Biomedical subjects

C G Gahmberg

Publications and source records attributed to C G Gahmberg.

At least 91 records · Page 5Linked to original sources

PubMed1986

Calmodulin may decrease cell surface sialic acid and be involved in the expression of fibronectin during liver regeneration.

The decrease of sialic acid in plasma membrane glycoproteins and the expression of cell surface fibronectin were studied during the pre-replicative phase of liver regeneration. The aim of this study was to correlate these cell-surface events to the intracellular surge of calmodulin observed a few hours after partial hepatectomy. The fact that calmodulin decreased the specific activity of UDP-N-acetyl-D-glucosamine 2'-epimerase, a key regulatory enzyme in the biosynthesis of glycoprotein sialic acids, and that trifluoperazine prevented the desialylation indicates that the membrane desialylation is a calmodulin-dependent process. On the other hand, Western blotting using anti-rat fibronectin antibody in trifluoperazine-treated animals suggests that calmodulin may also be involved in the surface expression of fibronectin in regenerating hepatocytes.

Animals↗

PubMed1986

Identification of a cell-surface glycoprotein mediating cell adhesion in EBV-immortalized normal B cells.

Phorbol ester treatment markedly enhanced aggregation of Epstein-Barr virus (EBV)-immortalized normal B cells. Monoclonal antibody (MAb) 60.3, recognizing a leukocyte common antigen, completely inhibited intracellular adhesion, whereas MAbs reacting with leukocyte common antigen T200, C3b receptor, T-cell-associated antigen TA-I, C3d (EBV) receptor, brain-granulocyte/T-lymphocyte antigen, transferrin receptor, surface immunoglobulin, Class-I or Class-II transplantation antigens or a B-cell-specific antigen (BI) showed no inhibitory effect. Both the spontaneous and phorbol-ester-enhanced cell aggregations were similarly inhibited by Fab fragments made from antibody 60.3. Phorbol esters also enhance binding between EBV-immortalized normal B cells and autologous or allogeneic blood mononuclear cells depleted of B lymphocytes. This process, which is independent of immunity to EBV, was similarly blocked by the antibody fragments. Antibody 60.3 precipitated 3 non-covalently associated surface glycopolypeptides with apparent MW of 90, 130 and 160/kDa from EBV-infected B cells. Although dissociation of a similar protein complex from granulocytes by sodium dodecyl sulfate treatment clearly indicated the presence of the epitope on the smallest component, the same treatment did not dissociate the protein complex from EBV-immortalized normal B cells. It is thus concluded that the 90-kDa glycopolypeptide, either alone or associated with the larger glycopolypeptides, mediates cell adhesion of the B cells.

Animals↗

PubMed1986

In vitro attachment of mono- and oligosaccharides to surface glycoconjugates of intact cells.

We have synthesized glycosylhydrazines of various mono- and oligosaccharides and coupled these to periodate- or galactose oxidase-treated human red cells and K562 erythroleukemia cells. The optimal conditions for this carbohydrate modification of cells have been established. This method makes it possible to specifically elongate oligosaccharide chains of cell surface glycoconjugates with desired carbohydrates. In this way, new antigenic and receptor properties can be conferred to cells, and the functional roles of carbohydrates in cell surface glycoconjugates can be studied. The method has been used to make red cells of blood group O reactive with anti-A and anti-B sera, and in rendering K562 cells or red cells of blood group O agglutinable with the alpha-N-acetylgalactosamine-specific Helix pomatia lectin.

ABO Blood-Group System↗

PubMed1986

Exposure of major neutral glycolipids in red cells to galactose oxidase. Effect of neuraminidase.

The exposure of several major red-cell glycolipids to galactose oxidase was studied by oxidizing the cells with the enzyme and reducing them with NaB2H4. After isolation, the deuterium label was detected by mass fragmentography. 60-70% globoside in human and porcine erythrocytes was exposed as measured by this method. In contrast, asialo-GM2 in guinea-pig erythrocytes and Forssman glycolipid in sheep erythrocytes were mainly in a cryptic state. Neuraminidase treatment increased the incorporation of deuterium label to asialo-GM2 4-8-fold. A similar effect was seen in Forssman glycolipid when sheep red cells were labeled with the neuraminidase/galactose oxidase/NaB3H4 method. In contrast, the increase in labeling was only about 10-40% in porcine and human globosides, which were efficiently exposed to galactose oxidase already in native red cells.

