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Biomedical subjects

A Slomiany

Publications and source records attributed to A Slomiany.

At least 109 records · Page 6Linked to original sources

Sulglycotide effect on the proteolytic and lipolytic activities of Helicobacter pylori toward gastric mucus.

OBJECTIVES: Infection with Helicobacter pylori is now recognized as a major factor in the etiology of gastric disease, and among the detrimental effects this bacterium exerts on the mucosal integrity is the elaboration of extracellular protease and lipase enzymes capable of mucus protein and lipids degradation. We present here evidence that the activities of these enzymes are inhibited by an gastroprotective agent, sulglycotide. METHODS: The grown colonies of bacterium were washed with saline, filtered through sterilization filter, and the filtrate used as the enzyme source. RESULTS: In the absence of sulglycotide, the H. pylori protease caused extensive degradation of human gastric mucus, while free fatty acids, glycerol monooleate and lysophosphatidylcholine were produced by the action of H. pylori lipase and phospholipase A enzymes. Introduction of sulglycotide to the incubation systems led to the reduction in the rate of mucus protein and lipid degradation. The rate of proteolysis inhibition was proportional to sulglycotide concentration up to 45 micrograms/ml, at which point a 43% reduction in mucus degradation was attained, whereas the maximum inhibition of lipase (39%) and phospholipase A (98%) activities occurred at a sulglycotide concentration of 100 micrograms/ml. CONCLUSIONS: This study indicates that sulglycotide is capable of counteracting the mucolytic activity of H. pylori, and thus may be of value in the therapy of H. pylori-associated gastric diseases.

Anti-Ulcer Agents↗

Inhibition of tyrosylprotein sulfotransferase by sphingosine and its reversal by acidic phospholipids.

Although tyrosylprotein sulfation has been implicated in the processing of several secretory proteins, nothing is known about the regulation of the enzyme responsible for this event. When poly(Glu6, Ala3, Tyr1) (EAY; M(r) 47,000) was employed as sulfate acceptor, the tyrosylprotein sulfotransferase (TPST) from Golgi membranes of submandibular salivary gland was used to study the effect of various lipids on the expression of its activity. The TPST activity in the Golgi membrane was 38 pmol (mg of protein)-1 (30 min)-1. Approximately 90% of the total activity present in Golgi membranes was extracted by NaCl and Triton X-100 treatment. The Km values of solubilized TPST for EAY and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) were 0.04 and 0.25 microM, respectively. Among the various lipids tested, sphingosine showed maximum inhibition of TPST activity followed by sphingomyelin and phosphatidylcholine (PC). Of the two sphingosine analogs tested, threosphinganine was as effective as sphingosine in TPST inhibition, while erythrosphinganine had little effect. In contrast, the acidic phospholipids phosphatidylinositol (PI) and phosphatidylserine (PS) and oleic acid showed slight stimulation. Half-maximal inhibition of TPST was obtained at 150 microM sphingosine (6 mol % when expressed as mole percent of sphingosine to total phospholipids plus Triton X-100). The inhibition was competitive with respect to EAY and uncompetitive with respect to PAPS. The inhibition caused by sphingosine could be reversed by PI, PS, and oleic acid but not by PC and sphingomyelin. Sphingosine inhibition of TPST activity was also observed in the enzyme isolated from several other tissues such as liver, lung, heart, and cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Effects of benzo(a)pyrene and nicotine on prostaglandin synthesis in buccal pouch and submandibular glands of the Syrian hamster.

Adult male Syrian hamsters were treated by swabbing the apex of the buccal pouch with corn oil (control, C), 1 mM benzo(a)pyrene (BP), nicotine (NC), or BP+NC in corn oil, twice daily, 5 days a week. After a 4-week treatment, the pouches and submandibular glands were dissected and used for the determination of endogenous prostaglandin (PG) production and studies on in vitro PG synthesis. Of the three PGs analysed (PGE2, PGF2 alpha and 6-keto-PGF1 alpha), PGE2 was predominant in the pouch and the glands. BP or NC alone had only a weak effect on PG synthesis in both tissues. However, the combination of BP and NC had a synergistic effect, causing diminished PG synthesis in both tissues. In buccal pouch, BP+NC significantly decreased the concentrations of endogenous PGE2 and PGF2 alpha (PGE2: 0.669 +/- 0.254 versus 1.698 +/- 0.460, PGF2 alpha: 0.273 +/- 0.090 versus 0.625 +/- 0.272 ng/g tissue; BP+NC versus C; mean +/- SD, n = 5, p < 0.05). Similarly significant results were also found for in vitro PG synthesis (PGE2: 0.541 +/- 0.249 versus 1.399 +/- 0.340, PGF2 alpha: 1.045 +/- 0.428 versus 2.133 +/- 0.510 ng/g tissue; BP+NC versus C; mean +/- SD, n = 5, p < 0.05). In submandibular glands, BP+NC significantly diminished the concentration of endogenous PGE2 (1.183 +/- 0.175 versus 2.379 +/- 0.488 ng/g tissue; BP+NC versus C; mean +/- SD, n = 5, p < 0.05). The synthesis of 6-keto-PGF1 alpha in both tissues, and the synthesis of PGF2 alpha in submandibular gland, were slightly decreased with all treatments.

