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Biomedical subjects

A Slomiany

Publications and source records attributed to A Slomiany.

At least 235 records · Page 13Linked to original sources
PubMed1988

Enzymatic sulfation of mucus glycoprotein in gastric mucosa. Effect of ethanol.

A sulfotransferase activity that catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate to carbohydrate chains of gastric mucus glycoprotein has been demonstrated in the antral and body mucosa of rat stomach. Subcellular fractionation studies revealed that the enzyme is associated with Golgi-rich membrane fraction. The sulfotransferase activity of this fraction in antral mucosa was about 35% lower than that in the body. Optimum enzyme activity was obtained with 0.5% Triton X-100 and 30 mM NaF at a pH of 6.8 using desulfated mucus glycoprotein substrate. The enzyme was equally capable of sulfation of the proteolytically degraded and reduced forms of the desulfated glycoprotein, but the acceptor capacity of the intact mucus glycoprotein was about 60% lower than that of the desulfated preparation. The enzyme preparation also catalyzed the transfer of sulfate to galactosylceramide. The sulfation of mucus glycoprotein, however, was not affected by the presence of this glycolipid, suggesting that the sulfotransferase involved in mucus glycoprotein sulfation is different from that responsible for the synthesis of sulfatoglycosphingolipid. The mucus glycoprotein sulfotransferase activity was inhibited by ethanol. The rate of inhibition was proportional to the concentration of ethanol up to 0.3 M and was of the competitive type. The apparent Km value of the enzyme for mucus glycoprotein was 10.5 X 10(-6) M (21 mg/ml), and the KI in the presence of ethanol was 4.7 x 10(-1) M. The 35S-labeled mucus glycoprotein product of the enzyme reaction gave in CsCl density gradient a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein led to the liberation of the label into reduced acidic oligo-saccharide fraction. Most of the label was found incorporated in three oligosaccharides. These were identified as tri-, tetra-, and pentasaccharides, each carrying a labeled sulfate ester group on the terminal N-acetyl-glucosamine residue. Based on the results of structural analyses, the most abundant oligosaccharide was characterized as SO3H----6GlcNAc beta 1----3Gal beta 1----3GalNAc-ol.

Animals↗
PubMed1988

Role of salivary epidermal growth factor in the maintenance of physicochemical characteristics of oral and gastric mucosal mucus coat.

The involvement of salivary epidermal growth factor (EGF) in the maintenance of oral and gastric mucosal mucus coat dimension and chemical characteristics was investigated using sialoadenectomized rats. Examination of the oral and gastric mucosal surface by phase contrast microscopy and Alcian blue uptake revealed that deprivation of salivary EGF caused a 31-36% reduction in mucus coat thickness and a 38-43% reduction in adherent mucin content. Chemical analyses indicated that the mucus coat of sialoadenectomized group exhibited a 21-28% increase in protein and a 67% decrease in covalently bound fatty acids, a 30% decrease in carbohydrates, and a 32-37% decrease in lipids. Sialoadenectomy also evoked changes in the chemical composition of mucus glycoprotein component of oral and gastric mucus coat reflected in the lower content of sulfate (25-26%), associated lipids (24-25%), and covalently bound fatty acids (67-75%). Intragastric supplementation of EGF had no effect on the physicochemical changes caused by sialoadenectomy in the oral mucosal mucus coat, while nearly complete restoration to normal characteristics occurred in the gastric mucosal mucus coat. The results suggest that salivary EGF is essential for the maintenance of mucus coat dimension and quality needed in the protection of alimentary tract epithelium.

Animals↗
PubMed1988

Role of associated and covalently bound lipids in salivary mucin hydrophobicity: effect of proteolysis and disulfide bridge reduction.