Animals↗

PubMed1986

Genetic assignment of GP90, leukocyte adhesion glycoprotein to human chromosome 21.

Intercellular adhesion among human leukocytes involves a cell-surface glycoprotein with an apparent molecular weight of 90,000 which forms complexes with higher-molecular-weight glycoproteins. A monoclonal antibody (60.3) against this glycoprotein blocks induced adhesion. Here we have shown that the antibody reacts with cell clones carrying human chromosome 21 in lymphocyte hybrids between an AKR mouse thymoma (BW5147) and human concanavalin A-activated peripheral blood lymphocytes. Cell sorting by FACS of a hybrid clone heterogeneous in the expression of the antigen identified by the 60.3 antibody yielded a positive fraction expressing the antigen and carrying human chromosome 21, and a negative fraction lacking both the antigen and chromosome 21. The gene coding for the cell adhesion glycoprotein is thus provisionally assigned to chromosome 21.

Antibodies, Monoclonal↗

PubMed1986

Studies on the defect underlying the lysosomal storage of sialic acid in Salla disease. Lysosomal accumulation of sialic acid formed from N-acetyl-mannosamine or derived from low density lipoprotein in cultured mutant fibroblasts.

Salla disease is a lysosomal storage disorder characterized by mental retardation and disturbed sialic acid metabolism. To study endogenous synthesis and breakdown of sialic acid, fibroblasts were incubated for 5 d in the presence and then in the absence of N-[3H]acetylmannosamine. Labeling of free sialic acid was 5-10 times higher in mutant than in normal cells. Radioactivity decreased in 4 d by 75% in normal but only by 30% in mutant fibroblasts. The labeling pattern was not normalized upon coculture of mutant and normal cells. To study the metabolism of extracellular sialic acid, low-density lipoprotein (LDL) was labeled in the sialic acid moiety (periodate-NaB3H4) or in the protein moiety (125I). Binding, internalization, lysosomal degradation, and exit of products of protein catabolism were similar in normal and mutant fibroblasts. Upon incubation with LDL labeled in the sialic acid moiety, mutant cells accumulated 2-3 times more free sialic acid radioactivity than normal fibroblasts, mostly in the lysosomal fraction. After a 24-h chase incubation, radioactivity in free sialic acid decreased by 70-80% in normal but only by 10-30% in mutant cells. In mutant fibroblasts, 40% of the radioactivity remained in lysosomes, whereas no labeled free sialic acid was detected in lysosomes from normal fibroblasts. We conclude that in Salla disease, fibroblast endogenous synthesis of sialic acid and lysosomal cleavage of exogenous glycoconjugates is normal, but free sialic acid cannot leave the lysosome. These findings suggest that the basic defect in Salla disease is deficient transport of free sialic acid through the lysosomal membrane.

Cells, Cultured↗

PubMed1986

B lymphoblastoid cell lines with normal and defective O-glycosylation established from an individual with blood group Tn.

Individuals with the Tn blood group contain terminal serine/threonine-linked N-acetylgalactosamine residues in their blood cells. This is due to lack of UDP-D-galactose: D-N-acetyl galactosamine beta-D-galactosyl transferase from part of their red cells and probably from their leukocytes. We have established B lymphoblastoid cell lines from such an individual by in vitro infection of his lymphocytes with Epstein-Barr virus. The original line contained a mixture of cells reactive and nonreactive with Helix pomatia lectin (Hp). These cells were subcloned after staining with fluorescent Hp by a fluorescence-activated cell sorter (FACS) into homogeneous, phenotypically stable lines of Hp-positive (Hp+) and Hp-negative (Hp-) cells. The molecular differences between the membrane glycoproteins were characterized by carbohydrate-specific surface labeling techniques, Hp affinity chromatography, polyacrylamide slab gel electrophoresis and glycopeptide/oligosaccharide analysis. The major O-glycosidic membrane glycoprotein (GP105) was retained on Hp-Sepharose columns only from Hp+ cells, whereas the common leukocyte antigen (GP160-200) was partially retained on Hp columns from both lines. These proteins were isolated by immune precipitation with monoclonal antibodies and characterized. The results show that the GP105 glycoprotein from Hp+ cells contains terminal N-acetylgalactosamine residues but also more complex oligosaccharides. The common leukocyte antigen showed different electrophoretic mobilities in Hp+ and Hp- cells. UDP-galactose D-N-acetyl galactosamine beta-galactosyl transferase was almost absent in the Hp+ cells. These cell lines are useful for studies on the functional role and regulation of the biosynthesis of O-glycosidic carbohydrates.

