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)

To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E. coli PQ37 density and centrifugation steps on the expression of beta-galactosidase (beta g) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs). Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37 degrees C before use. Initially, we used 0-5000 ng (= 0-20 nmole) benzo[a]pyrene (B[a]P) as a reference compound for the test procedure in the presence of standard S9 mix. Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol. We observed the highest beta g and ap activities and/or IFs using only 11-27 microliters 9000 x g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20-50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8-8.0. 60-300 micrograms NADP/assay (10-50% of standard) was sufficient for optimum activation of PAHs. In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12-18 x 10(6) cfu/assay). Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts. The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.
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PMID:Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons. 767 15

The chemiluminescent reaction of lucigenin with various reducing sugars and reducing compounds has been studied. It was found that dihydroxyacetone gave the most intense chemiluminescence (CL). We have developed highly sensitive chemiluminescent methods for alkaline phosphatase (ALP) based on the production of dihydroxyacetone using NADP+ or glycerol-3-phosphate as substrate. The detection limits for ALP using each substrate were 1.25 x 10(-19) mol/assay and 2.5 x 10(-19) mol/assay, and the coefficient of variation (n = 7) was 2.8% and 3.7%, respectively. We have also applied the method using NADP+ as substrate in enzyme immunoassays (EIA) for cholecystokinin (CCK) and human chorionic gonadotropin (hCG). CCK-8 (octapeptide sulphated form of a carboxy terminal fragment of CCK) concentrations released from alimentary canal of rat were assayed using the chemiluminescent EIA (CLEIA) and a fluorimetric EIA (ALP label). The correlation between CCK-8 values obtained by these methods was y = 1.04x + 18.21, r = 0.946, n = 28. hCG values in serum and in urine were measured. The correlation between hCG values in serum samples obtained using the CLEIA and a time-resolved fluoroimmunoassay (TR-FIA), and in urine samples obtained using the CLEIA and the fluorimetric EIA using ALP were satisfactory. The correlations were y = 1.00x - 0.04, r = 0.997 (n = 51) and y = 1.00x - 0.03, r = 0.999 (n = 10), respectively.
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PMID:A new highly sensitive chemiluminescent assay of alkaline phosphatase using lucigenin and its application to enzyme immunoassay. 776 11

IgG antibodies against the 2,3-di-o-acyltrehalose glycolipid of Mycobacterium tuberculosis were determined in a set of 49 sera from patients with pulmonary tuberculosis and 65 negative control subjects. We compared a conventional ELISA method using a beta-galactosidase anti-human IgG conjugate developed with ONPG, with an amplification ELISA system constituted of an anti-human IgG biotinylated conjugate, a streptavidin-alkaline phosphatase complex, and NADP as a substrate. The resulting NAD was measured by using a redox enzymatic recycling system of alcohol dehydrogenase, diaphorase and iodonitrotetrazolium as chromogen. With specificity set at 92.31% in both methods, we obtained a sensitivity of 42.86% in the conventional method and a sensitivity of 61.22% in the amplified method. We conclude that by using a more sensitive method we can detect cases that otherwise could be identified as false negatives.
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PMID:Development of an enzyme-linked immunosorbent assay (ELISA) combined with a streptavidin-biotin and enzyme amplification method to detect anti-2,3-di-o-acyltrehalose (DAT) antibodies in patients with tuberculosis. 782 39

Conventional total parenteral nutrition regimens (TPN-C) involve concentrations of dextrose/protein which necessitate administration of 2.5-4 liters/day to meet target nutritional needs. Although this is frequently acceptable, certain clinical settings mandate a volume-restricted (VR) approach. This study compares a VR TPN regimen (TPN-VR) involving the use of 25% dextrose and 9.5% amino acid with D17.5 AA 5.0 (TPN-C). The two groups were compared for adequacy of nutritional delivery, balance, and tolerance. Twenty patients received the TPN-VR (Group 1) and 20 patients received TPN-C (Group 2). The groups were comparable in age, sex, injury severity, and APACHE 2 scores. Harris-Benedict (BEE) x 2 and 2 g/protein/kg of ideal body weight were delivered by the second day of TPN. A 27% reduction in administered fluid was achieved in Group 1 (P < 0.001). Metabolic cart data in both groups demonstrated that delivered calories exceeded REE. The average RQ in Group 1 was 0.84 and in Group 2 was 0.90 (P > 0.1). There was no significant difference between the two groups in nitrogen balance, mean serum bilirubin levels, PT and PTT, serum albumin levels, and triglycerides (P > 0.20). SGPT and alkaline phosphatase levels were significantly higher in Group 2 (P < 0.001). Group 2 received an average of 22% more carbohydrate than Group 1 and 45% required insulin compared to 25% in Group 1 (P < 0.01). In summary, TPN-VR is comparable to TPN-C in terms of effectiveness of delivery, nutritional balance, and tolerance.
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PMID:Effectiveness and tolerance to highly concentrated vs conventional TPN formulas. 841 4

