Release Of Glycoconjugates Research Articles

Determination of the Importance of In-Mouth Release of Volatile Phenol Glycoconjugates to the Flavor of Smoke-Tainted Wines

The volatile phenols guaiacol, 4-methylguaiacol, syringol, 4-methylsyringol, o-, m-, and p-cresol, as well as their glycoconjugates, have previously been shown to be present in elevated concentrations in smoke-tainted wine. Sensory descriptive analysis experiments, with addition of free volatile phenols in combination with their glycosidically bound forms, were used to mimic smoke taint in red wines. The addition of volatile phenols together with glycoconjugates gave the strongest off-flavor. The hydrolysis of glycosidically bound flavor compounds in-mouth was further investigated by in vitro and in vivo experiments. The results indicate that enzymes present in human saliva are able to release the volatile aglycones from their glycoconjugates even under low pH and elevated ethanol conditions, confirming that in-mouth breakdown of monosaccharide and disaccharide glycosides is an important mechanism for smoke flavor from smoke affected wines, and that this mechanism may play an important general role in the flavor and aftertaste of wine.

Differential Release and Phagocytosis of Tegument Glycoconjugates in Neurocysticercosis: Implications for Immune Evasion Strategies

Neurocysticercosis (NCC) is an infection of the central nervous system (CNS) by the metacestode of the helminth Taenia solium. The severity of the symptoms is associated with the intensity of the immune response. First, there is a long asymptomatic period where host immunity seems incapable of resolving the infection, followed by a chronic hypersensitivity reaction. Since little is known about the initial response to this infection, a murine model using the cestode Mesocestoides corti (syn. Mesocestoides vogae) was employed to analyze morphological changes in the parasite early in the infection. It was found that M. corti material is released from the tegument making close contact with the nervous tissue. These results were confirmed by infecting murine CNS with ex vivo–labeled parasites. Because more than 95% of NCC patients exhibit humoral responses against carbohydrate-based antigens, and the tegument is known to be rich in glycoconjugates (GCs), the expression of these types of molecules was analyzed in human, porcine, and murine NCC specimens. To determine the GCs present in the tegument, fluorochrome-labeled hydrazides as well as fluorochrome-labeled lectins with specificity to different carbohydrates were used. All the lectins utilized labeled the tegument. GCs bound by isolectinB4 were shed in the first days of infection and not resynthesized by the parasite, whereas GCs bound by wheat germ agglutinin and concavalinA were continuously released throughout the infectious process. GCs bound by these three lectins were taken up by host cells. Peanut lectin-binding GCs, in contrast, remained on the parasite and were not detected in host cells. The parasitic origin of the lectin-binding GCs found in host cells was confirmed using antibodies against T. solium and M. corti. We propose that both the rapid and persistent release of tegumental GCs plays a key role in the well-known immunomodulatory effects of helminths, including immune evasion and life-long inflammatory sequelae seen in many NCC patients.

The DHE cell line as a model for studying rat gastro-intestinal mucin expression: effects of dexamethasone

The expression of mucin genes was evaluated in rat intestinal cell lines in order to establish an in vitro model for investigating the regulation of intestinal mucin expression in this species. Two rat intestinal cancer cell lines (DHE, LGA) and three nontumoral rat intestinal cell lines (IEC6, IEC17, IEC18) were screened. The mRNA expression of rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC mucin genes was studied by semiquantitative RT-PCR, real-time RT-PCR and Northern-blot analysis. Results were correlated with immunohistochemical expression of rat gastric and intestinal mucin proteins, and secretion of glycoconjugates was examined by enzyme-linked lectin assay. We showed that mRNA of rMucl and rMuc2 were constitutively expressed in all IEC cell populations but periodic acid Schiff staining of these cells did not reveal the presence of glycoproteins. DHE cells expressed rMuc1-5AC mRNA and LGA expressed the same mucins but the level of rMuc4 was much lower. Mucin mRNA expression also differed in relation with the length of cultivation. Immunocytochemical studies revealed the presence of gastric and intestinal mucins in the two tumoral cell lines. Functional experiments showed that bethanechol, A23187 and PMA stimulated release of glycoconjugates in DHE but not in LGA cells. Treatment of DHE cells with dexamethasone (10(-7) mol/l) enhanced rMuc2 mRNA but decreased rMuc1 and rMuc5AC mRNA. Real-time RT-PCR showed that the expression of rMuc1 and rMuc5AC genes was reduced by more than tenfold after 24 h. The increased expression of rMuc2 gene was confirmed by Northern blot analysis. In conclusion, DHE cells provide a valuable cellular model for research on rat mucin secretion and expression.