B-Lymphocytes↗

PubMed1985

Biosynthesis of the major human red cell sialoglycoprotein, glycophorin A. O-Glycosylation.

The biosynthesis of the major human red cell sialoglycoprotein, glycophorin A, was studied in the erythroleukemia cell line K562 with emphasis on O-glycosylation. The cells were pulse-chase labeled with [35S] methionine, and either directly immune precipitated with anti-glycophorin A antiserum or detergent-solubilized extracts first passed through columns containing the N-acetylgalactosamine-specific lectin from Helix pomatia or the glucose/mannose specific lectin from lentil beans. From the sugar-eluted fractions anti-glycophorin A antiserum was used to identify precursor molecules. After 5 min of labeling the first glycophorin A precursors were seen. The largest had an apparent molecular weight of 37,000, and bound to lentil lectin-Sepharose, but not to H. pomatia lectin-Sepharose. The lentil lectin-reactive glycophorin A molecules increased to Mr = 39,000 during chase and obtained sialic acids after 9 min of chase reflecting terminal N- and O-glycosylation. After 5-6 min of labeling two H. pomatia-interacting glycophorin A precursors with apparent molecular weights of 24,000 and 30,000 were obtained. These did not bind to lentil lectin-Sepharose. During chase also these molecules increased in size to Mr = 39,000. The immune precipitation of all antiglycophorin A-reactive precursor molecules was inhibited by purified red cell glycophorin A. The carboxylic ionophore, monensin, caused the accumulation of incompletely O-glycosylated glycophorin A molecules, which bound to H. pomatia lectin-Sepharose. These were degraded by treatment with endo-beta-N-acetylglucosaminidase H reflecting incomplete processing of the N-glycosidic oligosaccharide.

Cell Line↗

PubMed1985

Tumourigenicity, cell-surface glycoprotein changes and ornithine decarboxylase gene pattern in Ehrlich ascites-carcinoma cells.

We selected a 2-difluoromethylornithine-resistant Ehrlich ascites-carcinoma cell line that grows in the presence of 20 mM-difluoromethylornithine. These cells contain 10-20 times the normal amount of hybridizable sequences for ornithine decarboxylase (EC 4.1.1.17) in their genomic DNA. We used these gene-amplified cells, their revertant counterparts (grown in the absence of the drug after an established gene amplification) and tumour cells grown in the presence of putrescine to investigate the changes of ornithine decarboxylase gene pattern and simultaneously occurring phenotypic changes, such as tumourigenicity and the expression of cell-surface glycoproteins. In the tumour cells reverted back to the normal gene frequency, not only did the amplified sequences disappear, but there were also signs of gene re-arrangements seen as a "gene jump', when a signal evidently moved to a heavier restriction fragment. Similar gene re-arrangement likewise occurred in cells exposed to putrescine. Although the wild-type tumour cells and the gene-amplified cells readily grew in the peritoneal cavity of mice, the revertant cells and the putrescine-treated cells had lost their tumourigenicity in mice. Gene-amplified tumour cells and the revertant cells showed distinct changes in their surface glycoprotein pattern in comparison with the parental cell line. These findings indicate that alterations of ornithine decarboxylase gene pattern/dosage may be associated with phenotypic changes possibly related to the tumourigenicity of these carcinoma cells.

Animals↗

PubMed1985

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.

Animals↗

PubMed1985

Identification of the major human sialoglycoprotein from red cells, glycophorin AM, as the receptor for Escherichia coli IH 11165 and characterization of the receptor site.

