Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.6.4 (chondroitinase)
2,039 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cytoplasmic sialomucin in Paget cells of both mammary and extramammary Paget's diseases was examined, using a new method proposed by Volz et al. (1987a, b). The staining methods used involved an electrolyte-Alcian Blue (pH = 5.8) and periodic acid Schiff. Oxidation was performed with 0.4 mmol/l periodate in 1 mol/l HCl at 4 degrees C or 50 mmol/l periodate in distilled water at room temperature for 1 h. Methylation, saponification, borohydride reduction, and digestion with diastase, neuraminidase (Vibrio cholerae) or chondroitinase ABC, were also employed. The cytoplasmic mucin was found to exhibit positive reaction for the above staining which were variously altered by the chemical modification procedures and diminished in intensity or abolished by digestion with neuraminidase. These results suggest that the cytoplasmic mucin in Paget cells is sialomucin without side-chain substituent in genital Paget's disease, and that with a substituent at C7 in mammary Paget's disease.
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PMID:Histochemical analysis of sialomucin in Paget cells of mammary and extramammary Paget's disease. 137 8

The use of a miniature column chromatographic assay (using Sepharose CL-4B columns) for measuring mucin production in guinea pig gastric mucous cell cultures is described. The assay was based upon the ability of radiolabelled precursors ([14C]serine and [3H]galactose) to incorporate with high specificity into mucins which thereby appeared in the excluded material. Rates of excluded material radiolabelling by both precursors were constant for incubations up to 24 hours, and substantially reduced by cycloheximide co-incubation (25 microM). Labelled excluded material was completely degraded by mild alkaline borohydride treatment, only partially degraded by HNO2 (pH 1.5), and not degraded by chondroitinase ABC. Thus the major radiolabelled product measured in this system was mucin, although we found that it was less glycosylated than gastric mucins obtained from other sources. In addition, the technique employed to separate and measure mucin production proved rapid and consistent.
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PMID:Chromatographic measurement of mucin production in cultures of gastric mucous cells. 172 30

The synthesis and secretion of mucin-like high-molecular glycoprotein was studied in 2 human colon cancer cell lines that spontaneously differentiate in culture (Caco-2 and T84) and in 2 cell lines that do not spontaneously differentiate (LS174T and HT29). Mucin, quantitated by 3H-glucosamine labelling and chromatography on Sepharose CL-4B was found to be produced by all 4 cell lines. The mucinous nature of the labelled high-molecular glycoprotein was verified by enzymatic degradation treatments (heparinase, hyaluronidase, chondroitinase ABC, and N-glycanase), alkaline-borohydride treatment, inhibition of labelling by the glycosylation inhibitor benzyl-alpha-GalNAc, and by CsCl-density-gradient centrifugation. In all 4 cell lines, an inverse correlation of mucin synthesis with cell density was demonstrated. In Caco-2 cells, the spontaneous post-confluent enterocytic differentiation with increased brush-border enzyme expression was associated with a decrease in mucin synthesis and in the activities of polypeptidyl GalNAc transferase and beta 1,3-galactosyltransferase activity. Using cDNA probes for 2 distinct human intestinal mucins (MUC2 and MUC3), we found that all 4 colon cancer cell lines expressed mucin message, but the types of mucin mRNA expressed differed. These data indicate that mucin-like glycoproteins can be synthesized by cell lines derived from non-mucinous colon cancer, whether or not they undergo spontaneous differentiation in culture. These cell lines may serve as in vitro models for studying apomucin heterogeneity and control of mucin gene expression.
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PMID:Mucin synthesis and secretion in relation to spontaneous differentiation of colon cancer cells in vitro. 172 5

