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M Ugorski

Publications and source records attributed to M Ugorski.

At least 37 records · Page 2Linked to original sources
PubMed1996

[Sialosyl-Le(a) and Sialosyl-Le(x) antigens in adhesion of cancer cells and tumor progression].

Sialosyl-Le(a) and sialosyl-Le(x) are tumor-associated carbohydrate antigens present in different types of human tumors. They are commonly found on the cell surface of a variety of adenocarcinomas such as lung cancer, gastric cancer, pancreatic cancer and colorectal cancer, and in serum of cancer patients. Both antigens have been proposed as important diagnostic markers and they are used in detecting and monitoring of these diseases. Recently, it has been shown that sialosyl-Le(a) and sialosyl-Le(x) carbohydrate structures are ligands for selectins, newly described family of adhesion molecules. Selectins function as lymphocyte-homing and leukocyte enrollment receptors, or as activation dependent cell surface receptors of platelets and endothelial cells. Several lines of evidence suggest that sialosyl-Le(a) and sialosyl-Le(x) are responsible for adhesion of human cancer cells to endothelium. It has been shown that E-selectin and P-selectin present on endothelial cells mediate these interactions. The mentioned facts suggest that selectins and their carbohydrate ligands can play an important role in a selective homing of tumor cells during metastasis.

Animals↗
PubMed1995

Peanut agglutinin (PNA) binding glycoproteins on human urothelial cell lines of different grades of transformation.

PNA-reactive sites, representing mainly unmasked Thomsen-Friedenreich (TF) antigen, are predictors of invasive transitional cell carcinoma. The present studies were undertaken in order: 1) to quantify the expression of PNA-reactive sites on well-characterized human urinary bladder cell lines belonging to two different transformation grades (TGr II and TGr III); 2) to identify PNA-binding glycoproteins that are restricted in their expression to tumorigenic and invasive urothelial cell lines. Flow cytometry studies revealed significant differences between TGr II and TGr III cell lines. The mean fluorescence intensities of TGr III, tumorigenic and invasive cells, were in the range of 28.4 to 57.3 arbitrary units. The TGr II cells exhibited several fold lower fluorescence intensity: 9.8 arbitrary units for HCV 29 cell line and 13.1 arbitrary units for Hu 609 cells. Neuraminidase treatment, increasing PNA-binding to TGr II as well as in TGr III cell lines, revealed the presence of cryptic PNA-binding sites. The number of cryptic PNA-binding sites seemed to be similar in TGr II and in TGr III cells and, therefore, only add to the total number of PNA-binding sites on native, untreated cells. Binding of 125I-PNA to all cellular proteins resolved by SDS-PAGE and transferred to nitrocellulose showed multiple bands. The TGr III cells lines, after desialylation, were characterized by the presence of two major PNA-binding components represented by diffuse bands with apparent molecular mass about 68-79 kDa and 116-156 kDa, respectively, and two weakly stained bands with apparent molecular mass of 51 kDa and 60 kDa. Both cell lines representing TGr II expressed high molecular mass PNA-binding component of 207 kDa. They were further characterized by the weaker staining intensity of 116-156 kDa glycoproteins as compared to TGr III cell lines.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1995

Alterations in glycoconjugates related to the transformation grade of human uroepithelial cell lines.

To study the changes in carbohydrate expression associated with the progression of malignant phenotype, we have used the series of human urinary bladder epithelial cell lines. Non-tumorigenic and non-invasive cell lines, with infinite life span, were defined as transformation grade II (TGr II). Cell lines producing tumors in nude mice and invading fragments of embryonic chick hearts in vitro were defined as TGr III. In the present review the results obtained from our immunochemical, biochemical and structural studies on glycosphingolipids and glycoproteins of 10 human urothelial cell lines representing both grades of transformation are summarized and discussed in relation to the literature data. TGr III cell lines are characterized by decreased level of the GM2 ganglioside, presence of sialyl-Le(a) ganglioside, increased amount of more highly branched, tri- and tetraantennary N-acetyllactosamine-type glycans and higher number of binding sites for peanut lectin and wheat germ agglutinin lectin, as compared to non-tumorigenic and non-invasive TGr II cell lines.