Glycoconjugate secretion in human airways in vitro: effects of epithelium removal.

The aim of this study was to examine glycoconjugate secretion in human airways with and without an epithelium. Glycoconjugate release in supernatants derived from human airways in vitro was determined using an ELISA assay with an anti-human mucin monoclonal antibody (MAb 3D3). This monoclonal antibody reacted strongly with Le(b) antigen but also recognized in vitro Le(a) and Le(y) determinants. In 11 of the 34 different lung samples (32%) studied the glycoconjugate levels were below the threshhold of detection for this assay. The mean basal secretion of glycoconjugates in human airways in vitro was 100+/-28 microg/g tissue (Period I; n = 23 different lung samples). The amount of glycoconjugate measured in the medium derived from human isolated bronchial ring preparations did not change under control conditions during the course of the experimental procedure (Period I; 128+/-46 microg/g tissue and Period II; 159 +/-48 microg/g tissue; n = 13 paired lung samples). In the supernatants of airway preparations with an intact epithelium the amount of glycoconjugates detected was 90+/-38 microg/g tissue (Period I; n = 12 different lung samples) and removal of the epithelium did not alter this basal glycoconjugate release (94+/-60 microg/g tissue: Period I, n = 8 different lung samples). The absence of the epithelial layer was confirmed by histological evaluation. Methacholine (100 microM) induced a 10- and four-fold increase in glycoconjugate release from airways with and without an epithelium, respectively. In contrast, in preparations with an epithelium, LTD4 (10 microM) and anti-IgE (dilution: 1/1000) did not cause an increase of glycoconjugate release. The methacholine difference between airways with and without an epithelium was not significantly different (P > 0.10). However, a treatment with atropine (100 microM) prevented the increase of glycoconjugate release in preparations with an epithelium. These data derived from a limited number of experiments suggest that the epithelium may not regulate the basal or stimulated release of glycoconjugates from isolated human airways.

S13.6 Corticosteroid inhibition of the synthesis and release of ferret tracheal glycoconjugates

S13.6 Corticosteroid inhibition of the synthesis and release of ferret tracheal glycoconjugates

Ultrastructural localization of carbohydrates in Reichert's membrane of the mouse

In the present investigation, we examined the role of trophoblast and parietal endoderm cells in the synthesis of carbohydrate-containing components of Reichert's membrane. To eliminate the function of Reichert's membrane as a filter between maternal and embryonal tissues we carried out our examination under in vitro conditions. Parietal yolk sac from mouse embryos on day 9 post coitum (p.c.) were cultivated for 0 to 5 days. Because tannic acid enables a complex formation between carbohydrates and osmium we chose the fixation with this acid for the ultrastructural study. Electron microscopy showed that for assembly of Reichert's membrane, trophoblast cells produce and then release components that were detected as tannic acid-positive granules both in the Reichert's membrane and in the vacuoles of the trophoblast cells. To localize specific carbohydrates we used postembedding-gold-lectin histochemistry on LR-GoldR-embedded tissues. Strong binding sites for the lectins WGA (Triticum vulgare), RCA I (Ricinus communis) and Con A (Canavalia ensiformis) were observed in Reichert's membrane and trophoblast cells but not in the parietal endoderm cells. The LTA (Lotus tetragonolobus)-binding pattern was positive in the membrane and its adjacent cells but that of the LFA (Limax flavus) was negative in the parietal endoderm cells and very weak in Reichert's membrane and trophoblast cells. Our results demonstrate that trophoblast cells are involved in the construction of Reichert's membrane through the production and release of specific glycoconjugates.

