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)

Epithelial crypts from the bovine colon were obtained by using a combined mechanical and enzymatic isolation method, followed by differential D-sorbitol gradient centrifugation. By using this isolation technique, a pure fraction of epithelial crypts with minimal mesenchymal contamination was obtained. The crypts were seeded in collagen-coated plastic flasks. The attached epithelial cells proliferated and formed a confluent monolayer after 6 days in culture. Under low-serum culture conditions (1% fetal calf serum), the cells had a population doubling time of 21-22 hours. During the culture period, the colonocytes were characterised morphologically and enzymatically. The morphology of the cultured cells was confirmed by scanning electron microscopy and transmission electron microscopy. The presence of microvilli, tight junctions and desmosomes demonstrated the ability of the cultured cells to restore an epithelial-like cell monolayer. The epithelial origin of the cells was demonstrated by labelling the cells with antibodies against epithelial-specific cytokeratins 7 and 13. The functional integrity of the cells was evaluated by measuring various marker enzymes (gamma-glutamyltranspeptidase, acid phosphatase, alkaline phosphatase, NADH-dehydrogenase) and membrane-associated Na+-K+-ATPase activity. Membrane integrity was determined by measuring the leakage of lactate dehydrogenase into the culture medium. This new culture system for bovine colon epithelial cells could be used as an in vitro model of the colon epithelium in physiological and toxicological studies.
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PMID:Growth and characterisation of primary bovine colon epithelial cells in vitro. 1575 94

Nutrient and oxygen availability are key metabolic parameters for biopharmaceutical manufacturing. In order to enable mammalian cells to manifest their intracellular nutrient and oxygen levels we engineered a genetic sensor circuitry which converts signals impinging on the cellular redox balance into a robust reporter gene expression readout. Capitalizing on the Streptomyces coelicolor redox control system, consisting of REX modulating ROP-containing promoters in an NADH-dependent manner, we designed a mammalian dual sensor transcription control system by fusing REX to the generic VP16 transactivation domain of Herpes simplex, which reconstitutes an artificial transactivator (REDOX) able to bind and activate chimeric promoters assembled by placing a ROP operator module 5' of a minimal eukaryotic promoter (P(ROP)). When nutrient levels were low and resulted in depleted NADH pools REDOX-dependent P(ROP)-driven expression of secreted (human-secreted alkaline phosphatase; SEAP) or intracellular (Renilla reniformis luciferase; rLUC) reporter genes was high as a consequence of increased REDOX-P(ROP) affinity. Conversely, at hypoxic conditions leading to high intracellular NADH levels, strongly reduced REDOX-P(ROP) interaction mediated low-level transgene expression in Chinese hamster ovary (CHO-K1) cells. Other molecules (for example, 2,4-dinitrophenol, cyanide or hydrogen peroxide) which are known to imbalance the intracellular NADH/NAD+ poise could also be detected using the REDOX-P(ROP) sensor circuitry. REDOX's sensor capacity (nutrient and oxygen levels) operated seamlessly in transgenic CHO-K1 cell derivatives adapted for growth in serum-free suspension cultures and enabled precise monitoring of the population's metabolic state. As the first genetic metabolic sensor designed for mammalian cells, REDOX may foster advances in process development and biopharmaceutical manufacturing.
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PMID:A genetic redox sensor for mammalian cells. 1647 37

Human cerebral malaria is caused by a protozoan parasitic with no cure till date. The isolation of brain capillaries i.e. microvessels has permitted the in vitro study related to cerebral function. Microvessels were isolated from normal and P. yoelii infected mice brain cortex and subjected to biochemical characterization by the following enzyme markers viz alkaline phosphatase, gamma-glutamyI transpeptidase and monoamine oxidase and electron microscopically. Limited studies have been carried out in relation to drug metabolizing enzymes in cerebral microvessels of rodents. The present studies have been carried out in relation to status of drug metabolizing enzymes during P. yoelii infection in cerebral microvessels of mice. The data obtained depicted a clear cut impairment of cytochrome P450 (a terminal monooxygenase) and related indices viz b5, benzopyrene hydroxylase, aminopyrene-n-demethylase, aniline hydroxylase except NADH cytochrome e reductase which increased during P. yoelii infection in mice as compared to normal. Further the oral drug administration (arteether) treatment brought back the altered MFO system normal a week alter cessation of drug treatment.
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PMID:Studies on drug metabolizing enzymes during arteether treatment of Plasmodium yoelii nigeriensis infected mice cerebral microvessels. 1663

The membrane topologies of the six subunits of Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na+-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed.
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PMID:Membrane topology mapping of the Na+-pumping NADH: quinone oxidoreductase from Vibrio cholerae by PhoA-green fluorescent protein fusion analysis. 1704 Oct 63