Animals↗
PubMed1994

A molecular biologic approach to study the fine specificity of antibodies directed to the MN human blood group antigens.

The human MN blood group Ags on glycophorin A are linear complex glycopeptide Ags determined by a combination of amino acid polymorphisms and O-glycans. M Ag has Ser and Gly, and N Ag has Leu and Glu, at positions 1 and 5, respectively. Amino acids 2 to 4 are O-glycosylated. To analyze the fine specificity of Abs recognizing these Ags, recombinant glycophorin A molecules were expressed in Chinese hamster ovary (CHO) cells. The M-allele cDNA was used to generate the N-allele by site-directed mutagenesis. Two chimeric mutants were similarly constructed: Gly5-->Glu mimics the rare Mc phenotype; Ser1-->Leu is not found in human populations. Each type of glycophorin A was transfected into wild type CHO cells. In addition, the M-allele was expressed by mutant CHO cells defective in sialylation. The binding of M and N Abs and an anti-N lectin to recombinant glycophorin A was assessed by various methods. Two anti-N mouse mAbs and the anti-N lectin required leucine at position 1, whereas Glu5 was not essential. One anti-M mAb required both Gly5 and sialic acid. Three human anti-M sera required Ser1, whereas Gly5 was not essential. Four anti-M and -N mouse mAbs failed to bind recombinant glycophorin A, probably due to undersialylation of the recombinant glycoprotein. These results show that CHO cells expressing glycophorin A molecules varying in amino acid sequence and carbohydrate composition are useful for studying the fine specificity of Ab and lectin interactions with this glycoprotein. This is a novel approach and model system for investigating the immune response to linear complex glycopeptide Ags, a class of Ags that has received little attention previously.

Amino Acid Sequence↗
PubMed1994

Biochemical characterization of the O-glycans on recombinant glycophorin A expressed in Chinese hamster ovary cells.

Alterations in N- and O-linked glycosylation affect cell surface expression and antigenicity of recombinant glycophorin A expressed in transfected Chinese hamster ovary (CHO) cells. To understand these effects further, glycophorin A was purified by immunoaffinity chromatography from transfected wild type and glycosylation deficient CHO cells. The O-glycans were characterized both biochemically, using gel filtration and high performance anion exchange chromatography, and immunologically, using carbohydrate specific monoclonal antibodies to probe Western blots. The O-glycans of human erythrocyte glycophorin A consist mainly of short oligosaccharides with one, two, or three sialic acid residues linked to a common disaccharide core, Gal beta 1-3GalNAc alpha 1-Ser/Thr, with the disialylated structure being the most abundant. With the exception of the trisialylated derivative, the same structures were found on recombinant glycophorin A expressed by wild type CHO cells. However, in contrast to human erythrocyte glycophorin A, the monosialylated oligosaccharide was the most abundant structure on the recombinant protein. Furthermore, recombinant glycophorin A was shown to express a small amount of the Tn antigen (GalNAc alpha 1-Ser/Thr). Recombinant glycophorin A had the same O-glycan composition, whether purified from clones expressing high or moderate levels of the recombinant glycoprotein. This indicates that the level of expression of the transfected glycoprotein did not affect its O-glycan composition. Deletion of the N-linked glycosylation site at Asn26, by introducing the Mi.I mutation (Thr28-->Met) by site-directed mutagenesis, did not markedly affect the O-glycan composition of the resulting recombinant glycoprotein expressed in wild type CHO cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Recombinant Miltenberger I and II human blood group antigens: the role of glycosylation in cell surface expression and antigenicity of glycophorin A.