MACROMOLECULES EXUDED BY SYMBIOTIC DINOFLAGELLATES IN CULTURE: AMINO ACID AND SUGAR COMPOSITION1

ABSTRACTFive species of symbiotic dinoflagellates in culture exude water‐soluble glycoconjugates. Recovered glycoconjugates of three of the five species were composed of more carbohydrate than protein. The uronic acid content of three algal species that infect scyphistomae of the jellyfish Cassiopeia were higher than those of the two species that do not infect. Analyses of hydrolysates by high‐performance liquid chromatography indicated that the protein components were rich in aspartic acid (asparagine), glutamic acid (glutamine), serine, glycine, leucine, and threonine and contain proline and lysine. Histidine and methionine, when present, were in low concentration. Other sugars detected were glucose and galactose and their respective amines, ribose, mannose, and fucose. Neither sialic acid nor arabinose was detected. The release of glycoconjugates by symbiotic dinoflagellates appears to be a general phenomenon. Some components of these macromolecules could serve as signals that pass between symbionts and hosts, while other components, assuming host digestion, could be a source of previously undetected essential amino acids.

Nerve-induced secretion of glycoconjugates from cat submandibular glands: a correlative study with lectin probes on tissues and saliva.

Horseradish peroxidase-conjugated lectins were used on tissue sections to localize the main secretory glycoproteins in cat submandibular glands and on Western blots to evaluate their movement into saliva with selective nerve stimulation. Central acinar cells bound lectins from Arachis hypogaea (PNA) specific for the terminal disaccharide Gal beta 1, 3GalNac, Griffonia simplifolia (GSA I-B4) specific for terminal alpha Gal, and Lotus tetragonolobus (LTA) specific for fucose. Lectins from Limax flavus (LFA) specific for sialic acid and Dolichos biflorus (DBA) specific for terminal alpha GalNac reacted preferentially with demilunar cells, whereas apical granules in striated ducts were recognized principally by LTA. Parasympathetic stimulation promoted the release of lectin-reactive glycoconjugates from both central and demilunar cells. In contrast, sympathetic stimulation caused almost complete release of LTA-reactive granules in striated ducts and only moderate secretion from demilunar cells. Lectin blots of stimulated saliva discriminated many of the constituent bands, providing information about their glycosylation. Several bands were common to both parasympathetic and sympathetic saliva, and many bands gave wider ranges of lectin binding than anticipated from the histochemistry. The major component in parasympathetic saliva was a glycoconjugate of less than 12 KD which reacted with every lectin tested. Lectin blots of sympathetic saliva showed a prominent diffuse LTA-reactive band around 33 KD, which was attributed to tissue kallikrein. The identity and cellular origin of most bands in stimulated submandibular saliva are still unclear but the technique shows considerable promise for improving the recognition and characterization of individual glycoconjugates.

Vagal control of mucus glycoconjugate secretion into the feline trachea.