The hydrophobic properties of salivary mucus glycoprotein were investigated by fluorescence spectroscopy using bis(8-anilino-1-naphthalene-sulfonate). The mucin, purified from rat submandibular salivary gland, was subjected to removal of associated and covalently bound lipids, degradation with pronase, and reduction with beta-mercaptoethanol, and titrated with the probe. Analyses of fluorescence data revealed the presence of 49 +/- 5 hydrophobic binding sites in the intact mucin molecule, a 69% increase in the number of binding sites occurred following extraction of associated lipids, while the removal of covalently bound fatty acids caused a 25% decrease in the binding sites. Proteolytic destruction of the nonglycosylated regions of the glycoprotein essentially abolished the probe binding, whereas reduction produced glycoprotein subunits whose combined number of hydrophobic binding sites was 2.4 times greater than that of mucus glycoprotein polymer. The results suggest that associated and covalently bound lipids contribute to hydrophobic characteristics of salivary mucin and that the hydrophobic binding sites reside on the nonglycosylated regions of this glycoprotein buried within its core.

Animals↗
PubMed1988

Co-translational processing and intracellular transport of rat salivary mucus glycoprotein.

A preparation of peptidyl-tRNA from intact microsomes of mucin-synthesizing polysomes of sublingual salivary gland cells contained fatty-acylated galactosamine-free and galactosamine-enriched peptidyl-tRNA fractions, whereas trypsin-chymotrypsin treated microsomes yielded predominantly the acylated galactosamine-enriched peptidyl-tRNA complexes. Radioscanning and chemical analyses revealed that palmitate was substituted on all nascent peptides, except those shorter than 20 amino-acid residues. In contrast, the [35S]-methionine label was detected only on galactosamine-free peptides containing up to 70 amino acids. On SDS-polyacrylamide gel, the peptides released from galactosamine-enriched tRNA complexes separated into a multitude of bands ranging in size from 6000 to 60,000 dalton, whereas the total preparation afforded peptides ranging from 2000 to 60,000 dalton. Pulse-chase experiments, using radiolabelled methionine, palmitic acid and N-acetylgalactosamine, combined with chemical characterization of the radiolabelled fatty acids and carbohydrates from purified peptidyl-tRNA, confirmed that the N-terminal fatty acylation and the initial O-glycosylation with N-acetylgalactosamine are the co-translational processes taking place as soon as peptide is sufficiently large to be acylated, trimmed, and translocated to the luminal site of endoplasmic membrane.

Acylation↗
PubMed1988

Enzymatic sulphation of mucus glycoprotein in rat sublingual salivary gland.

Sulphotransferase activity catalysing the transfer of sulphate ester group from 3'-phosphoadenosine-5'-phosphosulphate to salivary mucus glycoprotein was located in detergent extracts of the Golgi-rich membrane fraction of rat sublingual salivary glands. Optimum enzyme activity was obtained with 0.5 per cent Triton X-100, 20 mM NaF and 2 mM MgCl2, at pH 6.8, using desulphated sublingual salivary mucus glycoprotein. The enzyme was equally capable of sulphation of the proteolytically degraded and desulphated glycoprotein, whereas the acceptor capacity of intact salivary mucus glycoprotein was about four times lower. The Golgi enzyme preparation also catalysed the sulphation of galactosylceramide. However, the sulphation of mucus glycoprotein was not affected by the presence of this glycolipid, suggesting that the sulphotransferase involved in mucin sulphation is different from that responsible for the synthesis of galactosylceramide sulphate. The apparent Km of the sublingual-gland mucus glycoprotein sulphotransferase for salivary mucin was 7.7 microM. The 35S-labelled glycoprotein product of the enzyme reaction gave, in CsCl density gradient, a band in which the 35S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein released the label into the reduced acidic oligosaccharide fraction. Upon thin-layer chromatography, two [35S]-oligosaccharides were detected. These were identified as penta- and heptasaccharides, each bearing a labelled sulphate ester group on the terminal N-acetylglucosamine residue. Based on the results of chemical and enzymatic analyses of the intact and desulphated compounds the following structures for these oligosaccharides are suggested: SO3----GlcNAc beta----Gal beta----GlcNAc beta----Gal beta----GlcNAc beta----(NeuAc----)GalNAc-ol and SO3----GlcNAc beta----Gal beta----GlcNAc beta----(NeuAc----)GalNAc-ol.

