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)

Among kidney tubular epithelial cell types, proximal tubule cells are one of the major renal targets for xenobiotics. Several in vitro culture models have been proposed for use of proximal tubule cells for in vitro pharmacotoxicology studies. This paper reports a comparative study of the response to cephaloridine exposure of two established cell lines from pig (LLC-PK1) and rabbit (LLC-RK1) kidneys and primary cultures of rat and rabbit proximal tubule cells. These cultured cells were first compared for their levels of activity of alpha-methylglucopyranoside transport, alkaline phosphatase, succinate dehydrogenase, and NADPH cytochrome c reductase, their glutathione-dependent activity levels, and their adenylate cyclase response pattern to stimulation by PTH and AVP. The results presented show major phenotypic differences between these four cellular models. The differences observed in glutathione-dependent mechanism activities and regulation may in part be responsible for the variability of the responses of these four cellular models when exposed to cephaloridine.
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PMID:Comparative impact of cephaloridine on glutathione and related enzymes in LLC-PK1, LLC-RK1, and primary cultures of rat and rabbit proximal tubule cells. 903 21

The homogenate from Caco-2 cells of day 13 or 15 after subculturing had high omega-hydroxylation activity of docosahexaenoic acid (22:6(n-3)) or arachidonic acid (20:4(n-6)). Activity, maximal at pH 8.0, was inhibited in the presence of CO or metyrapone and in the absence of NADPH. Omega-hydroxylation activity of lauric acid in the homogenate was not detected. Apparent Michaelis constant (Km) values for 22:6(n-3) and 20:4(n-6) were found to be 4 and 7 microM. Omega-hydroxylation activity considerably increased with growth up to day 13 and then decreased until day 20 even though alkaline phosphatase (ALP) and leucine-aminopeptidase (LAP) activity increased with growth to day 20. Metyrapone in cultures caused omega-hydroxylation, ALP and LAP activity to decrease, while sodium butyrate dose-dependently increased that of omega-hydroxylation, ALP and an endogenous endonuclease and decreased lactate dehydrogenase (LDH) activity in culture medium. The omega-hydroxylation system thus appears quite likely to be associated with cytochrome P450, differentiation and/or apoptosis rather than cytotoxic cell death of Caco-2 cells. Substrate specificity, however, differed from that of human cytochrome P450 4A11.
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PMID:Docosahexaenoic/arachidonic acid omega-hydroxylation system and differentiation in the human colonic adenocarcinoma cell line, Caco-2. 946 91

Cisplatin [cis-dichlorodiammineplatinum (II)] is a widely used chemotherapeutic drug that is toxic to the kidney. Concurrent administration of cysteine together with vitamin E, Crocus sativus and Nigella sativa reduced the toxicity of cisplatin in rats. When administered i.p. for 5 alternate days with 3 mg/kg cisplatin, cysteine (20 mg/kg) together with vitamin E (2 mg/rat) an extract of Crocus sativus stigmas (50 mg/kg) and Nigella sativa seed (50 mg/kg) significantly reduced blood urea nitrogen (BUN) and serum creatinine levels as well as cisplatin-induced serum total lipids increases. In contrast, the protective agents given together with cisplatin led to an even greater decrease in blood glucose than that seen with cisplatin alone. The serum activities of alkaline phosphatase, lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase and alanine aminotransferase of cisplatin-treated rats were significantly decreased, whereas the activities of glutathione reductase and isocitrate dehydrogenase were significantly increased. Addition of cysteine and vitamin E, Crocus sativus and Nigella sativa in combination with cisplatin partially prevented many changes in the activities of serum enzymes. In cisplatin-treated rats, the liver activities of isocitrate dehydrogenase and aspartate aminotransferase were significantly increased, whereas much greater changes were found in the kidneys, with increased activity of glucose-6-phosphate dehydrogenase and decreased activities of alkaline phosphatase, isocitrate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase and gamma-glutamyl transferase, as well as a decreased phosphorylation to oxidation ratio in the mitochondria, indicating reduced adenosine triphosphate production. Also, administration of cysteine and vitamin E, Crocus sativus and Nigella sativa together with cisplatin partially reversed many of the kidney enzymes changes induced by cisplatin. Cysteine together with vitamin E, Crocus sativus and Nigella sativa tended to protect from cisplatin-induced falls in leucocyte counts, haemoglobin levels and mean osmotic fragility of erythrocytes and also prevented the increase in haematocrit. The results of this study indicate a basis for the toxic effects of cisplatin, and suggest a possible way of counteracting the toxicity by introducing protective agents such sulphydryl compounds, other antioxidants and extracts of natural products. It also appears that cells adapt to the effects of cisplatin through the induction of systems that produce NADPH, which in turn compensates the decrease of free sulphydryl groups. We conclude that cysteine and vitamin E, Crocus sativus and Nigella Sativa may be a promising compound for reducing cisplatin-toxic side effects including nephrotoxicity.
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PMID:Protective effect of cysteine and vitamin E, Crocus sativus and Nigella sativa extracts on cisplatin-induced toxicity in rats. 960 69