It is stated that the ileocecal valve delays the passage of ileal contents into the cecum and acts as a barrier against reflux and ascension of colonic bacterial flora into the small bowel: its resection may lead to bacterial colonization of the ileum and to abnormalities of intestinal motility, transit and absorption. In this study twenty individuals subjected in pediatric age (1 day to 11 years) to ileocecal resection have been evaluated from 2 to 19 years after surgery. Three patients underwent limited ileocecal resection, in four this was associated with a significant ileal resection, in five with extensive right colon resection and in eight with extensive ileal and right colon resection. Growth, stool habit, hematology and serum biochemistry were examined; all patients also underwent abdominal ultrasonography. In all body weight and height were within normal limits; seven had moderate diarrhea up to 18 months after surgery and two who required extensive intestinal resection (40 and 30 cm of small bowel left) had diarrhea until about 36 months after surgery: now all of them have daily fecal evacuation. Hematological, biochemical, urinary and fecal studies proved normal except in one treated with TPN who presented transaminases slightly increased and in three suffering from mucoviscidosis in whom steatorrhea with moderate alterations of fats and elevation of alkaline phosphatase and transaminases were present. Urinary and gall stones were not seen in anyone. In conclusion from this study it can be postulated that removal of ileocecal valve can be done safely in children.
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PMID:[A follow-up study in individuals subjected to ileo-cecal resection in infancy and childhood]. 899 79

1-(5-Phospho-beta-D-ribosyl)2'-phosphoadenosine 5'-phosphate cyclic anhydride [2'-phospho-cyclic ADP-ribose, cAdo(2')P(5')PP-Rib] was prepared enzymatically from NADP+ using ADP-ribosyl-cyclase from Aplysia californica. The product was purified by HPLC and characterized by NMR and mass spectroscopy, by conversion to 1-(5-phospho-beta-D-ribosyl)adenosine 5'-phosphate cyclic anhydride (cADP-Rib) by alkaline phosphatase and by resistance to snake venom phosphodiesterase. cAdo-(2')P(5')PP-Rib dose-dependently released Ca2+ from an intracellular, non-endoplasmic reticular Ca2+ pool of permeabilized Jurkat and HPB. ALL T-lymphocytes. In contrast, the closely related compounds 1-(5-phospho-beta-D-ribosyl)3'phosphoadenosine 5'-phosphate cyclic anhydride and 1-(5-phospho-beta-D-ribosyl)cyclic 2',3'-phosphoadenosine 5'-phosphate cyclic anhydride did not induce Ca2+-release from permeabilized T cells. The Ca2+ pool sensitive to cAdo(2')P(5')PP-Rib partially overlapped with the Ca2+ pool sensitive to cADP-Rib recently described in T cells [Guse, A. H., da Silva, C. P., Emmrich, F., Ashamu, G. A., Potter, B. V. L. & Mayr, G. W. (1995) Characterization of cyclic adenosine diphosphate-ribose-induced Ca2+-release in T-lymphocyte cell lines, J. Immunol. 155, 3353-3359]. Control experiments suggest that the results were neither due to Ca2+ contaminations in the cADP-Rib preparation nor to catabolism of cAdo(2')P(5')PP-Rib to cADP-Rib.
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PMID:1-(5-phospho-beta-D-ribosyl)2'-phosphoadenosine 5'-phosphate cyclic anhydride induced Ca2+ release in human T-cell lines. 915 72