Animals↗
PubMed1988

Cotranslational fatty acylation of mucus glycoprotein. Addition of palmitic acid to peptidyl-tRNA occurs prior to peptide chain completion and its release.

1. The fatty acylation of mucus glycoprotein nascent peptides was investigated using [3H]palmitic acid and [35S]methionine-labeled peptidyl-tRNA of rat gastric mucous cells. 2. The mucus glycoprotein peptidyl-tRNA fraction was found to contain covalently bound palmitic acid in its complexes. 3. RNase digestion of the mucus glycoprotein peptidyl-tRNA released [3H]palmitic acid labeled peptides which, on SDS-polyacrylamide gel, separated into a multitude of bands ranging in size from 2000 to 60,000 Da. 4. The analyses of low molecular weight peptides revealed that palmitic acid was present in methionine-labeled peptides containing 30-43 amino acids and those of 18-25 amino acids or larger devoid of methionine, but was not identified in methionine-labeled peptides containing 10-15 amino acids. 5. The results indicate that the N-terminal fatty acylation of mucus glycoprotein nascent peptides is a cotranslational process which is occurring in an immediate vicinity of the signal peptide fragment.

Acylation↗
PubMed1988

Effect of ethanol on the in vitro sulfation of salivary mucin.

In vitro sulfation of mucus glycoprotein by sulfotransferase from rat submandibular salivary gland and the effect of ethanol on this enzyme activity was investigated. Subcellular fractionation studies revealed that the enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein is associated with Golgi-rich membrane fraction. The sulfotransferase enzyme exhibited optimum activity at pH 6.8 in the presence of 0.5% Triton X-100, 4 mM MgCl2, and 25 mM NaF. The enzyme was equally capable of sulfation of the desulfated intact as well as proteolytically degraded desulfated glycoprotein preparations, whereas the acceptor capacity of the intact mucus glycoprotein was 70% lower. The submandibular gland sulfotransferase activity was inhibited by ethanol. The rate of inhibition of mucus glycoprotein sulfation was proportional to the concentration of ethanol up to 0.4 M, at which concentration a 39% reduction in the sulfotransferase activity occurred. The apparent Km value of the enzyme for salivary mucus glycoprotein was 11.1 microM, and the Kl in the presence of ethanol was 0.93 M. The synthesized 35S-labeled glycoprotein gave on CsCl equilibrium density gradient centrifugation 35S-labeled peak which coincided with that of the glycoprotein. Alkaline borohydride treatment of this glycoprotein led to the liberation of the label into the acidic oligosaccharide alditol fraction. As the inhibition by ethanol of sulfotransferase enzyme occurred below its isosmotic concentration to plasma, the observed effect could also be detrimental to salivary mucus glycoprotein sulfation in vivo.

Adenine Nucleotides↗
PubMed1988

Protection against alcohol-induced gastric mucosal injury by geranylgeranylacetone: effect of indomethacin.

The mechanism of gastric mucosal protection by an antiulcer agent, geranylgeranylacetone (GGA), against ethanol-induced injury was investigated. The experiments were conducted with groups of rats with and without intraperitoneal indomethacin pretreatment. Animals received intragastrically either a dose of GGA (200 mg/kg) or a vehicle, followed 30 min later by 1 ml of absolute ethanol. The rats were sacrificed after 30 min and the gastric mucosa was subjected to macroscopic and histologic assessment and the measurements of adherent mucus, its dimension and chemical composition. In the absence of GGA, ethanol produced advanced macroscopic necrosis (greater than 38%) and the extensive necrotic lesions were visible upon histologic examination. Pretreatment with GGA significantly reduced (p less than 0.001) the extent and depth of mucosal necrotic lesions caused by ethanol, and this protection was not thwarted by indomethacin. Evaluation of the adherent mucus and its dimension by Alcian blue uptake and inverted microscope technique revealed that GGA was also capable of preventing the untoward effect of indomethacin on the adherent gastric mucus gel and its thickness. Results of chemical analyses established that in the absence of GGA indomethacin caused an increase in mucus protein (15%) and a decrease in its covalently bound fatty acids (67%) and lipids (36%). The decrease in lipids was particularly reflected in the content of phospholipids. Indomethacin, however, had no apparent effect on the composition of gastric mucus elaborated in the presence of GGA. The results suggest that gastric mucosal protective action of GGA is not mediated by endogenous prostaglandins but rather appears to involve the metabolism of mucosal lipids.