Ciprofibrates (racemate and both enantiomers, Raccip, R- and Scip) were administered orally in doses of 1 and 10 mg/kg once daily over 28 days to male inbred Fischer 344 rats, age 90-110 days at the beginning of the experiment. Body mass gain was observed in all groups. The 1 mg groups showed almost no difference to the control group. The 10 mg groups exhibited less body mass gain, most pronounced in the Scip group. Liver masses were increased in a dose dependent manner up to more than 200%, only the 10 mg Scip group was not significantly different from the 1 mg group which exhibited an increase in liver weight to about 175%. Also the kidney weights increased to 130%, whereas thymus and spleen weights were decreased in the high dose groups. Liver microsomal cytochromes P450 (P450) concentrations were not altered in the 1 mg groups and distinctly lowered in the 10 mg groups. Ethoxyresorufin and ethoxycoumarin O-deethylations were lowered in all experimental groups in a dose dependent manner, after administration of the high doses down to 30% of the control levels or less. Pentoxyresorufin O-depentylation, however, was increased in all 1 mg groups. In the high dose groups it was not altered. Ethylmorphine N-demethylation was decreased after administration of the high doses by about 50%, but only Scip decreased this reaction also after administration of the low dose. NADPH/Fe2+-stimulated microsomal luminol and lucigenin amplified chemiluminescence was increased, whereas hydrogen peroxide formation was depressed even by the low doses to 50% of the normal values, to about 25% by the high doses. Microsomal lipid peroxidation, however, was only slightly or not influenced. Glutathion concentrations (in the reduced and the oxidized form) were increased in a dose dependent manner by about 20 to 30%, the concentration of lipid peroxides was not significantly influenced. Thus, the effects of the enantiomers were not different and were similar to those of the racemate. In serum, cholesterol and triglycerides were only moderately lowered. Albumin concentrations were significantly enhanced in all groups, total proteins after 1 mg/kg Raccip only. Serum bilirubins were not altered, and among the indicator enzymes for liver damage only ALAT, alkaline phosphatase and the dehydrogenases were increased, in no case higher than twofold. Histologically distinct effects were seen after administration of both doses, more pronounced after 10 mg/kg, but with no differences between the enantiomers and Raccip: marked hypertrophy of the hepatocytes, reduced staining of the nuclei, strongly acidophilic granulated cytoplama, no basophilia of the cell bodies, loss of glycogen. These changes were most pronounced around the central veins. Hepatocyte apoptoses also were observed. By immunohistochemistry an increased staining was seen for all P450 isoforms tested (1A1, 2B1, 2E1, 3A2 and 4A1), predominantly perivenously and most pronounced after administration of the high doses without differences between Rcip, Scip or Raccip (preliminary results). By electron microscopy a moderate proliferation of peroxisomes after treatment with 1 mg/kg Cips with a ratio between mitochondria and peroxisomes of about 1:1 (controls: 10:1) was observed, and the peroxisomes were a more heterogeneous population. The relative portions of glycogen and both forms of the ER decreased. Treatment with 10 mg/kg Rcip, Scip or Raccip led to a strong increase in the number of peroxisomes, in some hepatocytes the ratio between mitochondria and peroxisomes was 1:3 with an increased heterogeneity among the peroxisomes evidenced by a broad range of electron densities. Most peroxisomes lacked a nucleoid. Thus, the biochemical effects differed only slightly and the morphological effects of the enantiomers were not different and were similar to those of the racemate.
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PMID:Ciprofibrate--racemate and enantiomers: effects of a four-week treatment on male inbred Fischer rats. A biochemical and morphological study. 978 2

Sexually mature female Wistar rats were given daily intragastric doses of ethinylestradiol (EE) and levonorgestrel (LE) used normally in women: (1) 0.03 mg EE and 0.05 mg LE; (2) 0.04 mg EE and 0.075 mg LE; (3) 0.03 mg EE and 0.125 mg LE. All groups were treated for 6 months in 5-day cycles (four-day treatment with a one-day break), i.e. for 36 sexual cycles. In rat kidneys, the activity of succinic dehydrogenase, NADPH-tetrazolium reductase, Mg(2+)-ATPase and alkaline phosphatase were decreased, while those of lactate dehydrogenase, acid phosphatase and glucose-6-phosphatase were enhanced. We have found a correlation between enzymatic changes and ultrastructural changes in epithelial renal cells. These changes may reflect: (1) inhibited oxidative processes associated with the mitochondrial and microsomal systems of electron transport; (2) a compensatory increase in anaerobic processes; (3) increased glyconeogenesis; (4) inhibited transport processes and increased cellular catabolism. The kidney cortex and medulla did not show any significant morphological changes after 6 months of treatment. The study has shown that EE/LE combinations produce histochemical and ultrastructural changes in the kidney, which are dependent on the doses of gestagens.
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PMID:Effects of ethinylestradiol and levonorgestrel on morphology, ultrastructure and histoenzymatic activity of rat kidney. 980 70