1. We examined the effects of frequencies and patterns of electrical stimulation of the peripheral cut ends of the vagus nerves on the release of mucus glycoconjugates into feline trachea in vivo. Mucus glycoconjugates, radiolabelled biosynthetically with [35S]sulphate and [3H]glucose, were washed from a tracheal segment in situ, and dialysed before being counted and assayed chemically by the periodic acid-Schiff (PAS) method. 2. Vagal stimulation with regular pulses (10 V, 2 ms duration) at 1, 2.25, 4.5, 9 and 18 Hz produced frequency-dependent increases in the output of mucus glycoconjugates. 3. The muscarinic agonist pilocarpine (0.1-10 microM), given intrasegmentally, produced dose-dependent increases in the output of mucus glycoconjugates. 4. Pretreatment with atropine, phentolamine and propranolol reduced but did not abolish the effects of vagal stimulation. Vagus nerve stimulation still caused frequency-dependent increases in the output of mucus glycoconjugates. 5. High frequency stimulations at 22.5 and 47.5 Hz given intermittently (1 s burst then 4 s rest), whether in the absence or presence of cholinergic and adrenergic blockade, produced similar secretory responses as the same number of pulses delivered in regular trains at 4.5 and 9.5 Hz. This suggests that neither cholinergic nor non-adrenergic, non-cholinergic (NANC) nerve mechanisms in this system are potentiated by high frequency, intermittent burst stimulation. 6. In the absence of atropine, regular vagal stimulation had a greater effect on heart rate than did the same number of pulses delivered in bursts. 7. High molecular weight glycoconjugates from secretions were taken from the void volume of a Sepharose CL-2B gel filtration column and separated further by density-gradient centrifugation. Macromolecular components were observed at two densities, a typical mucin at 1.52 g ml-1, and a high density atypical component at 1.63 g ml-1. In secretions collected during vagal stimulation, either in the absence or presence of cholinergic and adrenergic blockade, the ratio of low density to high density macromolecules was higher than in unstimulated secretions. This can be explained if both cholinergic and NANC nervous vagal mechanisms stimulate the output of typical (density = 1.52 g ml-1) mucins into the feline trachea.

Fertilization failure of frozen mouse oocytes is not due to premature cortical granule release.

The proportion of mouse oocytes that were fertilized in vitro after storage at -196 degrees C in the presence of 1.5 M dimethylsulfoxide (DMSO) was significantly lower than in unfrozen controls (39% vs. 81%). Sperm failed to penetrate the zona pellucida of approximately 80% of the frozen oocytes that remained unfertilized. Removal of the zona restored fertilization to control levels, indicating that changes induced in the zona during freezing and/or thawing were the primary cause of fertilization failure. Sperm-oocyte fusion, sperm nucleus decondensation, and the resumption of meiosis in frozen oocytes appeared to be delayed but subsequently fertilization progressed normally. No evidence was found to suggest zona modification by the premature release of fucosyl-rich glycoconjugates of cortical granule origin onto the surface of the plasma membrane of frozen oocytes stained immediately after thawing with fluorescently labeled Ulex europaeus lectin. Only a few frozen (less than 5%) and control (less than 3%) oocytes that failed to fertilize in vitro had fucosylated molecules on the plasma membrane. Prolonged exposure of fertilized oocytes to DMSO at 4 degrees C did not alter the pattern of lectin binding. In conclusion, fertilization is inhibited in frozen-thawed oocytes by as yet undefined modifications to the zona pellucida which do not involve the premature release of cortical granules.

Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion

Possible roles of eosinophil (EO) products in modulating the release of mucus from airway explants were investigated. Cell- and membrane-free lysates from purified human EOs (1 to 20 x 10(5)) caused a dose-dependent release of respiratory glycoconjugates (RGC) from cultured feline tracheal explants. Crude extracts from isolated EO granules also stimulated RGC release, suggesting that a granular protein might be responsible. Three proteins derived from EO granules, EO-derived neurotoxin, EO cationic protein (ECP), and major basic protein (MBP) were separated by sequential sizing and affinity chromatography. ECP (0.025 to 25 micrograms/ml) caused a dose-dependent increase in RGC release from both feline and human airway explants and also stimulated the release of the serous cell-marker, lactoferrin, from human bronchial explants. EO-derived neurotoxin (0.025 to 50 micrograms/ml) failed to affect RGC release, whereas MBP (50 micrograms/ml) significantly inhibited RGC release from feline explants. Thus, ECP stimulates RGC and lactoferrin release from airway explants, whereas MBP inhibits RGC release.

Human neutrophil elastase causes glycoconjugate release from the epithelial cell surface of hamster trachea in organ culture.