The two articles in this series are dedicated to bioaffinity electrodes with in situ detection of the product of the enzyme label after recognition by its conjugate immobilized on the electrode. Part 1 was devoted to direct electrochemical detection, whereas the present contribution deals with homogeneous chemical and enzymatic amplification of the primary electrochemical signal. The theoretical relationships that are established for these modes of amplification are applied to the avidin-biotin recognition in a system that involves alkaline phosphatase as enzyme label and 4-amino-2,6-dichloro-phenyl phosphate as substrate, generating 2,6-dichloro-4-aminophenol as electrochemically active product. Chemical amplification then results from the addition of NADH, which reduces the 2,6-dichloro-quinonimine resulting from the electrochemical oxidation of 2,6-dichloro-4-aminophenol. An increased amplification is obtained when the reduction of 2,6-dichloro-quinonimine involves diaphorase in solution with NADH as substrate. The excellent agreement between theoretical predictions and experimental data required a detailed theoretical analysis and the independent determination of the key kinetic parameters of the system. The theoretical analysis was extended to monolayer and multilayered films of auxiliary enzyme as well as to electrochemical amplification by means of closely spaced dual electrodes so as to offer a rational comparative panorama of the amplification capabilities of the various possible strategies. Confinement of the profile of the product, and/or its oxidized form, in the vicinity the electrode surface appears as a key parameter of amplification.
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PMID:Theory and practice of enzyme bioaffinity electrodes. Chemical, enzymatic, and electrochemical amplification of in situ product detection. 1849 54

Redox cycling of enzymatically amplified electroactive species has been widely employed for high signal amplification in electrochemical biosensors. However, gold (Au) electrodes are not generally suitable for redox cycling using a reducing (or oxidizing) agent because of the high background current caused by the redox reaction of the agent at highly electrocatalytic Au electrodes. Here we report a new redox cycling scheme, using nicotinamide adenine dinucleotide (NADH), which can be applied to Au electrodes. Importantly, p-aminophenol (AP) redox cycling by NADH is achieved in the absence of diaphorase enzyme. The Au electrodes are modified with a mixed self-assembled monolayer of mercaptododecanoic acid and mercaptoundecanol, and a partially ferrocenyl-tethered dendrimer layer. The self-assembled monolayer of long thiol molecules significantly decreases the background current of the modified Au electrodes, and the ferrocene modification facilitates easy oxidation of AP. The low amount of ferrocene on the Au electrodes minimizes ferrocene-mediated oxidation of NADH. In sandwich-type electrochemical immunosensors for mouse immunoglobulin G (IgG), an alkaline phosphatase label converts p-aminophenylphosphate (APP) into electroactive AP. The amplified AP is oxidized to p-quinoneimine (QI) by electrochemically generated ferrocenium ion. NADH reduces QI back to AP, which can be re-oxidized. This redox cycling enables a low detection limit for mouse IgG (1 pg mL(-1)) to be obtained.
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PMID:An electrochemical immunosensor using p-aminophenol redox cycling by NADH on a self-assembled monolayer and ferrocene-modified Au electrodes. 1893 39

The use of the alkaline phosphatase (AP) as an enzyme label and the amplification of its analytical response with a diaphorase (DI) secondary enzyme were investigated in an electrochemical hybridization assay involving arrays of carbon screen-printed DNA biosensors for the sensitive quantification of an amplified 406-base pair human cytomegalovirus DNA sequence (HCMV DNA). For this purpose, PCR-amplified biotinylated HCMV DNA targets were simultaneously bound to a monolayer of neutravidin irreversibly adsorbed on the surface of the electrodes and hybridized to complementary digoxigenin-labeled detection probes. The amount of hybrids immobilized on the electrode surface was labeled with an anti-digoxigenin AP conjugate and quantified electrochemically by measuring the activity of the AP label through the hydrolysis of the electroinactive p-aminophenylphosphate (PAPP) substrate into the p-aminophenol (PAP) product. The intensity of the cyclic voltammetric anodic peak current resulting from the oxidation of PAP into p-quinoneimine (PQI) was related to the number of viral amplified DNA targets present in the sample, and a detection limit of 10 pM was thus achieved. The electrochemical response of the AP label product was further enhanced by adding the diaphorase enzymatic amplifier in the solution. In the presence of the auxiliary enzyme DI, the PQI was reduced back to PAP and the resulting oxidized form of DI was finally regenerated in its reduced native state by its natural substrate, NADH. Such a bienzymatic amplification scheme enabled a 100-fold lowering of the HCMV DNA detection limit obtained with the monoenzymatic system.
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PMID:Bienzymatic-based electrochemical DNA biosensors: a way to lower the detection limit of hybridization assays. 1917 61