Organ culture of guinea pig trachea was performed in the presence of [35S]sulfate in order to characterize the sulfated glycoproteins released from the respiratory epithelium and mucosa. The sulfated macromolecules that were synthesized during a 6-h incorporation were separated by CsBr density-gradient centrifugation and gel-filtration chromatography successively. Most of the sulfated secreted macromolecules corresponded to a population of glycoproteins sensitive to reductive beta-elimination but resistant to both chondroitinase ABC and heparinase. These glycoproteins had different buoyant densities (ranging from 1.48 g/ml to 1.16 g/ml) and could be subfractionated according to molecular mass. A major part of the radioactivity was incorporated into high-molecular-mass mucins that were excluded from a Sepharose CL-2B column and did not penetrate into polyacrylamide gel in PAGE. However, a mixture of sulfated O-glycoproteins of much lower molecular mass was also characterized in addition to low amounts of chondroitin sulfate. Epithelial goblet cells are the predominant mucin-containing cells of the respiratory guinea pig trachea. Our results suggest that a wide range of sulfated O-glycoproteins are secreted by the guinea pig tracheal mucosa.
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PMID:Sulfated O-glycoproteins secreted by guinea pig trachea in organ culture. 189 37

Since the 1960s, the loss of sulfomucin from colonic epithelium has been considered to be an indicator of an early stage of carcinogenesis; yet, the biochemical basis for this phenomenon has never been elucidated. We recently prepared a monoclonal antibody (mAb) 91.9H that immunoprecipitates the normal colonic mucins metabolically incorporating [35S]-sulfate. This mouse IgG1 antibody did not cross-react with colon carcinoma mucins that lack sulfate groups. Using normal colonic epithelia unlabeled or radiolabeled with [35S]sulfate and [3H]glucosamine, we purified a high molecular weight glycoprotein that reacts with mAb 91.9H. This was achieved by a combination of DEAE-cellulose anion-exchange chromatography, consecutive treatments with chondroitinase ABC plus heparitinase and with sodium dodecyl sulfate plus 2-mercaptoethanol, and gel filtration on Sepharose CL-2B in the presence of 8 M urea. Antibody reactivity was found in acidic but not neutral high molecular weight glycoproteins. After Sepharose CL-2B fractionation, the mAb 91.9H-reactive fractions consisted of a component with an approximate molecular weight of 500,000-900,000. A purified sulfomucin contained protein, neutral sugar, amino sugar, sialic acid, and sulfate in an approximate ratio of 2.5:1.0:1.1:0.4:0.5. The polypeptide portion was rich in hydrophilic amino acids, particularly threonine. Binding of mAb 91.9H in solid-phase assays was inhibited to 50% by purified normal colon acidic mucin at doses of 5-50 micrograms/ml, depending on different preparations. Various glycosaminoglycans or sulfatides did not show inhibitory activity. Sulfomucin reactivity with mAb 91.9H, as determined by solid-phase-binding inhibition and by dot blot assays, was significantly reduced by chemical desulfation of sulfomucins with anhydrous hydrochloric acid, suggesting that sulfate groups served as a portion of the immunochemical determinant for this antibody. Sulfate residues were apparently linked to alkaline-sensitive carbohydrate chains, but alkaline-released carbohydrate chains did not react with mAb 91.9H. Immunohistochemical examinations showed that mAb 91.9H bound normal colonic epithelial cells, which also stained with high-iron diamine, more strongly than it bound colon carcinoma cells.
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PMID:Human colonic sulfomucin identified by a specific monoclonal antibody. 191 91

