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)

Hypochlorous acid (HOCl), generated from H2O2 and Cl- by myeloperoxidase in activated neutrophils, causes tissue damage during inflammation. We have developed a simple, sensitive (approximately 0.2 fmol on column) and specific GC-MS assay for the detection of 5-chlorouracil (5-ClUra), a signature product of HOCl-mediated damage to nucleobases. In this assay, 5-ClUra is released from isolated DNA by a digestion with nuclease P1, alkaline phosphatase, and thymidine phosphorylase (TP), or from chlorinated nucleosides in biological fluids by TP. The freed 5-ClUra is derivatized with 3, 5-bis-(trifluoromethyl)-benzyl bromide, which is detected by negative chemical ionization mass spectrometry. The assay can be used to simultaneously detect other halogenated uracils including bromouracil. Using this assay, we showed that 5-ClUra is generated by the reaction of low micromolar HOCl with (deoxy)cytidine, (deoxy)uridine, and DNA. In cell cultures, an increase of 5-ClUra was detected in DNA when cells were treated with sublethal doses of HOCl and allowed to proliferate. The elevation of 5-ClUra was markedly accentuated when physiologically relevant concentrations of (deoxy)uridine, (deoxy) cytidine, uracil, or cytosine were present in the medium during HOCl treatment. In the carrageenan-induced inflammation model in rats, chlorinated nucleosides was significantly increased, compared with controls, in the exudate fluid isolated from the inflammation site. Our study provides the direct evidence that chlorinated nucleosides are found in the inflammation site and can be incorporated in DNA during cell/tissue proliferation. These findings may be relevant to the carcinogenesis associated with chronic inflammation.
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PMID:5-Chlorouracil, a marker of DNA damage from hypochlorous acid during inflammation. A gas chromatography-mass spectrometry assay. 1281 Jul 14

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma x glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC). 2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ectophosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP. 3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide-diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole. 4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells. 5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.
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PMID:Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis. 1282 32

For the simultaneously visual detection of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type-1 (HIV-1), a qualitative DNA chip method, combining multiplex and nested polymerase chain reaction (PCR) with arrayed anchored primer PCR and a biotin-avidin alkaline phosphatase (Av-AP) indicator system, was developed. After pretreatment of infected blood samples and reverse transcription of the RNA virus genome, PCR was performed in a single tube by using the outer primer pairs. Second round nested multiplex PCR was performed on the DNA chip, on which the primers array had already been prepared. During the arrayed anchored multiplex PCR, 5[N-(N-biotinylaminocaproyl)-epsilon-3-aminoallyl]-2-deoxy-uridine-5-triphosphate (biotin-11-dUTP) was incorporated into the extended DNA chains in order to bind avidin alkaline phosphatase via avidin and biotin. To produce purple precipitates on the chips, the enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) was used in conjunction with the enhancer, nitro blue tetrazolium (NBT). Blood samples containing the three viruses were tested using this DNA chip and about 1 pg of specific viral DNA fragments were detected on the chip wells after nested PCR.
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PMID:A visual DNA chip for simultaneous detection of hepatitis B virus, hepatitis C virus and human immunodeficiency virus type-1. 1470 86

The nucleoside content of 32 elapid and viperid venoms was examined. Free purines, principally adenosine (ADO), inosine (INO), and guanosine (GUA), comprised as much as 8.7% of the solid components of some venoms. Thus, purines are far more abundant in some venoms than many proteinaceous toxins. Hypoxanthine (HYP) was found in about half of elapid and viperine venoms, in which it is a relatively minor constituent (<60 microg/g). Adenosine monophosphate (AMP) was tentatively identified in only three elapid and two viperid venoms. The pyrimidines, uridine (URI) and cytidine (CYT), were also found in most elapid and viperine venoms. In most of these, the amount of uridine was substantially greater than that of cytidine. Thymidine (THY) was not found in any venom, indicating that DNA from disintegration of glandular cells is not the source of venom nucleosides. In contrast to elapid and viperine venoms, most crotaline venoms are devoid of free nucleosides. Elapid and viperine venoms also contained other minor, low molecular weight constituents that could not be positively identified. Some had spectra identical to those of adenosine, nicotinamide adenine dinucleotide (NAD), inosine, xanthosine (XAN), and guanosine, while others had unique spectra. There is no apparent correlation between quantities of venom nucleosides and literature values for the three dominant venom enzymes that release endogenous nucleosides, 5'-nucleotidase (5NUC), phosphodiesterase (PDE), and alkaline phosphomonoesterase (PME).
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PMID:Taxonomic distribution and quantitative analysis of free purine and pyrimidine nucleosides in snake venoms. 1562 16

Size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analyses of Escherichia coli membranes expressing Streptococcus equisimilis hyaluronan synthase (seHAS) demonstrated an inherent artifact (10-100 MDa) that coeluted with hyaluronan (HA) and skewed the apparent weight-average mass of HA to erroneously high values. Briefly heating samples to 65-75 degrees C eliminated this artifact and increased the yield of recovered HA due to the release of HA chains that were attached to membrane-bound HAS. Inclusion of alkaline phosphatase, which removed uridine 5'-diphosphate (UDP) produced during the reaction, improved the linearity of HA synthesis-even at high substrate use. Surprisingly, the addition of EDTA, to chelate Mg(2+) ions, did not completely stop the HAS reaction at 30 degrees C or at 4 degrees C. The best conditions for stopping the reaction without altering SEC-MALLS profiles of the product HA were to chill samples on ice in the presence of both EDTA and UDP. Even with excess substrate, the maximum size of product HA decreased as the enzyme concentration increased. Therefore, the maximum HA size made by HAS was determined by extrapolation to zero enzyme concentration. Using the above conditions, membrane-bound seHAS synthesized a cohort of HA products that steadily increased in weight-average molar mass, reaching a final maximal steady-state size of 4 to 6 MDa within 2-4 h.
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PMID:Size exclusion chromatography-multiangle laser light scattering analysis of hyaluronan size distributions made by membrane-bound hyaluronan synthase. 1647 3

