Application of the 50% Hydrazine Solution Method for O-Glycans Release, their Chemical Labeling, and HPLC Separation.

Mucins are high molecular mass glycoproteins with oligosaccharides O-bonded to the protein core. beta-elimination is the most popular method used for releasing of O-glycans. However to such released glycoforms it is difficult to introduce a label to amplify a signal for oligosaccharide detection.In our study we used a combination of the beta-elimination and hydrazinolysis methods. Released glycoforms were labeled with para-amino benzooic acid ethyl ester (ABBE) and fractionated on HPLC column.This combined procedure seems to be a good tool for O-glycans analysis.

MUC 1 mucin content in gastric juice of duodenal ulcer patients: effect of Helicobacter pylori eradication therapy.

It has been suggested that Helicobacter pylori can interact via carbohydrate structures with gastric mucins. Particularly, the Lewis b structures of the secretory MUC 5AC mucin are considered to be putative receptors for bacterial adhesins. Also the epithelial MUC 1 mucin is implicated by some authors to have a major role in the mechanism of infection. The main objective of our study was to evaluate MUC 1 mucin levels in human gastric juice before and at the end of eradication therapy. Any possible changes could suggest the participation of MUC 1 in H. pylori infection. We assume that the amount of the soluble form of MUC 1 exfoliated to the juice correlates with MUC 1 expressed on epithelial cells. Gastric juice samples of 14 duodenal ulcer patients infected with H. pylori were assayed before and at the end of eradication. In all samples, DNA content was determined. Mucin fractions were isolated by gel exclusion chromatography. High molecular mass material containing MUC 1 was subjected to 4%-12% polyacrylamide gradient gels, electrotransfer to Immobilon P and immunodetection. In 12 infected patients (86%), the initial low level of MUC 1 mucin in gastric juice increased at the end of eradication. In comparison to the infected patients, neutral carbohydrate and DNA content in gastric juice diminished after treatment. Our results indicate that the bacterium affects the soluble form of MUC 1 mucin, thus suggesting a likely role of this mucin in the course of H. pylori infection.

Biosynthesis of MUC1 mucin in human endometrial adenocarcinoma is modulated by estradiol and tamoxifen.

Polymorphic epithelial mucin MUC1 is expressed by most epithelial cancers, although free natural MUC1 antibodies are present in the circulation of healthy subjects as well as in that of cancer patients. The role of MUC1 mucin molecules in cancer cells of endometrium is not precisely known. The results reported here demonstrate that MUC1 biosynthesis in human endometrial adenocarcinoma cells (Ishikawa line) is stimulated by estradiol hormone and inhibited by tamoxifen, which was measured by [(14)C]threonine or [(3)H]glucosamine incorporation into MUC1 protein. Tamoxifen applied in combination with estradiol also inhibited this process, but pre-incubation of cells with estradiol resulted in a decrease in the inhibitory effect of tamoxifen. Electroblotting and reactions with antibodies against MUC1 core protein epitopes confirmed the presence of MUC1 in cell lysates and culture media of Ishikawa cells. Reactions with lectins showed the presence of oligosaccharide structures demonstrating antigen-T activity and the presence of sialic acid residues. The results confirm that there is downregulation of MUC1 expression in cancer culture cells treated with selective estrogen receptor modulators, which may be essential for reducing the migration of cancer cells and the metastatic properties of tumor cells.

The role of mucus and its components in protection and repair within the alimentary tract mucosa: Polish experience.

The Gastroenterology Research Laboratory at New York Medical College, New York City, NY, directed by Prof. Dr. George B. Jerzy Glass and after his retirement by Prof. Dr. Bronislaw L. Slomiany and Prof. Dr. Amalia Slomiany served as a launching pad for successful careers in exploration of mucus for Dr. Andrzej Gindzienski and Dr. Krzysztof Zwierz and Janusz Badurski at the Medical School in Bialystok, Poland as well as Dr. Jerzy Sarosiek at Gastroenterology Research Laboratory, University of Virginia Health Sciences Center, Charlottesville, VA and currently, Gastroenterology Research Laboratory, Kansas University Medical Center, Kansas City, KS, USA. The dynamic and insightful research endeavors implemented at the Medical School of Bialystok revealed new information regarding enzymatic pathways of mucin synthesis especially its carbohydrate components such as hexosamines. These discoveries become instrumental in our understanding of the alimentary tract mucin synthesis and function in health and disease. Similarly innovative mucus research conducted across the Atlantic Ocean uncovered the novelty of mucin elaborated within the esophageal submucosal mucous glands in humans by demonstration that its chemical characteristics are different both from human salivary and gastric mucins. In addition, a novel method for the measurement of the thickness of the gastric mucus layer ex vivo in humans has also been developed. These pioneering works are continued at both mucus exploration centers attracting younger generation of investigators enticed by the mystery of the structure and function of the mucus barrier and its leading role in mucosal protection against injury as well as immediate and unequivocal contribution to mucosal repair and reconstitution process.

MUC1 expression in human breast cancer cells is altered by the factors affecting cell proliferation.

