Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human Caco-2 cells (passage 80 to 100) were seeded onto collagen-coated Millipore filter assemblies and these were maintained in culture either (a) floated on the surface of the medium or (b) submerged within the body of the medium. Structural and functional assessments were made over a 30-day period. After seeding, all cells assumed a flattened, squamous configuration and rapidly became confluent. Cells submerged within the medium formed polarised monolayers with well developed junctional complexes, abundant apical microvilli and increasing levels of alkaline phosphatase activity. Cells grown floated on the surface of the medium formed complex multilayers in which polarisation was confined to the surface layer. Junctional complexes and apical microvilli were similar to those seen in submerged monolayers but alkaline phosphatase activities were higher. Transepithelial electrical resistance increased rapidly from day 1, as the layers became confluent. Electrical resistance was higher and short-circuit current and potential differences were lower across monolayers than across multilayers. After 10 days in culture, the addition of D-glucose to the apical bathing solution, of all cell layers, caused a rapid rise in short-circuit current and potential difference. These changes were sodium-dependent and phlorizin-sensitive. Galactose and 3-O-methylglucose induced similar changes and the affinity constants for these hexoses ranked in the order reported for rat jejunum (Km glucose 2.44 +/- 0.52 mM; Km galactose 8.05 +/- 1.33 mM; Km 3-O-methylglucose 22.0 +/- 5.2 mM). Culture conditions had a marked effect on hexose maximum transport rates (glucose Vmax: submerged 2.94 +/- 0.20 microA/cm2; floated 9.94 +/- 0.82 microA/cm2, P less than 0.05) but affinity constants were unchanged. Apical to basolateral mannitol fluxes, used as an index of paracellular permeability, decreased from day 1 to day 5 and then remained steady. Fluxes across monolayers and multilayers were not significantly different. We conclude that sodium-dependent hexose transport occurs in cultured Caco-2 cell layers grown on permeable supports. Culture conditions, however, have a marked effect on both cell layer structure and function, and should be an important factor when considering Caco-2 cells as an in vitro model of enterocyte function.
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PMID:Active hexose transport across cultured human Caco-2 cells: characterisation and influence of culture conditions. 190 49

The present work investigates the ability of galactose to affect enterocyte differentiation during normal development in vivo. Energy intake has also been varied to take account of the fact that galactose is poorly metabolized in mice. Brush-border lactase, alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N, alkaline phosphatase and microvillus length were measured as markers of enterocyte differentiation in mice fed diets containing galactose (G diet), corn oil (E diet) or galactose + corn oil (G + E diet). Maintaining mice on a G instead of E diet reduced brush-border lactase activity and enterocyte migration rates; alpha-glucosidase, dipeptidylpeptidase-IV, aminopeptidase N and microvillus length expression increased and alkaline phosphatase activity remained unchanged. Feeding the G + E diet restored enterocyte migration rates, lactase, aminopeptidase N and dipeptidylpeptidase-IV activities to values found in mice fed the E diet. Galactose stimulation of alpha-glucosidase and microvillus length expression was, however, fully maintained in mice fed the G + E diet. Present results show that enterocyte differentiation is affected independently by varying dietary galactose and energy levels; that galactose effects always increase and energy effects usually decrease expression of enterocyte components and that energy stimulation of lactase activity is exceptional.
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PMID:Galactose effects on enterocyte differentiation in the mouse jejunum. 190 92

The effects of the histidine modifier, diethyl pyrocarbonate (DEPC), on brush-border membrane transport systems were studied in rat kidney. DEPC caused a strong inhibition of sodium-dependent phosphate and D-glucose uptake. Phosphate uptake remained linear up to 10 s in control and DEPC-treated membrane vesicles. The D-glucose carrier was more sensitive than the phosphate carrier with half-times of inhibition being 4 and 7 min, respectively. Sodium-independent phosphate and D-glucose uptake remained unaffected by DEPC. Intravesicular volume and two enzyme activities endogenous to the luminal membrane (alkaline phosphatase and aminopeptidase M) remained unaffected by DEPC. Increasing the preincubation pH from 5 to 9 increased phosphate transport inhibition caused by DEPC from 73 to 88% in the presence of DEPC. Hydroxylamine was able to completely reverse phosphate uptake inhibition by DEPC (100%), but only partially reversed the D-glucose uptake inhibition (16%). Sodium or substrate (D-glucose or phosphate) in the preincubation media were unable to protect their respective carriers from DEPC. Sodium-dependent transport of L-glutamine, L-phenylalanine, L-leucine, L-alanine, L-glycine, beta-alanine and L-proline were inhibited at different levels ranging from 70 to 90%. Three transport processes were found insensitive to DEPC modification: L-glutamate, L-lysine and D-fructose. None of the amino acid transporters was protected against DEPC by sodium and/or their respective substrates. Sodium influx was inhibited by DEPC (47%) in the absence of any substrate. Our results show a differential sensitivity of sodium-dependent transporters to DEPC and suggest an important role for histidine residues in the molecular mechanisms of these transporters. More experiments are in progress to further characterize the residue(s) involved in these transport inhibitions by DEPC.
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PMID:Kidney brush-border membrane transporters: differential sensitivity to diethyl pyrocarbonate. 191 27

