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ABSTRACT: Homeostatic cell physiology is preserved through the fidelity of the cell membranes restitution. The task is accomplished through the assembly of the precisely duplicated segments of the cell membranes, and transport to the site of their function. Here we examined the mechanism that initiates and directs the restitution of the intra- and extracellular membranes of gastric mucosal cell. The homeostatic restitution of gastrointestinal epithelial cell membrane components was investigated by studying the lipidomic processes in endoplasmic reticulum (ER) and Golgi. The biomembrane lipid synthesis during the formation of transport vesicles in the systems containing isolated organelle and the cell-specific cytosol (Cyt) from rat gastric mucosal epithelial cells was assessed. The results revealed that lipids of ER transport vesicle and the transmembrane and intravesicular cargo are delivered en bloc to the point of destination. En bloc delivery of proteins, incorporated into predetermined in ER lipid environment, ensures fidelity of the membrane modification in Golgi and the restitution of the lipid and protein elements that are consistent with the organelle and the cell function. The mechanism that maintains apical membrane restitution is mediated through the synthesis of membrane segments containing ceramide (Cer). The Cer-containing membranes and protein cargo are further specialized in Golgi. The portion of the vesicles destined for apical membrane renewal contains glycosphingolipids and phosphatidylinositol 3-phosphate. The vesicles containing phosphatidylinositol 4-phosphate are directed to endosomes. Our findings revealed that the preservation of the physiological equilibrium in cell structure and function is attributed to (1) a complete membrane segment synthesis in ER, (2) its transport in the form of ER-transport vesicle to Golgi, (3) the membrane components-defined maturation of lipids and proteins in Golgi, and (4) en bloc transfer of the new segment of the membrane to the cell apical membrane or intracellular organelle.
Journal of physiology and pharmacology: an official journal of the Polish Physiological Society 01/2005; 55(4):837-60. · 2.27 Impact Factor
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ABSTRACT: The gastric mucosal protective barrier consists of two essential elements: mucus glycoprotein, mucin, secreted by gastric mucosal cells, and the mucin binding protein (MBP), an integral component of the apical epithelial membrane. The studies described here provide evidence on the structure of MBP, its interaction with mucin, and the susceptibility to phospholipase C (PLC) and Helicobacter pylori protease.
The rat gastric mucosa was used to isolate mucin and the apical epithelial membranes. A buffered saline extract of the mucosal cells was used for the isolation of mucin and the 1% Triton X-100-insoluble gastric apical membranes for the preparation of MBP.
The studies on MBP, the mucosal mucin receptor revealed that the protein is anchored in apical membrane through glycosylphosphatidylinositol (GPI). The deamination of MBP with nitrous acid afforded phosphatidylinositols (PIs) and a water soluble, 97 kDa glycosylated protein. The in situ studies with untreated rat gastric mucosa and the mucosa depleted of mucin showed that MBP without mucin was susceptible to the proteolytic degradation with pepsin and H. pylori proteases, but was not released from the apical membrane by the treatment with bacterial PLC.
The study of carbohydrate ligands for MBP revealed binding of octa- and decasaccharides of gastric mucin. The severe impairment in mucin adhesion to MBP, induced by the diet containing ethanol, supports the conclusion that specific carbohydrate determinants participate in mucin attachment to MBP and epigenetic control of the processes that coordinates its interaction with apical mucosal epithelium in the formation of innate protective barrier.