6-Ketoprostaglandin F1 alpha↗

Calcium transport and calcium activated ATPase activity in microsomal vesicles of rat gastric mucosa.

1. Microsomal and plasma membrane vesicles, isolated from rat gastric mucosa, were found to exhibit Ca(2+)-dependent ATPase activities of 14.1 +/- 1.4 and 7.8 +/- 1.1 mumol/mg/hr, respectively. The optimum conditions for the microsomal Ca(2+)-ATPase was pH 6-7, and required Mg2+, while divalent cation such as Cu2+, Zn2+, Fe2+, Ba2+ and Cd2+ had no significant effect. 2. As in the case of Ca2+, Mg(2+)-ATPase, the Ca2+ uptake activity of the microsomal membrane required Mg2+. Both processes were stimulated by submicro molar concentrations of Ca2+ and the apparent Km for Ca2+, Mg2+ ATPase and Ca2+ uptake activities were 0.06 microM and 0.02 microM, respectively. 3. Divalent cations Ba2+ and Fe2+, inhibited both microsomal activities, while Zn2+ and Cd2+ showed no effect on them. However, the monovalent cation K+ did not stimulate Ca2+, Mg(2+)-ATPase and Ca2+ uptake activities. 4. The Ca2+ pumping ATPase of rat gastric mucosal microsome cross-reacted with a monoclonal antibody (mAb-5F10) against the human erythrocyte Ca2+ pump. The apparent molecular weight of mucosal Ca2+ pump was 98 kDa. 5. Close relationship between the kinetic parameters of Ca2+, Mg(2+)-ATPase and Ca2+ uptake activities, and the cross reaction of 98 kDa protein of mucosal microsome with erythrocyte Ca2+ pump antibody, strongly suggest the expression of Ca2+ pump in rat gastric mucosa.

Animals↗

Structural features of carbohydrate chains in human salivary mucins.

1. The structure of carbohydrate chains in the low and high molecular weight mucus glycoprotein forms from submandibular-sublingual saliva of individuals with blood group B was investigated. 2. Alkaline borohydride reductive cleavage of the glycoproteins yielded in each case a population of neutral (55%) and acidic (45%) oligosaccharide alditols ranging in size from 3 to 16 sugar units. 3. The predominant neutral oligosaccharides in both glycoprotein forms consisted of 16 and 15 sugar units arranged in triantennary fashion, and carried blood group B and I antigenic determinants. 4. Three of the oligosaccharides in each glycoprotein contained sialic acid and ranged in size from 3 to 12 sugar units. In two oligosaccharides sialic acid was linked to C3 of galactose and in one to C6 of N-acetylgalactosamine. The sulfated oligosaccharide in both glycoproteins was identified as a pentasaccharide with the sulfate ester group at C6 of N-acetylglucosamine. 5. The results demonstrate that contrary to the earlier view the low and high molecular weight mucus glycoprotein forms of human saliva contain identical carbohydrate chains.

ABO Blood-Group System↗

Regulation of buccal mucosal calcium channel activity by salivary mucins.

1. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of 45Ca2+ while only moderately (15%) affected by the intact low and high molecular weight mucin forms, was significantly inhibited, by the acidic low and high molecular weight salivary mucins which evoked 64 and 60% inhibition, respectively. 2. The inhibitory effect of salivary mucins was associated with the sialic acid and sulfate ester groups of the carbohydrate chains, as the removal of either group caused partial loss in the glycoproteins inhibition, and the complete loss in the inhibitory effect occurred following desialylation and desulfation. 3. The channel in the presence of epidermal growth factor (EGF) and ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the phosphorylated channels showed a 46% increase in 45Ca2+ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein. 4. The binding of EGF to calcium channel receptor protein was inhibited by the low and high molecular weight acidic mucins, causing 41.2 and 36.1% reduction, respectively. This reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoproteins' inhibitory capacity following removal of these groups. 5. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the EGF-controlled buccal mucosal calcium channel activity expression, a process of importance to the preservation of oral tissue integrity.