Glycophorin A is a heavily glycosylated glycoprotein (1 N-linked and 15 O-linked oligosaccharides) and is highly expressed on the surface of human red blood cells. It is important in transfusion medicine because it carries several clinically relevant human blood group antigens. To study further the role of glycosylation in surface expression of this protein, four mutations were separately introduced into glycophorin A cDNA by site-directed mutagenesis. Each of these mutations blocks N-linked glycosylation at Asn26 of this glycoprotein by affecting the Asn-X-Ser/Thr acceptor sequence. Two of these mutations are identical to the amino acid polymorphisms found at position 28 in the Mi.I and Mi.II Miltenberger blood group antigens. The mutated recombinant glycoproteins were expressed in transfected wild-type and glycosylation-deficient Chinese hamster ovary (CHO) cells. When expressed in wild-type CHO cells and analyzed on Western blots, each of the four mutants had a faster electrophoretic mobility than wild-type glycophorin A, corresponding to a difference of approximately 4 Kd. This change is consistent with the absence of the N-linked oligosaccharide at Asn26. Each of the four mutants was highly expressed on the surface of CHO cells, confirming that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not necessary for cell surface expression of this glycoprotein. To examine the role of O-linked glycosylation in this process, the Mi.I mutant cDNA was transfected into the IdlD glycosylation-deficient CHO cell line. When the transfected IdlD cells were cultured in the presence of N-acetylgalactosamine alone, only intermediate levels of cell surface expression were seen for Mi.I mutant glycophorin A containing truncated O-linked oligosaccharides. In contrast, when cultured in the presence of galactose alone, or in the absence of both galactose and N-acetylgalactosamine, Mi.I mutant glycophorin A lacking both N-linked and O-linked oligosaccharides was not expressed at the cell surface. This extends previous results (Remaley et al, J Biol Chem 266:24176, 1991) showing that, in the absence of O-linked glycosylation, some types of N-linked glycosylation can support cell surface expression of glycophorin A. The glycophorin A mutants were also used for serologic testing with defined human antisera. These studies showed that the recombinant Mi.I and Mi.II glycoproteins appropriately bound anti-Vw and anti-Hut, respectively. They also demonstrated that these antibodies recognized the amino acid polymorphisms encoded by Mi.I and Mi.II rather than cryptic peptide antigens uncovered by the lack of N-linked glycosylation.

Amino Acid Sequence↗
PubMed1992

The binding of human alloantibodies to recombinant glycophorin A.

Chinese hamster ovary (CHO) cells were transfected with the wild-type, M allele of glycophorin A cDNA. The binding of human alloantibodies to recombinant glycophorin A was assessed with a modified hemagglutination-inhibition assay. Patient sera were incubated with acetone powders derived from CHO cells, and the adsorbed supernatants were tested in standard hemagglutination assays. Five M antibodies and one sample containing anti-En(a) bound to transfected CHO cells expressing glycophorin A but did not bind to untransfected CHO cells. Three N antibodies as well as 21 other alloantibodies (representing other major red cell blood group specificities) bound to neither CHO cell line. The M allele specificity of recombinant glycophorin A was further verified by the demonstration that a high-titer D alloantibody maintained the same titer of agglutination after incubation with recombinant glycophorin A. Transfected CHO cells thus express an M blood group antigen that appears to be serologically equivalent to that found on human red cells. A panel of cell lines expressing mutant glycophorin A molecules with defined variations in amino acid sequence and carbohydrate composition will be useful in studies of the fine specificity of human glycophorin alloantibodies. This approach may also provide an abundant source of artificial antigens for clinical use in blood group serology.

Animals↗
PubMed1992

[The role of glycosphingolipids in the expression of neoplastic phenotype. III. Changes in glycosphingolipids related to the capacity of neoplastic cells for metastasis and tumor growth in vivo].

The role of glycosphingolipids in the expression of neoplastic phenotype. III. Alterations in glycosphingolipids of tumor cells related to their metastatic potential and tumorigenicity. This review describes changes in glycosphingolipids related to metastatic and tumorigenic properties of malignant cells. The possible effects of such alterations on malignant behavior of cells is discussed.

Animals↗
PubMed1991

Expression of human glycophorin A in wild type and glycosylation-deficient Chinese hamster ovary cells. Role of N- and O-linked glycosylation in cell surface expression.