The pyelonephritogenic Escherichia coli strain 1 H 11165 specifically agglutinates human erythrocytes carrying the M blood group antigen. The polymorphic forms of this antigen, M and N, are located in the NH2-terminal region of the major human red-cell sialoglycoprotein, glycophorin A. Radioactively labeled glycophorin A from M cells specifically bound to the bacteria. Purified glycophorin AM, but not glycophorin AN, efficiently inhibited for binding. Mild periodate treatment oxidized the NH2-terminal serine in glycophorin AM and this resulted in loss of binding to the bacteria. High concentrations of serine and alkali-labile oligosaccharides derived from glycophorin AM inhibited the binding, whereas the synthetic M-specific NH2-terminal pentapeptide Ser-Ser-Thr-Thr-Gly did not. Neuraminidase treatment of glycophorin AM did not destroy the binding. The most efficient inhibition of binding was observed with the N-terminal glyco-octapeptide obtained from glycophorin AM by CNBr cleavage. This peptide contains both the essential serine residue and the alkali-labile oligosaccharides, which both are recognized by the bacterium.

Amino Acid Sequence↗

PubMed1985

Pre-replicative changes of the rat sinusoidal plasma membrane glycoproteins during hepatic regeneration.

Cell-surface glycoproteins of rat liver sinusoidal plasma membranes from control and regenerating livers were studied. The glycoproteins were labeled using specific methods for sialic acid (NAIO4/NaB3H4) and galactosyl/N-acetyl galactosaminyl residues (galactose oxidase/NaB3H4 and neuraminidase-galactose oxidase (NaB3H4) and the solubilized proteins were analyzed by gel electrophoresis. The patterns obtained with regenerating livers were quantitatively different from controls. This shows that cell surface glycoproteins change during liver regeneration.

Adenosine Triphosphatases↗

PubMed1985

Assignment of gene for human cell-surface membrane antigen Trop-4 to chromosome 11.

The gene (named MF16) for a surface membrane antigen, Trop-4, is assigned to human chromosome 11 on the basis of studies using a mouse monoclonal antibody, immunofluorescence, fluorescence-activated cell sorting (FACS), immunoprecipitation, and mouse-human lymphocyte hybrids. The Trop-4 antigen is present on all human cell lines tested, on peripheral blood monocytes and granulocytes, and on a small fraction of peripheral blood lymphocytes, but is absent from erythrocytes. The Trop-4 monoclonal antibody precipitates an 85,000-dalton glycopolypeptide from hybrid cells containing human chromosome 11. However, in a human cell line expressing this antigen, a larger-molecular-weight species, 100-105,000 daltons was coprecipitated with the 85,000-dalton glycopeptide, and under nonreducing conditions a larger compound of 110-125,000 daltons was obtained. Although the Trop-4 antigen is of similar molecular weight to the Mab-4 and F10.44.2 antigens previously assigned to chromosome 11, it is shown to be different from them.

Animals↗

PubMed1985

Hormonally regulated expression of an apocrine epithelial antigen in the cell membranes of endometrial glands.

We have raised a rabbit antiserum against formalin-treated membrane glycoproteins isolated by lectin affinity chromatography from human milk fat globules. In immunofluorescence staining the antiserum reacts strongly and exclusively with the apical membrane of the glandular epithelial cells in normal endometrium during the proliferative phase. No membrane-bound antigen is seen during the secretory phase but some positively staining material is found in the glandular lumina and a weak cytoplasmic staining is also seen. The antigen is absent in postmenopausal endometrium but is found in abundance in the cell membrane of the glandular epithelium in endometria from postmenopausal women receiving estrogen treatment. The glandular epithelium of hyperestrogenism-induced endometrial hyperplasia is very strongly decorated by the antiserum. Molecular characterization of the antigen by immunoblotting under reducing conditions reveals two polypeptides of 110,000 daltons and 93,000 daltons from normal endometrium during the proliferative phase. A single band of 75,000 daltons, probably representing shed fragment, is seen in secretory endometrium. This hormonally regulated and strictly located protein differs from many other endometrial proteins in that it is estrogen-induced, while others are dependent on progesterone effect, and it constitutes an interesting histochemical marker of endometrial glands.

Antibodies↗

PubMed1985

Identification of a cell surface protein complex mediating phorbol ester-induced adhesion (binding) among human mononuclear leukocytes.