Calcium↗

Modulation of gastric mucosal calcium channel activity by mucus glycoprotein.

1. The effect of gastric mucus glycoprotein on the activity of calcium channel isolated from gastric epithelial cell membrane was investigated. The 45Ca2+ uptake into the vesicle-reconstituted channels, while only moderately (14%) affected by the intact mucus glycoprotein, was found significantly inhibited (59%) by the acidic glycoprotein fraction. This effect was associated with the sialic acid and sulfate ester groups of the glycoprotein, as their removal caused a loss in the inhibition. 2. The channel complex in the presence of epidermal growth factor (EGF) and ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels showed a 50% increase in 45Ca2+ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein. 3. The channel protein phosphorylation was inhibited by the acidic mucus glycoprotein, which also interfered with the binding of EGF to the channel protein. The inhibitory effect was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoprotein inhibitory capacity following their removal. 4. The results suggest that the acidic gastric mucus glycoproteins, by modulating the EGF-controlled calcium channel phosphorylation, play a major role in gastric mucosal calcium homeostasis.

Adenosine Triphosphate↗

Intracellular transport, organelle biogenesis and establishment of Golgi identity: impact of brefeldin A on the activity of lipid synthesizing enzymes.

1. The effect of brefeldin A (BFA) on generation of transport vesicles, synthesis of phosphoglycerides, sphingosine and ceramides, and utilization of the sphingolipid precursors in the formation of sphingomyelin and glycosphingolipids in Golgi was investigated. 2. In the presence of 5-10 micrograms/ml BFA, the incorporation of [3H]palmitate into glycerides, phosphoglycerides and sphingolipids decreased 45-60%, and the production of endoplasmic reticulum transport vesicles was reduced 30-50%. 3. In Golgi membranes, the presence of 5-10 micrograms/ml BFA in the mixture, assembled to generate Golgi vesicles, evoked inhibitory effect on the synthesis of sphingomyelin, glycosphingolipids and phosphatidylcholine. On average, the synthesis of the sphingolipids and phosphatidylcholine and production of Golgi transport vesicles declined to 30-40%. 4. Addition of 5-10 micrograms/ml BFA to the assay mixture prepared to measure the activity of cytidylyltransferase, phosphocholine diacylglyceroltransferase, and serine palmitoyltransferase, caused up to 50% inhibition of the enzymes involved in the synthesis of phosphatidylcholine and up to 70% inhibition of the enzyme generating 3-ketosphinganine. 5. The results suggest that BFA inhibits the synthesis of phosphoglycerides and sphingolipids. This, at first, is displayed in reduced production of endoplasmic reticulum and Golgi transport vesicles, while the depletion of sphingolipids abrogates the identity of Golgi membranes.

Acyltransferases↗

Differential expression of salivary mucin bacterial aggregating activity with caries status.

1. The low and high mol. wt mucin forms were isolated from saliva of caries-resistant (CR) and caries-susceptible (CS) individuals, and assessed for their bacterial aggregating potential towards S. mutans and S. sanguis, the common cariogenic microorganisms encountered in the oral cavity. 2. The high mol. wt mucin from both groups of subjects exhibited similar protein and carbohydrate content, but the level of covalently bound fatty acids was significantly lower in the CR group. The mucin from CR group showed only a weak inhibitory potential, and no inhibitory activity was observed with the mucin of CS group. 3. The low mol. wt mucins from both groups, while displaying compositional similarities, showed a marked variation in the bacterial aggregating activity. With both bacteria, the activity of the mucin from CR group was at least 128-fold greater than that of CS group. 4. The conversion of the high mol. wt mucin to a low mol. wt form through the action of salivary protease produced in both groups enhancement in mucin's bacterial aggregating capacity. This enhancement was, however, considerably less pronounced in the case of mucin from CS group. 5. The results for the first time demonstrate that the bacterial aggregating epitope of salivary mucins is expressed to a greater extent in CR individuals, and that this epitope is apparently more accessible to bacteria in the low mol. wt mucin form.

Carbohydrate Sequence↗

Control of mucin molecular forms expression by salivary protease: differences with caries.