Glycophorin A, the most abundant sialoglycoprotein on human red blood cells, carries several medically important blood group antigens. To study the role of glycosylation in surface expression and antigenicity of this highly glycosylated protein (1 N-linked and 15 O-linked oligosaccharides), glycophorin A cDNA (M-allele) was expressed in Chinese hamster ovary (CHO) cells. Both wild type CHO cells and mutant CHO cells with well defined glycosylation defects were used. Glycophorin A was well expressed on the surface of transfected wild type CHO cells. On immunoblots, the CHO cells expressed monomer (approximately 38 kDa) and dimer forms of glycophorin A which co-migrated with human red blood cell glycophorin A. The transfected cells specifically expressed the M blood group antigen when tested with mouse monoclonal antibodies. Tunicamycin treatment of these CHO cells did not block surface expression of glycophorin A, indicating that, in the presence of normal O-linked glycosylation, the N-linked oligosaccharide is not required for surface expression. To study O-linked glycosylation, glycophorin A cDNA was transfected into the Lec 2, Lec 8, and ldlD glycosylation-deficient CHO cell lines. Glycophorin A with truncated O-linked oligosaccharides was well expressed on the surface of ldlD cells (cultured in the presence of N-acetylgalactosamine alone), Lec 2 cells, and Lec 8 cells with monomers of approximately 25 kDa, approximately 33 kDa, and approximately 25 kDa, respectively. In contrast, non-O-glycosylated glycophorin A (approximately 19-kDa monomers) was poorly expressed on the surface of ldlD cells cultured in the absence of both galactose and N-acetylgalactosamine. Thus, under these conditions, in the absence of O-linked glycosylation, the N-linked oligosaccharide itself is not able to support appropriate surface expression of glycophorin A in transfected CHO cells.

Animals↗
PubMed1990

The sialosyl Lewis a ganglioside is present in tumorigenic human urothelial cell lines.

The sialosyl Lewis a antigen was identified in a ganglioside extract from a malignant human urothelial cell line (Hu 1703He) by fast atom bombardment mass spectrometry. The presence of this antigen in urothelial cell lines with varying tumorigenic properties was further studied using the 19-9 monoclonal antibody (MAb). The sialosyl Lewis a ganglioside was expressed only in the tumorigenic cell lines. Thus, the expression of this antigen is a marker of malignancy for human urothelial cell lines. Mass spectrometry also suggests that the fucosyl GM1 ganglioside was expressed in the Hu 1703He cell line. This was confirmed using the F12 MAb, specific for fucosyl GM1. However, the expression of this antigen was not confined to cell lines with tumorigenic properties.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1989

Glycosphingolipids in lectin-resistant variants of mouse Lewis lung carcinoma cells.

Neutral glycolipids and gangliosides from murine Lewis lung carcinoma cell line LL2 and its lectin-resistant variants, differing in metastatic properties, were studied by fast-atom-bombardment mass spectrometry (FAB-MS), exoglycosidase treatment and an immunostaining procedure. The neutral glycolipids identified in all cell lines studied included CMH, CDH, CTH, asialo GM2, globoside and a glycolipid with a preliminary structure of Hex-Hexl-4HexNAc-Hex-Hex-Cer. The major gangliosides were GM3, GM2, GM1 and GD1a. No qualitative differences in glycosphingolipid expression were found between the metastatic cell lines (LL2 and LL2AAA) and the weakly metastatic variants (LL25, LL28, LL230 and LL2RCA II). Some quantitative differences were observed between the cell lines, e.g., in the level of ganglioside-bound sialic acid, which was not apparently correlated with the metastatic capacities.

Animals↗
PubMed1989

Glycosphingolipids of human urothelial cell lines with different grades of transformation.

Neutral glycolipids and gangliosides from seven human urothelial cell lines, differing in grades of transformation (TGr), were characterized by fast atom bombardment mass spectrometry, exoglycosidase treatment and an immunostaining procedure. The major neutral glycolipids identified in all cell lines studied included CMH, CDH, CTH, globoside and paragloboside, the gangliosides were GM3, GM2, sialosylparagloboside and GD1a. The following observations were made: 1. GM2 was the major ganglioside in the TGrII cell lines (non-tumorigenic, non-invasive), but a minor component in the TGrIII cell lines (tumorigenic, invasive). 2. All components showed C16:0 and C24:0 as major fatty acids, but in the TGrIII cell lines the fatty acid composition of CMH and some of the gangliosides were more complex showing unsaturated and hydroxy-fatty acids as well.