The antigenic determinant recognized by monoclonal antibody SPan-1 is greatly elevated in sera of patients with pancreatic cancer but not in sera of normal individuals. Here we describe the mucin-like characteristics of the SPan-1 antigen isolated from culture medium and xenografts of the human pancreatic cancer cell line SW-1990. YPan-1, another pancreatic cancer associated monoclonal antibody, also reacts with the SPan-1 antigen. The SPan-1/YPan-1 antigens have densities of 1.4-1.5 g/ml and elute in the void volume of Sepharose CL-2B columns. They are resistant to degradation by chondroitinase ABC, nitrous acid, and hyaluronidase but susceptible to protease digestion and reductive beta-elimination. All these characteristics suggest that the SPan-1 and YPan-1 determinants are carried on mucinous antigens. Both SPan-1 and YPan-1 immunoreactivities are unaffected by boiling or by alkylation and reduction of the mucins while they are abolished by mild periodate oxidation or neuraminidase and are markedly decreased by wheat germ agglutinin. Thus, their antigenic determinants are composed principally of carbohydrates with sialic acid, an absolute requirement for reactivity. However, the epitope specificities of SPan-1 and YPan-1 are different since YPan-1 does not compete with SPan-1 for binding to antigen. Moreover, YPan-1 and SPan-1 can be distinguished from several other sialic acid requiring, cancer associated antibodies such as B72.3, CSLEX-1, DU-PAN-2, OC-125, and 19-9 by either their epitope characteristics or their tissue reactivity patterns.
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PMID:Mucin-like antigens in a human pancreatic cancer cell line identified by murine monoclonal antibodies SPan-1 and YPan-1. 245 32

Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.
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PMID:An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates. 262 58

Colon cancer cells in culture synthesize and secrete mucin glycoproteins, which carry a number of cancer-associated antigens. However, the structures and mechanisms of biosynthetic processing are not well understood. Mucins synthesized and secreted by LS174T human colon cancer cells were compared to those in LS174T xenografts in athymic mice. Mucins radiolabeled with glucosamine or sulfate were purified by gel filtration and cesium chloride density gradient centrifugation. The mucins were of high molecular weight and were resistant to chondroitinase ABC, hyaluronidase and HNO2 treatment. They were, however, susceptible to pronase digestion and mild alkaline treatment. Using radiochemical precursors, the cellular mucin was shown to contain fucose, galactose, N-acetylgalactosamine, N-acetylglucosamine, N-acetylneuraminic acid, and sulfate. Oligosaccharides released by beta-elimination had N-acetylgalactosaminitol as the reduced amino sugar and also unreduced galactosamine, indicating that there is N-acetyl-galactosamine O-glycosidically attached to protein core and also peripheral N-acetyl-galactosamine not directly linked to protein. DEAE-cellulose chromatography of mucins showed two major peaks with both intracellular and secreted mucins, but xenograft mucins also had more acidic components. Sulfate-labeled mucins were shifted to less acidic peaks by neuraminidase digestion, which indicates that the same mucin molecules are both sialylated and sulfated. We conclude that the intracellular mucins of cultured colon cancer cells, those secreted into the medium, and those in nude mouse xenografts are chemically similar, but differ in sialic acid and sulfate content. This experimental model system, LS174T cells maintained in culture and as nude mouse xenografts, may be useful for further biosynthetic and structural studies of colon cancer mucin.
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PMID:Comparison of metabolically labeled mucins of LS174T human colon cancer cells in tissue culture and xenograft. 273 49

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.
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PMID:The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta. 381 52

The middle ear effusion specimens were obtained by myringotomy and aspiration from 4 children of 4-7 years old, who had been diagnosed as patients with secretory otitis media on the basis of conductive hearing loss and tympanogram. In cases 1 and 2, their ear fluids were macroscopically serous, while those of cases 3 and 4 were mucous. These ear fluids were digested with pronase and the digests were analyzed by cellulose acetate membrane electrophoresis with alcian blue and high-iron-diamine stainings. All samples were found to contain glycopeptides possibly derived from sulfated mucin-type glycoproteins with small amounts of glycosaminoglycans. The glycoconjugates from cases 3 and 4 were further examined after hyaluronidase and chondroitinase ABC treatments, followed by heparitinase digestion. The resultant glycopeptide fractions appeared to be electrophoretically homogeneous and their chemical compositions suggested that they were typical mucin-type glycopeptides. Furthermore, they contained sulfates. The data suggest that in secretory otitis media, one of the major components of middle ear effusions is sulfated mucin-type glycoprotein.
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PMID:Sulfated glycopeptides from middle ear effusions of secretory otitis media. 407 40


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