1. A protease activity capable of degradation of the high mol. wt salivary mucus glycoprotein to a low mol. wt glycoprotein form was identified in human submandibular gland secretion. 2. The protease exhibited optimum activity at pH 7.0-7.4, and gave on SDS-PAGE under reducing conditions two major protein bands of 48 and 53 kDa. The enzyme showed susceptibility to PMSF, alpha 1antitrypsin, and egg white and soybean inhibitors, a characteristic typical to serine proteases. 3. The activity of the protease towards the high mol. wt mucus glycoprotein was found to be 3.8-fold higher in submandibular gland secretion of caries-resistant individuals than that of caries-susceptible. Furthermore, the enzyme from both groups displayed greater activity against the mucus glycoprotein of caries-resistant subjects. 4. Since the low mol. wt salivary mucus glycoprotein form is more efficient in bacterial clearance than the high mol. wt mucin, the enhanced expression of this indigenous salivary protease activity towards mucin may be the determining factor in the resistance to caries.

Animals↗

Activation of gastric mucosal calcium channels by epidermal growth factor.

1. Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. 2. The complex following labeling with [3H]PN200-110 was reconstituted into phosphatidylcholine vesicles which exhibited active 45Ca2+ uptake into intravesicular space as evidenced by La3+ displacement and osmolarity measurements. The 45Ca2+ uptake was independent of sodium and potassium gradients indicating the electroneutral nature of the process. 3. The gastric mucosal channels on epidermal growth factor binding in the presence of ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa subunits of calcium channel. 4. The phosphorylated channels following reconstitution into vesicles displayed at 48% greater 45Ca2+ uptake, thus indicating the tyrosine kinase involvement in EGF dependent activation of calcium channel. 5. The results point towards the importance of epidermal growth factor in the maintenance of gastric mucosal calcium homeostasis.

Animals↗

Nitecapone effect on the synthesis and secretion of gastric sulfomucin.

1. The effect of a new antiulcer agent, nitecapone, on the synthesis and secretion of sulfomucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [3H]proline, [3H]glucosamine and [35S]sulfate. 2. The drug, while showing no discernible effect on the apomucin synthesis, evoked a dose-dependent increase in mucin glycosylation and sulfation, which at 225 microM nitecapone, attained its maximum of 1.8 and 2.2-fold stimulation, respectively. 3. The analysis of mucin secretory responses revealed that nitecapone caused a concentration-dependent enhancement in sulfomucin secretion attaining maximum increase of 1.5-fold at 150 microM nitecapone. 4. The stimulatory effect of nitecapone on sulfomucin secretion was accompanied by 1.4-fold increase in mucosal cAMP level, and showed sensitivity to protein kinase A inhibitors, thus pointing towards the involvement of protein kinase A in mediation of gastric sulfomucin secretory responses to nitecapone. 5. The ability of nitecapone to enhance sulfomucin synthesis and secretion could be of importance to the gastroprotective action of this agent.

Animals↗

Effect of ebrotidine on the synthesis and secretion of gastric sulfomucin.

1. The effect of a new antiulcer agent, ebrotidine, on the synthesis and secretion of sulfomucin in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in DMEM containing [3H]proline, [3H]glucosamine and [35S]Na2SO4 as markers for mucin synthesis, glycosylation and sulfation, in the presence of 0-150 microM ebrotidine. 2. The drug, while showing no discernible effect on the apomucin synthesis, evoked a dose-dependent increase in mucin glycosylation and sulfation, which at 100 microM ebrotidine, attained its maximum of 2.4 and 2.7-fold stimulation, respectively. 3. The analysis of mucin secretory responses revealed that ebrotidine caused a concentration-dependent enhancement in sulfomucin secretion which attained its maximum increase of 3.3-fold at 100-120 microM ebrotidine. Furthermore, the sulfomucin elaborated in the presence of ebrotidine exhibited a higher content of a large molecular-weight mucus glycoprotein form, the assembly of which is intimately associated with the sulfation event. 4. The results suggest that the ability of ebrotidine to enhance gastric sulfomucin synthesis and secretion may play an important role in the gastroprotective mechanism of action of this agent.

Animals↗

Inhibition of Helicobacter pylori glycosulfatase activity towards human gastric sulfomucin by a gastroprotective agent, sulglycotide.

1. A glycosulfatase activity towards human gastric sulfomucin was identified in the extracellular material elaborated by Helicobacter pylori, a pathogen implicated in the etiology of gastric disease. 2. The purified enzyme displayed an apparent molecular weight of 30 kDa, and exhibited maximum activity at pH 5.7 in the presence of 0.3% Triton X-100 and 100 mM CaCl2. 3. The H. pylori glycosulfatase activity towards human gastric sulfomucin was inhibited by a gastroprotective agent, sulglycotide. The inhibitory effect was proportional to the concentration of sulglycotide up to 20 micrograms/ml, at which a 98% decrease in mucin desulfation occurred. However, the drug lost the inhibitory effect following its chemical desulfation. 4. The results demonstrate that sulglycotide is a potent inhibitor of H. pylori glycosulfatase and, hence, may be of value in the treatment of gastric disease associated with this bacterial infection.

