoprotein I: primary structure of a glycoprotein immunologically crossreactive with carcinoembryonic antigen.
We have isolated and sequenced four overlapping cDNA clones from a normal adult human colon library, which together gave the entire nucleotide sequence for biliary glycoprotein I (BGP I). BGP I is a member of the carcinoembryonic antigen (CEA) gene family, which is a subfamily in the immunoglobulin gene superfamily. The deduced amino acid sequence of the combined clones for BGP I revealed a 34-residue leader sequence followed by a 108-residue N-terminal domain, a 178-residue immunoglobulin-like domain, a 108-residue region specific to BGP I, a 24-residue transmembrane domain, and a 35-residue cytoplasmic domain. The nucleotide sequence of BGP I exhibited greater than 80% identity with CEA and nonspecific crossreacting antigen (NCA) in the leader peptide, N-terminal domain, and immunoglobulin-like domain. The BGP I-specific domain, designated A', was 56.7% and 55.8% identical at the nucleotide level and 42.6% and 39.6% identical at the amino acid level to the immunoglobulin-like domain of NCA and the first immunoglobulin-like domain of CEA, respectively. Beyond nucleotide position 1375 the 3' region of the BGP I cDNA was found to be specific for BGP I. Hybridization of a probe from this region to electrophoretic blots of RNAs from different human tissues showed a predominant 2.8-kilobase (kb) message accompanied by weaker bands 4.1 and 2.1 kb in size. The same probe gave a single band in Southern blot analysis of restricted total human DNA. Using a coding region probe from the BGP I domain A', we observed 4.1- and 2.1-kb messages. Lack of the 2.8-kb band suggested that different forms of BGP I may be generated by posttranscriptional modification of the same gene. We propose that BGP I diverged from NCA by acquiring an immunoglobulin-like domain substantially different from the domains found in NCA or CEA and also a new cytoplasmic domain. The latter feature should result in a substantially different membrane anchorage mechanism of BGP I compared to CEA, which lacks the cytoplasmic domain and is anchored via a phosphatidylinositol-glycan structure. Protein structural analysis of BGP I isolated from human bile revealed a blocked N terminus, 129 amino acids of internal sequence that are in agreement with the translated cDNA sequence, and five glycosylation sites in the peptides sequenced.
Amino Acid Sequence↗
[The Tumor Immunology Study Group: tumor immunology of the gastrointestinal tract--experimental and clinical experiences].
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Biomarkers, Tumor↗
Carcinoembryonic antigen (CEA) and two crossreacting antigens of 165 kD and 105 kD isolated from meconium exhibit identical amino terminal sequences.
Two antigens of 165 kD and 105 kD crossreacting with the carcinoembryonic antigen (CEA) were isolated from human meconium by perchloric acid extraction, differential immunoaffinity chromatography, and size exclusion HPLC. The sequences of the 30 amino terminal amino acids of both antigens are identical with the corresponding sequence of CEA.
Amino Acid Sequence↗
Rapid expression of c-fos specific messenger RNA after wounding of a BALB/c-3T3 fibroblast monolayer.
A grid of damaged areas was introduced into serum-starved BALB/c-3T3 fibroblast monolayers. The expression of c-fos specific messenger RNA was examined by the use of the cytoplasmic dot hybridisation technique 15, 30 and 45 min after wounding. The expression of c-fos messenger RNA was highest after 30 min, while microglobulin was constantly expressed. These results show that the unspecific event of wounding induces the specific expression of c-fos messenger RNA, an effect similar to that of growth factors during serum stimulation.
Animals↗