It is known that human neutrophil elastase (HNE) treatment of hamster tracheal explants causes the release of glycoconjugates, most of which appear to have the characteristics of mucus glycoproteins. This study was designed to determine the origin of HNE-induced glycoconjugate release from 1-day-old cultures of adult hamster trachea. After confirming that HNE treatment released glycoconjugates from cultures labeled with tritiated glucosamine, light microscopic autoradiograms and electron micrographs were prepared. Untreated cultures and cultures treated with inactivated HNE served as controls. HNE treatment caused a 40 to 50% decrease in the silver grain count on the external apical surfaces of secretory cells (p less than 0.05) and ciliated cells (p less than 0.01). Silver grain counts in secretory and ciliated cell cytoplasm, submucosa, and nontissue background were not significantly different from controls. The percentage of nongranulated secretory cells and the number of secretory granules in granulated secretory cells were similar in the HNE-treated and untreated controls. There was no evidence of constitutive release of radiolabeled glycoproteins, or of discharge of secretory granules from the secretory cells. We conclude that HNE releases mucins and other glycoconjugates from the external surfaces of both secretory and ciliated cells in tracheal organ culture.

Synthesis and release of glycoconjugates bearing N-linked oligosaccharides by ovarian carcinoma cells isolated from effusions.

Ovarian carcinoma cell clusters were isolated from patient effusions. The cell isolates were incubated in vitro with radioactive glycoconjugate precursors. Radiolabelled glycoconjugates released to culture media were analyzed for molecular mass heterogeneity and lectin binding activity. From 10 to 50% of the released glycoconjugates were present as a heterogeneous array of glycoconjugates of molecular mass greater than 250 kilodaltons. The remaining glycoconjugates were dispersed in a molecular mass range extending down to approximately 15 kilodaltons. Concanavalin A-Sepharose affinity chromatography revealed the presence of N-linked oligosaccharides. Interaction of glycoconjugates with lentil and pea lectins indicated the presence of L-fucose residues linked to asparagine-bound N-acetylglucosamine. Precipitation of glycoconjugates with ricinus communis agglutinin I showed the presence of nonreducing terminal N-acetyllactosamine residues. Collectively, the data indicate that ovarian carcinoma cells release to culture medium fucosylated glycoconjugates bearing complex-type oligosaccharides. The synthesis and release of these glycoconjugates showed no significant differences among different histologic types of ovarian carcinoma; however, modulation as a function of tumor progression may occur.

Dog tracheal epithelial cells in culture synthesize sulfated macromolecular glycoconjugates and release them from the cell surface upon exposure to extracellular proteinases.

To determine whether glycoconjugates can be released into airways by surface epithelial cells that do not contain secretory granules and, if so, whether extracellular proteinases can affect this release, we studied dog tracheal epithelial cells after 8-10 days in culture. Ultrastructurally, these cells showed an extensive cell surface coat and no secretory granules. Cells were pulse labeled with radioactive sulfate (Na2 35SO4, 50 microCi/ml/24 h) and washed free of the unbound label. Release of sulfated products was then measured at 20-min intervals under basal conditions and again after 20 min of incubation with various extracellular proteinase. We found that these cells synthesized sulfated products and released them spontaneously and continuously into the medium. In addition, trypsin, Pseudomonas aeruginosa elastase, thermolysin, Staphylococcus aureus proteinase, mast cell chymase, plasmin, and kallikrein (each at 10(-7) M except plasmin, at 5 X 10(-6) M) increased the release of sulfated products to 77-667% over baseline release (p less than 0.01, n = 5 dogs for each); preliminary results showed that human neutrophil elastase was also very potent. The sulfated products released by trypsin had an apparent molecular weight of greater than or equal to 10(6) da as determined by gel filtration on Sepharose Cl-4B. Over 50% of these 35S-labeled products were digested to low-molecular-weight products (500-2000 da) upon incubation with endo-beta-galactosidase or with keratanase, suggesting that they are glycoconjugates containing poly(N-acetyllactosamine)-type carbohydrate chains. Decrease in cell staining by lectins specific for poly(N-acetyllactosamine), which accompanied the release of glycoconjugates, indicates that these sulfated glycoconjugates were released by proteinases from the apical cell surface. We conclude that cultured tracheal epithelial cells synthesize and transport sulfated macromolecular glycoconjugates to apical cell surfaces. These glycoconjugates are released from cell surfaces when exposed to extracellular proteinases. We therefore suggest that macromolecular glycoconjugates in airway secretions can originate not only from secretory granules but also from epithelial cell surfaces during airway inflammation.