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. In the present study, we have investigated if the factors affecting cells proliferation could influence MUC1 mucin biosynthesis and shedding from cell surface into the culture medium in two human breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ER-). Using MCF-7 line we found that estradiol at a concentration of 10(-7) M increased [3H]glucosamine incorporation into mucin in cell lysate approximately twofold in comparison with control cultures, and a similar increase was observed in the culture medium. The selective estrogen receptor modulator, tamoxifen (at concentrations of 10(-6) M and 10(-5) M) had a little inhibitory effect. MDA-MB-231 cells in culture were stimulated with phorbol ester PMA, the protein kinase C activator. We noted that PMA greatly stimulated MUC1 synthesis and its shedding to culture medium and that this effect was abolished by protein kinase C specific inhibitor--bisindolylmaleimide.

Identification of a pepsin-sensitive type III-like collagen in breast cancer MCF-7 cells.

Electrophoretic analysis of [3H]proline-labeled culture medium proteins of MCF-7 cells revealed the presence of disulfide-bonded, bacterial collagenase-sensitive component which comigrated with pro(alpha)1 chains of type III and type I collagens. However, it was pepsin- and trypsin-sensitive. Within 1 min of pepsin-digestion, a component with a size of alpha1 chain of type I or III collagen was produced which degraded after 5 min of digestion. Similarly, the pepsin-sensitive band was completely degraded by trypsin at 30 degrees C within 5 min. We examined CNBr peptides of the collagenous band and demonstrated that it was alpha1 chain of type III collagen. When MCF-7 cells were cultured in the presence of 2 nM estradiol, a marked increase in the level of collagen secreted into medium was found. The identified proteinase-sensitive type III-like collagen as major protein of extracellular matrix, would be expected to be more susceptible to degradation which might contribute to tumor progression.

Isolation of MUC 1 antigen from human gastric juice.

Episialin (MUC 1 antigen) is a membrane bound mucin. It is located on the surface of the epithelium. It can also be detected in soluble form in body fluids. In our paper we present method of the purification of this antigen from human gastric juice. MUC 1 antigen was isolated from human gastric juice by the gel filtration on Sepharose 4B column and affinity chromatography with HMFG-1 antibody coupled to CNBr activated Sepharose 4B. Using presented methods for purification of MUC 1 antigen from gastric juice we received after immunostaining one diffuse band of about 400 kDa molecular weight. Our results revealed that presented method of isolation of MUC 1 antigen from gastric juice is a valuable and sufficient procedure for purification of soluble form of this antigen.

The participation of ribosome-UDP-GalNAc complex in the initiation of protein glycosylation in vitro.

The gastric epithelial cells ribosome-UDP-GalNAc complex is a donor of UDP-GalNAc as the substrate for N-acetylgalactosaminyltransferase, which catalyse the transfer of GalNAc residue to the polypeptide, existing on polysomes. It was observed that the deglycosylated porcine mucin and synthetic peptide (PTSSPIST) can be also glycosylated with participation of N-acetylgalactosaminyltransferase and ribosome-UDP-GalNAc complex. The probability of the ribosome-UDP-GalNAc complex as an intermediate in the O-glycosylation is considered.

Disulfide-bound proteolytic fragments of gastric mucin are 100- and 140-kDa proteins.

Pig gastric mucus was tested for its autodegradative proteolytic degradation at pH 7.0, in the presence or absence of proteinase inhibitors and SDS. Samples of crude mucus were incubated at room temperature for 48 and 96 h in sodium azide stabilized buffer, pH 7. 0, and urea-extracted mucin was purified. Electrophoretically homogenic mucin preparation was reduced and alkylated with iodo[(14)C]acetamide, and analyzed for labeled products. On 7.5% SDS/PAGE protein bands at 80 and 120 kDa were noted, but radioactivity was incorporated into 100- and 140-kDa bands, with increasing intensity from T(0) to T(96), and into high molecular mass mucin subunits. The results confirmed the autodegradative properties of gastric mucin and demonstrated that the 100- and 140-kDa fragments are the main proteolytical products of pig gastric mucin and are disulfide bound with the rest of the molecule.

Cytoplasmic inhibitor of eEF-2 ADP-ribosylation catalyzed by diphtheria toxin or endogenous transferase in rat liver cells.

eEF-2 (100 kDa) isolated from rat liver cells undergo ADP-ribosylation in the presence of diphtheria toxin or endogenous ADP-ribosyltransferase, which was co-purified with the factor. We separated the fraction free of elongation factor and endogenous transferase, which strongly inhibited the ADP-ribosylation of eEF-2. This fraction did not affect the activity of the elongation factor. The lack of endogenous transferase activity (which is potentially lethal for the cell) in the postribosomal supernatant could be the result of its inhibition. eEF-2 (65 kDa) which is probably responsible for the process of translocation (Gajko, A. et al. (1999) Biochem. Biophys. Res. Commun. 255, 535-538) was protected from ADP-ribosylation and its irreversible inactivation in the presence of the rat liver extract fraction.

Deglycosylated mucin as a substrate in enzymatic O-glycosylation in vitro.