We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+-dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells.
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PMID:Morphological and biochemical investigation of nitric oxide synthase and related enzymes in the rat and pig urothelium. 1033 Apr 50

Data is reported on the reproducibility and purity of alveolar type II cell isolations from 4 species. Human and pig type II cells were isolated using a tissue slice method to remove blood and contaminating cells, whilst rat and hamster cells were isolated using the method of protease instillation. All cells were purified on Percoll gradients and by differential attachment. Cell type purity was assessed by phase contrast microscopy, electron microscopy (EM), percentage of cells alkaline phosphatase (AP) positive and percentage of cells staining strongly for NADPH dependent nitro blue tetrazolium reductase (NBT). These enzymes are considered as markers for type II and Clara cells respectively. The purity of all cell preparations was enhanced following 24 h culture on a biomatrix and whilst plating efficiency was similar for all species, the human tissue consistently yielded the highest purity of type II cells. All cells with lamellar bodies did not contain AP, and activity was variable between species. Further studies are needed to determine if NBT is equally nonspecific as a cell marker enzyme. In summary, sufficient type II cells of high purity can be isolated thus permitting interspecies comparative studies to investigate the effects of selective and non-specific pulmonary toxins, but more specific marker enzymes are required to identify Type II and Clara cells.
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PMID:A comparative study of the isolation of type II epithelial cells from rat, hamster, pig and human lung tissue. 1073 56

Native human cytochrome P4501A1 (CYP1A1) was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase. The chimeric P450 construct was placed under the transcriptional control of the native phoA promoter in a prokaryotic expression vector. Induction of the hemoprotein by heterologous expression in E. coli following growth in a phosphate-limited medium resulted in abundant synthesis of recombinant CYP1A1 as detected by reduced CO-difference spectra. Furthermore, the signal-appended CYP1A1 was translocated across the bacterial inner membrane by the sec-dependent pathway and processed to yield authentic, heme-incorporated P450 within the periplasmic space. In vitro and whole-cell metabolic activity studies showed that the periplasmically-located CYP1A1 competently catalysed NADPH-dependent benzo[a]pyrene 3-hydroxylation and 7-ethoxyresorufin O-deethylation. The means to localise cytochromes P450 in the periplasm offers an ability to produce high levels of protein, attributable to the less hostile nature of the compartment, and therein the enzymes for posttranslational assembly of heme with the translocated protein.
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PMID:Targeting of active human cytochrome P4501A1 (CYP1A1) to the periplasmic space of Escherichia coli. 1116 32

Human sterol 14alpha-demethylase (P45051; CYP51) catalyzes the oxidative removal of the C32 methyl group of dihydrolanosterol, an essential step in the cholesterol biosynthetic pathway. The reaction is dependent upon NADPH cytochrome P450 reductase (CPR) that donates the electrons for the catalytic cycle. Here we used a recombinant yeast CPR to investigate the abilities of four different forms of cytochrome b(5) to support sterol demethylation activity of CYP51. The cytochrome b(5) derivatives were genetically engineered forms of the native rat cytochrome b(5) core-tail: the soluble globular b(5) core (core), the core linked at its N-terminus with the secretory signal sequence of alkaline phosphatase (signal-core), and the signal sequence linked to the native b(5) (signal-core-tail). The rat core-tail enzyme greatly stimulated sterol demethylation, whereas the signal-core-tail was only marginally active. In contrast, the core and signal-core constructs were completely inactive in stimulating the demethylation reaction. Additionally, cytochrome b(5) enhanced sterol demethylation by more than threefold by accepting electrons from soluble yeast CPR and in its ability to reduce P450. We show that the nature of transient linkage between the hemoproteins and the redox partners is most likely brought about electrostatically, although productive interaction between cytochrome b(5) and CYP51 is governed by the membrane-insertable hydrophobic region in the cytochrome b(5) which in turn determines the correct spatial orientation of the core. This is the first report showing the stimulation of CYP51 by cytochrome b(5).
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PMID:Human sterol 14alpha-demethylase activity is enhanced by the membrane-bound state of cytochrome b(5). 1167 68

In wheat, non-phosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) was found to be encoded by one gene giving rise to a single protein. However, Western blots revealed two different subunits of about 58 and 60 kDa in endosperm and shoots. The latter was attributed to in vivo phosphorylation of shoot GAPN. No modification occurred in leaves, where the enzyme is composed by a single 58 kDa polypeptide. GAPN partially purified from shoots and endosperm was dephosphorylated in vitro with alkaline phosphatase. Phosphorylated GAPN exhibited similar affinity for substrates but a lower V(max) compared to the non-phosphorylated enzyme. Results suggest that reversible phosphorylation of GAPN could regulate NADPH production in the cytosol of heterotrophic plant cells.
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PMID:Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is post-translationally phosphorylated in heterotrophic cells of wheat (Triticum aestivum). 1238 87


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