Animals↗
PubMed1988

Evidence for weakening of gastric mucus integrity by Campylobacter pylori.

The role of Campylobacter pylori in weakening the mucus component of the gastric mucosal barrier was investigated. The extracellular material elaborated by C. pylori, cultured from antral mucosal biopsy specimens of patients undergoing gastroscopy, caused a rapid degradation of mucus glycoprotein polymer to glycopeptides comparable in size to those produced by the action of pepsin. Viscosity measurements showed that incubation of C. pylori filtrate with gastric mucus led to a gradual loss in mucus viscosity, which at the end of 48 h decreased by 36%. The C. pylori filtrate also had a detrimental effect on the ability of mucus to retard the diffusion of H+. After 12 h of incubation the permeability of mucus to H+ increased by 10%, whereas a 32% increase in permeability was observed after 48 h. The results suggest that the degenerative changes produced in gastric mucus gel by C. pylori may be a contributing factor in the pathogenesis of gastritis and peptic ulcer.

Animals↗
PubMed1988

Gastric mucin hydrophobicity: effects of associated and covalently bound lipids, proteolysis, and reduction.

The hydrophobic properties of gastric mucus glycoprotein were investigated using the fluorescent probe, bis(8-anilino-1-naphthalenesulfonate). The glycoprotein was subjected to removal of associated and covalently bound lipids, peptic degradation, and disulfide bridge reduction. Fluorescence titration data revealed the presence of 55 hydrophobic binding sites in the intact mucin molecule, 71 binding sites in the glycoprotein devoid of associated lipids, and 53 binding sites in the glycoprotein devoid of associated lipids and covalently bound fatty acids. Proteolytic digestion of the glycoprotein with pepsin essentially abolished the probe binding, while reduction of disulfide bridges resulted in glycoprotein subunits whose combined number of binding sites was about 3 times greater than that of the mucin polymer. The binding of the probe to mucus glycoprotein varied with the pH of the medium, being highest at pH 2.0 and lowest at pH 9.0. The results indicate that lipids contribute to the hydrophobic character of gastric mucin and that hydrophobic binding sites reside on the nonglycosylated regions of the glycoprotein polymer buried within its core.

Animals↗
PubMed1987

Enhancement of the lipid content and physical properties of gastric mucus by geranylgeranylacetone.

The effects of intragastric administration of geranylgeranylacetone (GGA) on the content, composition and physical properties of the mucus component of the gastric mucosal barrier were investigated. One group of rats received twice daily for 3 consecutive days a dose of 100 mg/kg body weight of GGA, while the control group was subjected to daily doses of the vehicle. Sixteen hours following the last dose, the animals were killed, and their stomach was cut open and subjected to measurements of the adherent mucus gel content, analysis of its lipids and molecular forms of elaborated mucin, and evaluation of the viscosity and H+ retardation capacity. The results revealed that GGA elicited a 62% increase in the adherent mucus gel and caused a marked decrease in the proportion of the lower molecular weight mucin. Furthermore, the mucus of the GGA group exhibited a 67% higher content of covalently bound fatty acids and contained 46% more total lipids which were greatly (143%) enriched in phospholipids. The physical measurements demonstrated that mucus elaborated in the presence of GGA also exhibited 2.3 times higher viscosity and had a 32% greater ability to retard the diffusion of H+ than the mucus of the control group. The results suggest that GGA exerts a profound effect on the lipid content and the properties of gastric mucus associated with the maintenance of the mucosal integrity.