To obtain the apomucin as a substrate for glycosyltransferases activity testing, the pig gastric mucin deglycosylation by chemical method was carried out. Resulted apomucin, rich in Ser, Thr and Pro residues, was as good carbohydrate acceptor in enzymatic O-glycosylation in vitro, as synthetic peptide, analogue of MUC2 mucin tandem repeats sequence.

Structure and biosynthesis of human salivary mucins.

Human salivary glands secrete two types of mucins: oligomeric mucin (MG1) with molecular mass above 1 MDa and monomeric mucin (MG2) with molecular mass of 200-250 kDa. Monomers of MG1 and MG2 contain heavily O-glycosylated tandem repeats located at the central domain of the molecules. MG1 monomers are linked by disulfide bonds located at sparsely glycosylated N- and C-end. MG1 are synthesized by mucous cells and MG2 by the serous cells of human salivary glands.

Activity of partially purified UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase with different peptide acceptors.

As part of investigations on the role of the UDP-GalNAc-ribosome complex in the initial O-glycosylation of proteins, we have isolated from porcine gastric mucosa GalNAc-transferase, mucin and apomucin, and its three fractions containing carbohydrate in the amounts: I - 1.6%, II - 0.65% and III - 0.00% (wt/wt) of apomucin mass. Amino acid analysis showed that fractions I and II contained slightly higher amounts of serine and threonine as compared to native mucin and apomucin. The short peptide Pro-Thr-Ser-Ser-Pro-Ile-Ser-Thr was the most effectively glycosylated. Our apomucin preparations are also good acceptors of GalNAc and can be used for testing of O-glycosylation in vitro.

[Content of certain sugars and polyols in cataractous nucleus in non-diabetics and diabetic patients with type II diabetes]. / Zawartosc wybranych cukrów i polioli w jadrach zmetnialych soczewek u osób bez cukrzycy i z cukrzyca typu II.

PURPOSE: Sugars and polyols content assessment in cataractous nucleus in diabetics and non-diabetics. MATERIAL AND METHODS: Cataractous nucleus was obtained during extracapsular cataract extraction derived from 52 diabetic and 58 non-diabetic patients. The content of glucose, fructose, sorbitol and mioinositol in examined material was determined using iquid gas chromatography. RESULTS: Average contents of sorbitol, glucose and fructose in cataractous nucleus diabetic patients (ZSC) are significantly higher than in non-diabetics (ZS). No changes were observed in mioinositol content in both studied groups. CONCLUSION: The role of the polyol pathway in the development of diabetic cataract is considerable.

Possibility of the transformation of eEF-2 (100 kDa) to eEF-2 (65 kDa) in the peptide elongation process in vitro.

Two active eEF-2 polypeptides of approximately 100 and 65 kDa were copurified from rat liver cells and separated. The fate of eEF-2 (100 kDa) during its binding to ribosomes and in the translocation step of the peptide elongation process was investigated. It was shown that eEF-2 (100 kDa) did not change its form during the process of binding to the ribosomes. In the postribosomal supernatant, obtained from the postincubation mixture of the elongation process, only eEF-2 (65 kDa) was found. These results suggest that the form of eEF-2 (100 kDa), when bound to the ribosome during the elongation process, is transformed to eEF-2 (65 kDa).

Low molecular mass products of depolymerization of purified mucin--attempts at isolation and characterization.

Samples of crude mucin were incubated at room temperature for 48 and 96 h in a sodium azide containing buffer, pH 7.0. Then each sample was purified, reduced and alkylated with iodo[14C]acetamide. Electrophoretic analysis demonstrated that radioactivity was incorporated into the mucin subunits and proteins of 100 and 140 kDa. The results of our experiments suggest that the released proteins can be a part of mucin molecule, cleaved by proteolysis and reduction of disulfide bridges.

Effect of the components extracted from the needles of Taxus baccata on protein biosynthesis in a cell-free rat liver system.

Various species of Taxus contain taxanes that promote polymerization and stabilization of microtubules. They have been reported as antineoplastic compounds with highly effective chemiotherapeutic application. A decrease in incorporation of the radiolabelled precursors into DNA, RNA and proteins in vivo has been reported too. The preliminary results have shown that also the other compounds present in the aqueous extract from Taxus baccata needles, participate in the inhibition of the protein biosynthesis. The binding site of eEF-2 on the ribosome seems to be the target of this inhibition process.

Preparation of gastric mucin "STP-domains" using pronase digestion and chemical deglycosylation.

In this work we present a method of mucin protein backbone fragments preparation, which are rich in serine, threonine and proline amino acid residues. The purified native gastric mucin was reduced, the pronase digested and chemically deglycosylated. The obtained apomucin preparations contained slight quantities of residual carbohydrates (0-1%). In O-glycosylation reaction in vitro, with the participation of GalNAc-transferase, the amounts of incorporated [14C]GalNAc to apomucin fractions were about twofold greater in relation to the deglycosylated non-digested mucin subunits. We also demonstrate the usefulness of immunoblotting technique of apomucin preparations detection.