The aim of this study was first to search for isofolliculia in right and left ovaries during postnatal development of Ouled Djellel ewe lambs, a non-seasonal breed of sheep. In addition, the contribution of different sizes of large antral follicles to this phenomenon was studied, and finally the variations in both plasma FSH and LH levels during this period of life were determined. Plasma was collected from groups of four ewe lambs at 0 (<24h), 1 week, and every 2 weeks from 4 to 14 weeks of age. Thereafter, each group was slaughtered, right and left ovaries recovered, weighed and their length and width measured. One ovary was fixed in Bouin-Holland's solution and prepared for histological study. The other one was immediately frozen and cut in a cryostat and prepared for histochemical study. This latter method was used to detect the activity of alkaline phosphatase, lactate dehydrogenase, NADH(2)-tetrasolium reductase, and glucose-6-phosphate dehydrogenase enzymes. Number and size of antral follicles and their contribution to isofolliculia were determined from ovarian sections of both studies. Isofolliculia was seen in right and left ovaries of Ouled Djellel ewe lambs at 4, 6, 8, and 10 weeks of age. This phenomenon was characterized by the presence of large antral follicles almost equal in size and total enzymatic inactivity in the interstitium. Weight and dimensions of right and left ovaries increased rapidly from birth to 4 weeks of age, and then rose gradually to week 8 and then rising again to week 10, followed by a decline at 12 and 14 weeks of age. All large antral follicles contributed to isofolliculia in right and left ovaries but, the percentage of antral follicles <2mm at 4, 6, 8, and 10 weeks of age were significantly greater than the percentage of follicles > or =2 and <3mm and the contribution of follicles > or =3mm was the lowest. FSH levels increased slowly from birth to 6 weeks of age then, increased rapidly to week 10, followed by a decline at weeks 12 and 14. LH was low at birth and the level increased slowly to 8 weeks of age, followed by a further rapid increase at 10 weeks of age. All parameters studied did not show any significant differences between the right and left ovary. It was concluded that isofolliculia occurred between 4 and 10 weeks of age in left and right ovaries of Ouled Djellel ewe lambs. This phenomenon was characterized by the increase of both ovarian weights and dimensions, and of plasma FSH and LH levels. All large antral follicles > or =1mm in diameter contributed to isofolliculia but the contribution of antral follicles <2mm was greater than the contribution of antral follicles > or =2.
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PMID:Circulating gonadotropins levels and contribution of different large antral follicles to isofolliculia in sheep. 1957 3

A signal amplificatory electrochemical immunoassay with biotin-streptavidin conjunction and multienzymatic-based substrate recycling was developed in this work. Biotinylated secondary antibody (bio-IgG) was preliminarily assembled onto the immunosensor interface based on the sandwich format. Streptavidin was then loaded based on biotin-streptavidin conjunction. Owing to four identical binding sites of streptavidin to biotin, amounts of biotinylated alkaline phosphatase (bio-AP) were attached, and this improved the catalytic performance of the proposed immunosensor. Under the enzyme catalysis of AP, the electroinactive p-aminophenylphosphate (PAPP) substrate was rapidly hydrolyzed into the electroactive p-aminophenol (PAP) product, which next oxidized at the electrode surface into p-quinoneimine (PQI). In the presence of diaphorase (DI), PQI was reduced back to PAP, leading to a reversible cycle of PAP. Then the oxidized state of DI was regenerated into its reduced native state by its natural substrate, nicotinamide adenine dinucleotide (NADH). With the several amplification factors mentioned above, a wider linear ranged from 10(-14) to 10(-5) gml(-1) was acquired with a relatively low detection limit of 3.5 x 10(-5) gml(-1) for human IgG. In addition, the nonspecific adsorption of proposed immunosensor was also investigated here.
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PMID:An electrochemical enzyme bioaffinity electrode based on biotin-streptavidin conjunction and bienzyme substrate recycling for amplification. 2050 24

Monolayer cultures of porcine urinary bladder epithelial cells were obtained by isolating cells from bladders of freshly slaughtered pigs. The cultures were investigated morphologically and characterized according to their growth characteristics and enzymatic functions. Ultrastructural investigations demonstrated that the cells regain their in vivo polarization with apically situated membrane vesicles, tight junctions and desmosomes between neighbouring cells when they have built up a confluent monolayer. Membrane integrity and high cell viability was indicated by low levels of lactate dehydrogenase activity released into the medium. The chromosome set of the cells was stable during the first 5 wk of culture. Under standard culture conditions activities of the enzymes alkaline phosphatase and acid phosphatase were stable over a period of 4 wk. gamma-glutamyl transpeptidase activity, indicating a dedifferentiation process, did not increase. Activity of NADH-dehydrogenase dropped, indicating a decrease in ER as a consequence of adaptation to the culture conditions. By measuring concentration dependent increase of sister chromatid exchanges (SCEs) induced by N-methyl-N'-nitro-N-nitrosoguanidine and 2-acetylaminofluorine, the suitability of this model for genotoxicity tests was estimated. These results indicate that the model can be used as a target for bladder cancer inducing agents to investigate the effects of such agents on the level of the target organ in further genotoxicity studies.
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PMID:A cell culture model of isolated porcine urinary bladder epithelial cells for genotoxicity studies. 2069 6


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