Anti-Ulcer Agents↗

Helicobacter pylori lipopolysaccharide inhibition of gastric mucosal laminin receptor: effect of sulglycotide.

1. The effect of cell-wall lipopolysaccharide from Helicobacter pylori, a bacterium implicated in the etiology of gastric disease, on the gastric mucosal laminin-receptor was investigated. 2. The receptor, isolated from gastric epithelial cell membrane by affinity chromatography on laminin-coupled Sepharose, was radioiodinated and incorporated into liposomes which exhibited specific affinity towards laminin-coated surface. 3. The binding of liposomal receptor to laminin-coated surface was inhibited by H. pylori lipopolysaccharide, which at 50 micrograms/ml caused a nearly complete (97%) inhibition in binding. 4. The inhibitory effect of the lipopolysaccharide was prevented by a cytoprotective agent, sulglycotide, that evoked a 92% restoration in binding at 40 micrograms/ml. 5. The results demonstrate that through its lipopolysaccharide H. pylori is capable of disrupting the gastric mucosal integrity and that this detrimental effect could be successfully countered by sulglycotide.

Animals↗

Expression of prostaglandin E2 receptor in hamster buccal pouch: effect of benzo (a) pyrene and nicotine.

Prostaglandin E2 (PGE2) plays an important role in the maintenance of oral mucosal integrity. In this study, we characterized PGE2 receptor binding in the buccal mucosa of Syrian hamster and assessed the effect of nicotine (NC) and benzo (a) pyrene (BP), the two major ingredients in cigarette smoke, on this receptor. Adult male animals were treated for 4 weeks by apical swabbing of the buccal pouch with corn oil (control, C), 1 mM NC, BP, or NC + BP in corn oil, twice a day, 5 days a week. The results obtained with the untreated group revealed the presence of a specific PGE2 receptor consisting of two binding sites (high affinity with Kd = 1.52 nM and Bmax = 37 fmol/mg protein and low affinity with Kd = 813 nM and Bmax = 1.29 pmol/mg protein). The treatment with NC, BP, and NC + BP caused a significant decrease in PGE2 receptor binding (specific binding: 10.20 +/- 0.42, 6.84 +/- 1.32**, 6.58 +/- 0.67** and 5.88 +/- 1.03** fmol/mg protein; C, NC, BP, and NC+BP, respectively; Mean +/- SD, n = 5, **p < 0.01). The data suggest that decreased receptor binding for PGE2 in the buccal mucosa may be the cause for the adverse effect of cigarette smoke on the health of oral mucosa.

6-Ketoprostaglandin F1 alpha↗

Inhibition of gastric mucosal mucin receptor by Helicobacter pylori lipopolysaccharide.

A gastric mucosal mucin receptor has been isolated from the epithelial cell membrane by affinity chromatography on wheat germ agglutinin. The receptor protein displayed an apparent molecular weight of 97kDa and exhibited binding specific to gastric mucin in a concentration-dependent manner. The binding of mucin to the mucosal receptor was inhibited by lipopolysaccharide from H. pylori. The inhibitory effect was proportional to the concentration of lipopolysaccharide up to 30 micrograms/ml, at which concentration a 91% decrease in binding occurred. The results suggest that H. pylori lipopolysaccharide is capable of disrupting the integrity of mucus perimeter of gastric mucosal defense by interfering with epithelial cell-mucin binding.

Animals↗

Modulation of buccal mucosal calcium channel activity by salivary mucins.

The effect salivary mucus glycoproteins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The channel complex following reconstitution into phospholipid vesicles exhibited an active 45Ca2+ uptake and responded to calcium channel activator, BAY K8644, and the antagonist, PN200-110. The uptake of 45Ca2+, while only moderately (15%) affected by the intact mucus glycoprotein was significantly inhibited (60-64%) by the acidic mucus glycoprotein fractions. This effect was associated with the sialic acid and sulfate ester groups of the carbohydrate chains. The channel complex in the presence of epidermal growth factor (EGF) and ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170kDa proteins, and the vesicles containing the phosphorylated channels showed a 46% increase in 45Ca2+ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein. The binding of EGF to buccal mucosal calcium channel receptor protein was also inhibited (36-41%) by acidic mucus glycoprotein. The reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoprotein inhibitory capacity following removal of these groups. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the buccal mucosal calcium channel activity, a process of importance to the preservation of soft oral integrity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