Molecular Medicine 02/2001; 7(1):1-10. · 3.76 Impact Factor
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ABSTRACT: The outcome of chronic ethanol consumption recorded in liver by in situ staining of the genomic DNA in fragmented nuclei indicates the course of cellular events that has been coined as apoptosis or programmed cell death. Hence, we designed the study to determine which ethanol-induced modification of the cellular make-up is responsible for the hastening the cell damage. The in vitro assays were performed with cellular organelles and cytosol prepared from hepatocytes derived from rats subjected to 9 weeks of chronic alcohol consumption. The results were compared with the pair-fed controls receiving isocaloric liquid diet. In the initial phase of the studies, we established that the process of apoptosis was not triggered by the aberrant activity of neutral or acidic sphingomyelinase. The hepatocytes derived from alcohol and control diets manifested equal enzymatic activity. The de novo synthesis of sphingoid bases and ceramides in the alcohol-derived sample of endoplasmic reticulum was reduced, but the in situ apoptosis was up to 36-fold higher than in the control. Also, the isolated hepatocytes contained a 2- to 4-fold higher amount of nucleosomal fragments in the cytosolic extracts. The endosomes from liver hepatocytes of ethanol-consuming rats, in the presence of the cytosol and mitochondria from pair-fed controls, disclosed 2 to 3 times higher apoptotic potential than sample consisting of ethanol-derived fractions only, and 3 to 5 times higher than the control-derived fractions. The substantial increase in apoptosis, as recorded in the amount of DNA fragments released to the cytosol from the fresh nuclei, was also recorded when the microsomal membranes of endoplasmic reticulum and Golgi were incubated in the conditions with preserved intracellular transport. The maximal 20-fold increase of apoptotic activity was recorded in the incubation mixtures of ethanol-derived endoplasmic reticulum-Golgi membranes with control-derived cytosol in the presence of the ATP generating system. Results infer that the intracellular transport vesicles, generated from ethanol-modified membranes in the presence of the substrates that are available in the cytosol of the control hepatocytes, activate the apoptotic activity in the in vitro system. This interpretation is supported by the results of analysis of the clathrin-coated transport vesicles that, in contrast to nonclathrin transport vesicles, contain a sizable accumulation of ceramides that are known to induce apoptosis.
Alcoholism Clinical and Experimental Research 03/1999; 23(2):334-43. · 3.34 Impact Factor
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ABSTRACT: Membrane biogenesis, expressed in endoplasmic reticulum (ER) by formation of transport vesicles, was studied in the liver of ethanol-fed and pair-fed rats. In ER of ethanol-fed animals, the endogenous synthesis of phosphatidylcholine (PC) and its contribution to ER transport vesicles were reduced by 50%, as compared to that in pair-fed controls. Reduction of PC synthesis and of its presence in ER-transport vesicles was also observed in pair-fed controls when the native cytosol was replaced with that from ethanol-fed animals. In contrast, preincubation of ER membranes from ethanol-fed animals with cytosol from controls led to the stimulation of PC synthesis in ER and its contribution to ER-transport vesicles. Analysis of water soluble metabolites of [methyl-14C]choline phosphate revealed the accumulation of CDP-choline precursor in samples derived from ethanol-fed rats. Concomitantly, the endogenous synthesis of phosphatidylinositol (PI) in the ER of ethanol-fed animals was stimulated up to 400-500%, but declined when the cytosol from ethanol-fed rats was replaced with that from the controls. The restoration of PC synthesis, the normalization of PI synthase activity, and, similar to control, the contribution of PC to ER-transport vesicles in ethanol-fed animals was achieved when ER membranes were preincubated with diglycerides or the cytosol was treated with ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Conversely, addition of CaCl2-EGTA buffer containing 3 microM free Ca2+ to control samples, led to a reduction in PC synthesis. The studies on the effect of free Ca2+ on PI synthase and phosphatidic acid (PA) phosphatase activity established that in the presence of 1-3 microM free Ca2+, PI synthase activity remained constant, whereas that of PA phosphatase was reduced by 40% at 1 microM Ca2+, and no activity was detected when free Ca2+ was adjusted to 3 microM. The results suggest that modified membrane biogenesis in the liver of ethanol-fed rats is connected to the elevated free Ca2+ in the cytosol, which appears to regulate phosphatase activity. Accumulation of CDP-choline, decreased activity of PA phosphatase, and increased contribution of PI lipids to ER-transport vesicle membrane suggest that in ethanol-fed animals diglycerides are depleted and PA is utilized in a CDP-diacylglycerol pathway, thus leading to the generation of a different group of phospholipids and consequently modified ER-transport vesicle membrane.
Archives of Biochemistry and Biophysics 05/1994; 310(1):247-55. · 2.93 Impact Factor
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ABSTRACT: 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.
International Journal of Biochemistry 10/1993; 25(9):1281-9.
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ABSTRACT: 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.
International Journal of Biochemistry 07/1993; 25(6):891-901.
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ABSTRACT: The effect of gastric mucin on the activity of calcium channel isolated from gastric epithelial cell membrane was investigated. The uptake of 45Ca2+ into the vesicle-reconstituted channels, while only moderately (14%) affected by the intact 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. The channel complex in the presence of epidermal growth factor (EGF) and ATP responded by an increase in protein tyrosine phosphorylation, and the vesicles containing the phosphorylated channels showed a 50% increase in 45Ca2+ uptake. 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, and the loss of the inhibitory capacity occurred following their removal. The results indicate that the acidic gastric mucins, by modulating the EGF-controlled calcium channel phosphorylation, play a major role in gastric mucosal calcium homeostasis.