Oral administration of embelin (75 mg/kg per day, daily for 15 and 30 days) to male rats caused significant elevation in the uptake of D-glucose, L-alanine, L-leucine and calcium in small intestinal segments. Embelin also produced significant increases in intestinal brush border membrane-associated enzymes (sucrase, lactase, maltase, alkaline phosphatase and leucine aminopeptidase) in both intestinal homogenates and partially purified brush border membrane preparations. Significant increases were also noted for microsomal glucose-6-phosphatase and cytosolic lactate dehydrogenase. Increase in brush border membrane-associated total lipids, phospholipids, cholesterol, triacylglycerol, unesterified fatty acids and ganglioside sialic acid were seen but not in the cholesterol/phospholipid molar ratio. All these changes returned to control or near control levels following withdrawal of the drug.
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PMID:Effects of embelin, a male antifertility agent, on absorptive and digestive functions of rat intestine. 192 15

The renal brush border membrane vesicles (BBMV) were used to elucidate the early biochemical functional status during the course of experimental M. leprae infection in mice. The activities of the characteristic brush-border enzymes viz: alkaline phosphatase, leucine amino peptidase and gamma-glutamyl transpeptidase were found to be significantly decreased (p less than 0.001) at 3 and 6 months after infection. The transport of nutrients viz: D-glucose, L-alanine, L-lysine and L-aspartate across BBMV showed similar pattern. The activity of brush border enzymes and transport of nutrients across the membrane returned to normal at 9 months post-infection suggesting regeneration of the brush border membrane.
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PMID:Renal brushborder membrane vesicle. Study of marker enzymes and uptake of nutrients in Mycobacterium leprae infected mice. 198 18

Although embryonic chick small intestinal segments provide a very limited amount of tissue for preparation of enterocyte brush border membrane vesicles (BBMV), we were able to develop a procedure for isolation of BBMV from cultured 20-d-old embryonic chick jejunum in high yield by modifying a divalent cation precipitation method. Total yield of the brush border marker enzyme alkaline phosphatase in the vesicle fraction as compared to the crude homogenate was approximately 40%, and the specific activity of the enzyme was increased 25-fold on the average. The brush border membrane vesicle fraction was only contaminated with other cellular organelles (basolateral membranes, mitochondria, lysosomes or endoplasmic reticulum) to a minor extent. Functional integrity of the brush border vesicles was indicated by Na+ gradient-driven electrogenic D-glucose transport leading to concentrative transfer (overshoot) of the sugar into an osmotically active intravesicular space. When jejuna were cultured for 48 h in the presence of 10(-6) mol/L insulin, the initial rate of Na(+)-dependent D-glucose uptake by brush border membrane vesicles as well as Na(+)-dependent [3H]phlorizin binding to brush border membranes was approximately twice as high as in vesicles from untreated controls. This strongly suggests that insulin could enhance intestinal absorption of D-glucose by increasing the intrinsic activity of the Na(+)-dependent D-glucose transport system at the luminal membrane of enterocytes.
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PMID:A high yield preparation of brush border membrane vesicles from organ-cultured embryonic chick jejunum: demonstration of insulin sensitivity of Na(+)-dependent D-glucose transport. 199 47

In the present experiments, selective quenching by trinitrophenyl groups as well as steady-state fluorescence polarization and differential polarized phase fluorescence techniques, using three different lipid soluble fluorophores, were used to directly examine the fluidity of the exofacial and cytofacial leaflets of rat small intestinal brush-border membranes. These studies revealed that the fluidity of the exofacial hemileaflet was greater than the cytofacial hemileaflet. Differences in the distribution of phosphatidylcholine and phosphatidylethanolamine, as assessed by phospholipase A2 treatment and trinitrophenylation of aminophospholipids, were, at least partially, responsible for the asymmetrical fluidity of the hemileaflets. Moreover, in vitro addition of benzyl alcohol (final concn 25 mM) preferentially fluidized the exofacial leaflet and concomitantly decreased leucine aminopeptidase activity but did not affect the activities of maltase, sucrase, alkaline phosphatase, or gamma-glutamyltranspeptidase. In vivo addition of the membrane-mobility agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanate] (A2C) (final concn 7.5 microM) preferentially fluidized the cytofacial leaflet and increased Na(+)-gradient-dependent D-glucose transport but not Na(+)-gradient-dependent L-leucine transport.
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PMID:Characterization and modulation of rat small intestinal brush-border membrane transbilayer fluidity. 201 33