Antibodies, Monoclonal↗
PubMed1989

Wheat germ agglutinin binding sites on human urothelial cells of different grades of transformation.

125I-Wheat germ agglutinin (WGA) binding parameters of human urothelial cell lines of different grades of transformation (TGrII and TGrIII) were compared. The values of association constant (Ka) and the number of binding sites/cell for HCV29 (TGrII) cell line were about 3 x 10(6) M-1 and over 4 x 10(7), respectively. Two TGrIII cell lines, HCV29T and Hu549 revealed lower values for Ka, and considerably higher numbers of binding sites/cell (about 3 x 10(8) and 2 x 10(8), respectively). Binding of 125I-WGA to total cellular proteins resolved by SDS-PAGE and transferred to nitrocellulose showed multiple diffused bands in the range of 58-180 kDa. Some of these bands were characteristic for TGrII cells (124 kDa) or TGrIII cells (135 and 148 kDa).

Animals↗
PubMed1988

Interaction of membrane-bound Fc gamma receptors with the cytoskeletal matrix.

Binding of ligands to cell surface receptors may induce an interaction of the receptors with the cell cytoskeleton. This interaction may decrease the solubility of the receptors in nonionic detergents. We studied effect of binding of various 125I-labeled immunoglobulin ligands to Fc gamma receptors on guinea pig peritoneal macrophages and human placental syncytiotrophoblast plasma membranes on an interaction of these receptors with the cytoskeletal matrix. The receptor-cytoskeleton association was evaluated by measurement radioactivity of bound ligands in pellets and supernatants obtained after lysis of cells or membranes in a nonionic detergent NP-40. Binding of soluble immune complexes or crosslinking of IgG bound induces much stronger insolubilization of the receptors than binding of monomeric or aggregated IgG. It shows that the interaction of the receptors with the cytoskeletal matrix strongly depends on the degree of cross-linking of the Fc gamma receptors by ligands bound. The observed effects were IgG Fc region-specific. Isolated, purified putative Fc gamma receptors from guinea pig peritoneal macrophages and from human placental syncytiotrophoblast plasma membranes do not interact with free G or F actin. We also studied association of the guinea pig peritoneal macrophage Fc gamma receptor with the cytoskeleton, before and after shedding of macrophage membrane proteins. The results obtained showed that the macrophages have only one class of Fc gamma receptors interacting with the cytoskeletal matrix. Effect of a cytoskeleton-destabilizing buffer and DNAse I on release of the receptors from the cytoskeleton suggests that insolubilization of ligand-Fc gamma R complexes was caused, at least partially, by an interaction of the receptors with actin filaments in the cytoskeleton. The results presented in this paper suggest that the cytoskeleton might play a role in transmission of signals from Fc gamma receptors to the cells. They underline the role of immune complexes as physiological ligands for Fc receptors and correlate well with activation of cells via their Fc receptors (e.g. superoxide burst) observed by other authors after treatment of the cells with immune complexes, but not with monomeric or aggregated IgG.

Animals↗
PubMed1986

Immunoglobulins of colostrum--VI. Comparative studies of cytophilic properties of bovine serum and colostral IgG.

In our previous studies, using physical-chemical and serological methods, substantial differences between bovine serum and colostral IgG, especially IgG2, have been shown. The structural differences were localized in the Fc region of immunoglobulins studied. The present comparative studies were undertaken to determine whether structural differences in the Fc region of bovine serum and colostral IgG are reflected in the interaction of these immunoglobulins with the guinea-pig peritoneal macrophage Fc gamma receptor. It was found that binding of bovine serum and colostral IgG1 was a saturable process and only quantitative differences in the mode of binding to the Fc receptor were observed. There is, however, a big difference in cytophilic activity of bovine IgG2--no saturable and reversible binding is observed in the case of bovine serum IgG2.

Animals↗