Biochemistry and molecular biology international 02/1993; 29(1):5-15.
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ABSTRACT: To investigate the effect of ethanol on the intracellular transport in gastric epithelial cells, the in vitro system, generating transport vesicles which transfer mucus glycoprotein apopeptide (apomucin) from rough endoplasmic reticulum (RER) to Golgi, was assembled. The vesicles, generated from gastric mucous cell RER microsomes and labeled with [3H]palmitic acid, were isolated from the maternal RER and characterized. The electron microscopy revealed that these RER products consisted of 80 to 100 nm smooth membrane vesicles, while the immunochemical analyses showed that they contain apomucin but were devoid of the characteristic integral proteins of the RER membrane. Incubation of apomucin transporting vesicles with Golgi in the presence of UDP-[3H]galactose resulted in the formation of glycosylated mucin and fusion of the vesicles with Golgi. Formation of ER transport vesicles was dependent on the supply of lipid precursors, and the activity of phosphoglyceride and sphingolipid synthesizing enzymes. In the presence of 60 and 120 mM ethanol, the vesicles were formed, but their lipid composition was modified. The results suggest that ethanol-induced membrane alterations are initiated at the early stages of the membrane biogenesis and are first reflected in the lipid composition of the intracellular transport vesicles.
Alcoholism Clinical and Experimental Research 01/1993; 16(6):1152-61. · 3.34 Impact Factor
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ABSTRACT: The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.
Archives of Biochemistry and Biophysics 11/1992; 298(1):167-75. · 2.93 Impact Factor
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ABSTRACT: Colonization of gastric mucosa by Helicobacter pylori, a bacterium implicated in the etiology of gastric disease, involves the cell surface sulfated glycosphingolipid receptors for the attachment. Evidence has also been obtained recently that sulfated mucus glycoproteins have the ability to interfere with this process. Here, we show that H. pylori displays glycosulfatase activity, and report the specificity of this enzyme toward gastric mucosal sulfated glycoproteins and glycolipids. With 35S-labeled human gastric sulfated mucin as substrate, the enzyme activity was identified in the extracellular material elaborated by the bacterium. The glycosulfatase exhibited maximum activity at pH 5.7 in the presence of Triton X-100 and CaCl2, and gave on SDS-PAGE a protein band of 30 kDa. Specificity studies revealed that the enzyme effectively caused desulfation of N-acetylglucosamine-6-sulfate and galactose-6-sulfate present in carbohydrate chains of gastric mucins, as well as that of glucose-6-sulfate, a constituent of mucus glyceroglucolipids. However, the H. pylori glycosulfatase was ineffective toward galactosyl- and lactosylceramide sulfates which serve as receptors for this bacterium attachment and contain the sulfate ester group at C-3 of galactose. The glycosulfatase activity toward human sulfated gastric mucin was inhibited by sucralfate. The inhibitory effect was proportional to the concentration of sucralfate up to 120 micrograms/ml, at which a 78% decrease in mucin desulfation occurred. The results demonstrate that H. pylori, through its glycosulfatase activity, affects the sulfated mucin and glyceroglucolipid content of the protective mucus layer, and that antiucler drug sucralfate is able to counteract the detrimental action of this enzyme.
The American Journal of Gastroenterology 10/1992; 87(9):1132-7. · 7.28 Impact Factor
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ABSTRACT: The effect of intragastric administration of GM1-ganglioside on the expression of gastric mucosal epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors was investigated. Gastric mucosal cell membranes were isolated from the stomach of groups of rats, one receiving twice daily for 3 consecutive days a dose of 0.25 mg/100g GM1-ganglioside, and the other only vehicle. Binding assays revealed the presence of both types of receptors, activation of which led to the elevation of tyrosine kinase activity as evidenced by a marked increase in membrane protein tyrosine phosphorylation patterns. The specific receptor binding in the control group was 2.47 fmol/mg protein for EGF and 1.46 fmol/mg protein for PDGF, whereas the respective binding values in the GM1-ganglioside treated group increased by 45% and 38%. The results suggest that GM1-ganglioside is capable of enhancement of gastric mucosal EGF and PDGF receptor activities.
Biochemistry international 07/1992; 27(1):97-104.
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ABSTRACT: 1.1. 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.2.2. 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.3.3. 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.4.4. 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.5.5. 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.
International Journal of Biochemistry.