Glucocorticoids promote the development of many organs including intestine. At the cellular level, the activity of glucocorticoids is regulated by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) which converts active glucocorticoids to inactive metabolites. As 11 beta HSD is also expressed in the intestine, this enzyme may be an important regulator of intestinal maturation. To investigate this, we have performed the systematic study of the development of intestinal 11 beta HSD activity and its cofactor preference as well as of the effect of 11 beta HSD inhibition by carbenoxolone on postnatal development of sucrase, alkaline phosphatase and Na,K-ATPase in the intestine. The activity of 11 beta HSD was low in ileum of suckling rats and significantly increased during the weaning period. In colon, the activity was already high in suckling rats and gradually rose during the postnatal development. 11 beta HSD activity was undetectable in jejunum both in young and adult rats. At 14.5 nM corticosterone, colonic 11 beta HSD utilized predominantly NAD as a cofactor, but displayed significant sensitivity also to NADP. Ileal 11 beta HSD had similar sensitivity to both cofactors. With NAD as a cofactor, ileal 11 beta HSD had a Km (59 +/- 10 nM) compatible with the colonic enzyme (81 +/- 14 nM). Carbenoxolone administration to suckling and weanling rats in vivo did not result in any changes of sucrase activity in jejunum and ileum, alkaline phosphatase activity in ileum and distal colon or Na,K-ATPase activity in ileum. However, carbenoxolone significantly increased Na,K-ATPase activity in distal colon. Our results indicate that the high-affinity type of 11 beta HSD is expressed not only in colon but also in ileum and that 11 beta HSD is an important factor in the regulation of tissue levels of active glucocorticoids in developing colon but not in the small intestine.
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PMID:The role of 11 beta-hydroxysteroid dehydrogenase in maturation of the intestine. 937 10

Nicotinic acid-adenine dinucleotide phosphate (NAADP), a molecule derived from beta-NADP, has been shown to promote intracellular calcium release in sea urchin eggs. However, there is little information regarding the role of NAADP in the regulation of intracellular calcium fluxes in mammalian cells. We found recently that several mammalian tissues have a high capacity for NAADP synthesis, as assessed by sea urchin egg bioassay. To determine the functional significance of NAADP production by mammalian tissues, we sought to determine whether NAADP is capable of inducing calcium release from microsomes prepared from cultured cells. We found that NAADP, but not beta-NADP, activates a specific microsomal calcium release system in mesangial cells isolated from rat kidney; NAADP was without effect in renal tubular epithelial cells. NAADP-induced calcium release is not affected by inhibitors of the inositol 1,4,5-trisphosphate or ryanodine channels. However, NAADP-elicited calcium release was inhibited by L-type calcium channel blockers and by alkaline phosphatase treatment of NAADP. NAADP also promotes specific microsomal calcium release in rat vascular smooth muscle cells, cardiac myocytes, fibroblasts and a human leukaemia cell line, indicating that the capacity for NAADP-induced calcium release is widespread in mammalian cells. We propose that NAADP may be an important regulator of intracellular calcium in many mammalian tissues.
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PMID:Nicotinic acid-adenine dinucleotide phosphate (NAADP) elicits specific microsomal Ca2+ release from mammalian cells. 1117 Oct 49

A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.
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PMID:Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay. 1175 40

Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel nucleotide derived from NADP that has now been shown to be active in releasing Ca(2+) from intracellular stores in a wide variety of cells ranging from plant to human. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, no assay has been reported for NAADP to date. In the present study, a widely applicable assay for NAADP with high sensitivity is described. NAADP was first dephosphorylated to nicotinic acid-adenine dinucleotide by treatment with alkaline phosphatase. The conversion was shown to be stoichiometric. NMN-adenylyltransferase was then used to convert nicotinic acid-adenine dinucleotide into NAD in the presence of high concentrations of NMN. The resultant NAD was amplified by a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD cycled through these coupled reactions, a molecule of highly fluorescent resorufin was generated. The reaction could be performed for hours, resulting in more than a 1000-fold amplification. Concentrations of NAADP over the 10-20 nM range could be routinely measured. This novel cycling assay was combined with an enzymic treatment to provide the necessary specificity for the assay. NAADP was found to be resistant to NADase and apyrase. Pretreatment of samples with a combination of the hydrolytic enzymes completely eliminated the interference from common nucleotides. The versatility of the cycling assay can also be extended to measure nicotinic acid, which is a substrate in the synthesis of NAADP catalysed by ADP-ribosyl cyclase, over the micromolar range. All the necessary reagents for the cycling assay are widely available and it can be performed using a multi-well fluorescence plate reader, providing a high-throughput method. This is the first assay reported for NAADP and nicotinic acid, which should be valuable in elucidating the messenger functions of NAADP.
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PMID:A novel cycling assay for nicotinic acid-adenine dinucleotide phosphate with nanomolar sensitivity. 1211 13


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