Phorbol esters rapidly induce aggregation of human mononuclear leukocytes in vitro. Previous studies have indicated that cell surface proteins are involved. We report now that the monoclonal antibody 60.3, either as purified IgG or as Fab' fragments, to an antigen common to leukocytes completely inhibited the phorbol ester-induced intercellular adhesion (binding). No inhibition of cell aggregation was observed with monoclonal antibodies to common leukocyte antigen T 200, T-cell-associated antigen, monocyte-granulocyte antigen, brain granulocyte-T-lymphocyte antigen, transferrin receptor, mature T-cell antigens (mol.wt either 67,000 or 19,000/29,000), T helper/inducer cell antigen, sheep erythrocyte receptor, class I or class II antigens, or T cytotoxic/suppressor cell antigen. The antibody 60.3 did not inhibit stimulation of the cells since the characteristic phorbol ester-induced morphological changes and phorbol ester-enhanced cap formation of membrane glycoproteins were readily observed. Two major cell surface polypeptides with apparent molecular weights of 90,000 and 160,000 were immunoprecipitated. We conclude that this protein complex, or at least one of its components, mediates adhesion among mononuclear leukocytes.

Antibodies, Monoclonal↗

PubMed1985

Identification of a cell-surface glycoprotein mediating adhesion in human granulocytes.

Previous studies have shown that monoclonal antibody 60.3 reacting with a surface antigen common to human leukocytes inhibits phorbol ester-induced adhesion among blood mononuclear cells and precipitates from these cells three surface polypeptides with apparent molecular weights of 90,000, 130,000 and 160,000. Now we report that the same antibody, either as purified IgG or Fab fragments, also inhibits the extensive adhesion among granulocytes induced by phorbol ester. Inhibition of cell aggregation was not observed with monoclonal antibodies to C3b receptor, common leukocyte antigen T200, C3bi receptor, brain granulocyte-T lymphocyte antigen, IgG Fc receptor, class I transplantation antigen, or a granulocyte-specific antigen. Intercellular adhesion induced by either the chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (FMLP) or the ionophore A23187 was also inhibited by antibody 60.3. However, this antibody did not affect phorbol ester-induced superoxide (O2-) generation or lysozyme release. Two major surface glycopolypeptides with apparent molecular weights of 92,000 and 155,000 were immunoprecipitated from granulocytes. Dissociation of the protein complexes obtained from blood mononuclear cells and granulocytes indicated the presence of the epitope on the 90,000-92,000 molecular-weight components. It is thus concluded that the smallest glycopolypeptides mediate adhesion in human granulocytes and mononuclear leukocytes.

Antibodies, Monoclonal↗

PubMed1985

Identification of non-T non-B lymphocyte leukaemia patients with favourable prognosis by cell surface glycoprotein analysis.

Leukaemic cells from 15 children with ALL were isolated and their surface glycoproteins labelled with 3H after treatment with neuraminidase and galactose oxidase followed by NaB3H4. The labelled glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. The cells could be classified into four groups: 1) Surface glycoprotein pattern resembling that of normal T lymphocyte (one patient); 2) Surface glycoprotein pattern showing some T cell characteristics (5 patients); 3) Surface glycoprotein profile with strongly labelled glycoproteins of apparent molecular weight of 120 000 and 130 000, and frequently a glycoprotein with an apparent molecular weight of 210 000 and visible HLA-coded glycoproteins (6 patients); 4) Surface glycoprotein pattern not resembling those of the other groups (3 patients). Follow-up showed that patients in group 3 have remained in their initial remission (5/6) for the complete study period of 5-6 yr, whereas all patients in the other groups have relapsed and/or died. We conclude that the surface glycoprotein analysis enables the identification of patients with non-T non-B ALL with favourable prognosis.

Actuarial Analysis↗

PubMed1985

Late expression of M and N antigens on glycophorin A during erythroid differentiation.

The M/N blood groups are carried by the major human red cell sialoglycoprotein, glycophorin A. O-glyosidic carbohydrate is needed for the activity, but the M/N specificity is due to amino acid replacements in the NH2-terminal portion of the molecule. We have used monoclonal antibodies specific for M and N blood groups to study their expression during normal erythropoiesis. Here we report that the M/N blood group activities are very weakly or not at all expressed before the polychromatic normoblast stage. Using polyclonal anti-glycophorin A antiserum, it was shown that glycophorin A molecules are already abundantly present on the earliest morphologically recognizable erythroid precursor, the proerythroblast. These findings can be explained by our previous observation that the O-glycosylation of glycophorin A gradually increases during erythroid maturation.

Antibodies, Monoclonal↗