Intestinal extraction of circulating glutamine across the basolateral membrane is diminished in the tumor-bearing rat (TBR). This study was designed to investigate the effects of progressive malignant growth on brush border glutamine transport in order to gain further insight into the adaptive/regulatory changes in intestinal glutamine metabolism that occur in the tumor-bearing rat. Fischer 344 rats (225 +/- 5 g) were implanted with fibrosarcoma cells and were studied at various time points after implantation when the tumors comprised 7%, 20%, and 29% of total body weight. Control and tumor-bearing rats were pair-fed throughout the study. Jejunal brush border membrane vesicles (BBMVs) were prepared by magnesium aggregation/differential centrifugation and transport of radioactively labeled L-glutamine, L-leucine, L-alanine, and D-glucose by BBMVs was measured using a Millipore filtration technique. BBMVs were enriched 15-fold in alkaline phosphatase, indicating brush border vesicle purity. Uptake of all substrates occurred into an osmotically active space, exhibited overshoots, and had similar 1-hr equilibrium values. The rate of glutamine uptake by BBMVs from all tumor-bearing rats was significantly greater than controls, regardless of tumor size. The increase in transport activity was not due to a change in carrier affinity but rather to an increase in maximal transport velocity. In rats with small tumors (7% of body weight), the Vmax was 431 +/- 40 pmole/mg protein/10 sec compared to 259 +/- 30 in control animals (P less than 0.01). In marked contrast, the mean transport of alanine was diminished in BBMVs from TBR (31 +/- 3 pmole/mg protein/10 sec in TBR vs 23 +/- 2 in controls, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Selective stimulation of brush border glutamine transport in the tumor-bearing rat. 202 Jan 90

There are at least three stages in the targeting of soluble lysosomal enzymes: transfer of N-acetylglucosaminyl 1-phosphate to high-mannose oligosaccharide side chains, removal of N-acetylglucosamine and recognition of the "uncovered" mannose 6-phosphate residues. Defects in the transfer reaction cause mucolipidoses II and III. Those in the subsequent stages of the targeting may result in similar clinical disorders. To differentiate between possible defects of the targeting in cultured cells we have developed a procedure for a combined detection of the phosphorylation, uncovering of the transferred phosphate residues and the targeting of lysosomal enzymes. For this purpose cultured cells are metabolically labelled with [32P]phosphate and a lysosomal enzyme, such as cathepsin D, is isolated from the labelled cells and the medium by immunoprecipitation. The immunoprecipitates are dissolved with sodium dodecylsulphate and incubated in the presence and absence of calf intestine alkaline phosphatase. We show that the treatment of the denatured protein results in hydrolysis of phosphomonoester groups and that the phosphodiester and the peptide bonds remain intact. The initial and the residual radioactivity associated with the lysosomal enzyme which represent the total phosphate and the phosphodiester groups, respectively, are determined by gel-electrophoresis, fluorography and densitometry. This procedure extends one of the previously established methods for the diagnosis of mucolipidoses II and III.
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PMID:Determination of the phosphorylation, uncovering of mannose 6-phosphate groups and targeting of lysosomal enzymes. 207 12

Administration of Embelin, an experimental antifertility agent, to male rats (20 mg/kg body wt/day, daily for 15 and 30 days), caused an elevation in the uptake of D-glucose, L-alanine, L-leucine, and calcium in the small intestinal segments. An increase was also noted in the intestinal brush border membrane (BBM)-associated enzymes, sucrase, lactase, maltase, alkaline phosphatase, and leucine aminopeptidase in both the intestinal homogenates and partially purified BBM preparations, particularly after 30-day administration of the drug. Embelin treatment also caused a significant increase in the microsomal glucose-6-phosphatase and the cytosolic enzyme, lactate dehydrogenase. In the Embelin-treated animals BBM-associated total lipids, phospholipids, cholesterol, triacylglycerol, unesterified fatty acids, ganglioside-sialic acids as well as the cholesterol/phospholipids molar ratio showed a considerable increase. All these changes in the Embelin-treated animals were restored back to the normal or near normal biochemical makeup when the drug therapy was withdrawn and the animals were allowed to recover for another 15 and 30 days, respectively.
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PMID:Changes in glucose/amino acid/calcium uptake and brush-border membrane-associated enzymes in rat small intestine after the administration of embelin (plant benzoquinone), an antifertility agent. 211 47


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