The release of glycoconjugates by rat parathyroid glands in tissue culture II studies on uptake and incorporation of isotopically labeled glucosamine and characterization of the secreted material

Abstract 1. 1. The uptake and incorporation of [3H] glucosamine into rat parathyroid glands were examined in organ culture. Both uptake and incorporation were stimulated by a low Ca2+ concentration (0.75 mM) and inhibited by a high Ca2+ concentration (2.25 mM). 2. 2. Uptake of [3H]glucosamine into the trichloroacetic acid-soluble pool was rapid, and the majority of the radioactivity was present as glucosamine 6-phosphate, N-acetylglucosamine or UDP-N-acetylglucosamine. Addition of actinomycin D and cycloheximide did not inhibit this uptake process. 3. 3. There was incresing incorporation of labeled glucosamine into trichloroacetic acid-insoluble material with time. Only small amounts of label were lipid soluble. When the trichloroacetic acid-precipitate of the gland was solubilized with dodecysulphate, it eluted with the void volume of Sephadex G-100, but after treatment with trypsin, it was degraded to material which eluted just before the salt volume. Brief treatment of intact parathyroid glands which had been incubated with [3H]glucosamine with trypsin released a peak migrating on Sephadex G-100 similar to that of the smallest peak secreted by the glands during culture. 4. 4. The medium from cultures of parathyroid glands incubated with [3H] glucosamine showed labeled macromolecular material in three peaks. The largest, Peak 1, was released continuously throughout 10 days of culture. The two smaller peaks, Peak 2, which migrated in the region of ovalbumin, and Peak 3, which migrated in the region of parathyroid hormone, were released in increasing amounts for 4 days in a low-Ca2+ medium. High Ca2+ inhibited their release. Bioassay showed that the maximum parathyroid hormone activity was secreted at 4 days in these cultures. 5. 5. Rat liver, thyroid or salivary gland culture in the presence of [3H]glucosamine released labeled macromolecular material which migrated in the void volume and with serum albumin, but no Peak 3 material. 6. 6. Further incubation of Peaks 1 or 2 with serum or culture medium and dual-labeling studies failed to demonstrate any precursor product relation between Peak 3 and Peaks 1 or 2. 7. 7. Peaks 1, 2, and 3 were all nearly insoluble in lipid solvents. Peaks 1 and 2 were easily digested by trypsin and pronase, and Peak 3 was partially digested by papain and contained labeled serine as well as glucosamine. 8. 8. On gel filtration, Peak 1 was heterogeneous having a molecular weight range of 100 000 to 150 000 while Peak 2 was more homogeneous, with a molecular weight of 45 000 to 46 000 daltons. On DEAE-cellulose or CM-cellulose Peaks 1 and 2 were heterogeneous consisting of basic and acidic as well as neutral moieties. 9. 9. Peak 3 was homogeneous on Sephadex G-100 and P-30 gel chromatography and by scurose gradient ultracentrifugation. Its calculated molecular weight was 6200, while labeled rat parathyroid hormone had an average weight of 7500. 10. 10. Peak 3 was eluted from DEAE-cellulose with 0.02 M phosphate before hyaluronate or chondroitin sulfate, Peak 3 was not digested by testicular hyaluronidase or bacterial chondroitinase nor precipitated by cetyl trimethyl ammonium bromide. It was not labeled when parathyroid glands were cultured with 3 5SO42− or [14C]glucuronate. 11. 11. Isotopically labeled fucose, mannose, and galactosamine, as well as glucosamine, were incorporated into Peak 3, and analysis of the hydrolysis products revealed multiple labeled sugars.