Animals↗
PubMed1987

Fatty acid acylation of mucin by gastric mucosa: effects of sofalcone and sucralfate.

The effects of antiulcer drugs, sofalcone and sucralfate, on the activity of gastric mucosal mucus glycoprotein fatty acyltransferase were investigated. The acyltransferase enzyme, contained in the detergent extracts of the microsomal fraction of rat gastric mucosa, was incubated with the deacylated gastric mucin and palmitoyl-CoA substrates in the presence and absence of drugs, and the formed fatty acid acylated glycoprotein product was quantitated. In the absence of drugs, the enzymatic activity increased proportionally with increased concentrations of both substrates and of enzyme, and gave an apparent Km value of 5.6 X 10(-7) M. Introduction of sofalcone to the reaction mixtures led to an enhancement in the rate of mucus glycoprotein acylation. The rate of enhancement was proportional to sofalcone concentration up to 1.0 X 10(-5) M, with an apparent Km value of 3.7 X 10(-7) M. In contrast to sofalcone, the acyltransferase activity was inhibited by sucralfate. The rate of inhibition of mucus glycoprotein acylation by sucralfate was of the competitive type and at 1.0 X 10(-4) M reached a value of 25%. The apparent KI value calculated from the double-reciprocal plots for sucralfate was 9.1 X 10(-7) M. As the acylation of mucin with fatty acids plays an important role in the maintenance of gastric mucosal integrity, the results suggest that stimulation of the fatty acyltransferase enzyme by sofalcone may be one of the beneficial effects of this drug towards ulcer healing.

Acylation↗
PubMed1987

Campylobacter pyloridis degrades mucin and undermines gastric mucosal integrity.

The role of Campylobacter pyloridis, a spiral bacteria associated with gastritis and peptic ulcers in weakening the mucus component of gastric mucosal barrier was investigated. The colonies of bacteria, cultured from antral mucosal biopsies of patients undergoing gastroscopy, were washed with saline, passed through sterilization filter and the filtrate was examined for protease and glycosylhydrolase activities. The obtained results revealed that the filtrate exhibited a strong proteolytic activity not only towards the typical protein substrates such as albumin but also towards gastric mucin. Optimum enzymatic activity for degradation of mucin was attained at pH 7.0 and the protease activity was found in a low m.w. (less than 50K) protein fraction. The filtrate showed little glycosylhydrolase activity and did not cause the hydrolysis of mucin carbohydrates. The data suggest that C pyloridis infection weakens the gastric mucosal defense by causing proteolytic degradation of mucin component of the protective mucus layer.

Campylobacter↗
PubMed1987

Role of carbohydrates in the viscosity and permeability of gastric mucin to hydrogen ion.

The effect of carbohydrate removal on the viscosity of gastric mucin and its ability to impede the diffusion of hydrogen ion was investigated. The mucin, purified from dog gastric mucus, was subjected to partial or extensive deglycosylation with specific exoglycosidases and then used in the measurements. The obtained results revealed that removal of peripheral fucose or N-acetylglucosamine caused in each case only about 5% reduction of the glyco-protein viscosity. An 18% drop in the viscosity, however, occurred following removal of sialic acid, while extensive deglycosylation (removal of 86% carbohydrate) reduced the glycoprotein viscosity by 40%. The ability of mucin to retard the diffusion of hydrogen ion increased by 7% following removal of fucose or N-acetylgalactosamine, a 28% increase was obtained following removal of sialic acid, while the permeability to hydrogen ion of the extensively deglycosylated glycoprotein decreased by 42%. The results suggest that carbohydrates contribute significantly to the viscoelastic and permselective properties of gastric mucin.

Acetylgalactosamine↗