Tonoplast vesicles isolated from stalk parenchyma tissue of sugarcane plants transport sucrose via a uridine diphosphate glucose (UDPGlc)-dependent group translocator. No sucrose transport via an ATP-dependent system could be detected. The products of UDPGlc uptake in the vesicles were sucrose and sucrose phosphate which, upon hydrolysis with alkaline phosphatase and invertase, showed that both hexose moieties are derived from UDPGlc.
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PMID:UDP-Glucose-Dependent Sucrose Translocation in Tonoplast Vesicles from Stalk Tissue of Sugarcane. 1666 26

Extracellular nucleotides, signaling through P2 receptors, may act as local regulators of bone cell function. We investigated the effects of nucleotide agonists [ATP, ADP, uridine triphosphate (UTP), and uridine diphosphate] and pyrophosphate (PPi, a key physiological inhibitor of mineralization) on the deposition and mineralization of collagenous matrix by primary osteoblasts derived from rat calvariae. Our results show that extracellular ATP, UTP, and PPi strongly and selectively blocked the mineralization of matrix nodules; ADP and uridine diphosphate were without effect. Significant inhibition of mineralization occurred in the presence of relatively low concentrations of ATP, UTP, or PPi (1-10 microm), without affecting production of fibrillar or soluble collagen. In cultures treated with 10 microm ATP or UTP, the expression and activity of alkaline phosphatase, which promotes mineralization by hydrolyzing PPi, was inhibited. The potent inhibitory actions of ATP and UTP on bone mineralization are consistent pharmacologically with mediation by the P2Y(2) receptor, which is strongly expressed by mature osteoblasts. In support of this notion, we found 9-17% increases in bone mineral content of hindlimbs of P2Y(2)-deficient mice. We also found that osteoblasts express ectonucleotide phosphodiesterase/pyrophosphatase-1, an ectonucleotidase that hydrolyzes nucleotide triphosphates to yield PPi, and that addition of 10 microm ATP or UTP to osteoblast cultures generated 2 microm PPi within 10 min. Thus, a component of the profound inhibitory action of ATP and UTP on bone mineralization could be mediated directly by PPi, independently of P2 receptors.
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PMID:Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate. 1756 59

Here, the extracellular interconversion of nucleotides and nucleosides was investigated in rat hippocampal slices and synaptosomes by an HPLC-UV technique. Adenosine 5'-triphosphate (ATP) was converted to adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), adenosine, inosine, and hypoxanthine in the slices, whereas ADP elicited parallel and concentration-dependent formation of ATP and AMP. The specific adenylate kinase inhibitor diadenosine pentaphosphate decreased the rate of decomposition of ADP and inhibited the formation of ATP. No substantial changes in the interconversion of ADP to ATP and AMP were found in the presence of dipyridamole, flufenamic acid, the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium (PPADS), and the alkaline phosphatase substrate para-nitrophenylphosphate. Negligible levels of nucleotides were generated when uridine 5'-diphosphate (UDP), AMP or adenosine were used as substrates. Ecto-adenylate kinase activity was also observed in purified synaptosomes. In summary, we demonstrate the presence of an ecto-adenylate kinase activity in the hippocampus, which is a previously unrecognized pathway that influences the availability of purines in the central nervous system.
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PMID:Extracellular interconversion of nucleotides reveals an ecto-adenylate kinase activity in the rat hippocampus. 1772 17

Several studies have reported differing data on the effect of exogenous nucleosides and nucleotides on the proliferation and differentiation in various intestinal cell lines and explants. To study whether exogenous nucleosides modulate intestinal cell differentiation, IEC-6 cells were differentiated in the presence or absence of a nucleoside mixture (cytidine, uridine, guanosine and inosine, 30 microM each), and the concentrations of nucleoside derivatives were determined by HPLC. Cell differentiation was assessed by electron microscopy, alkaline phosphatase activity and Rnd3 gene expression. The present results showed that uridine, guanosine and inosine were cleared from culture media (up to 32, 63 and 100 % in proliferating cells, and 31, 80 and 94 % in differentiated cells, respectively) whereas cytidine concentrations increased. Differentiation of IEC-6 cells was associated with a significant increase in intracellular nucleotide concentrations. Clearance of nucleosides correlated with a significant increase in the intracellular nucleotide pool in proliferating and differentiated IEC-6 cells. Intracellular guanosine nucleotides increased 2.5- and 5-fold in nucleoside-supplemented proliferating and differentiated cells, respectively. At 24 h, nucleoside-supplemented differentiated IEC-6 cells had significantly higher energy charge and GTP levels than non-supplemented ones. These modifications paralleled changes in cell differentiation as indicated by increased alkaline phosphatase activity, prolonged microvilli formation and accelerated down-regulation of Rnd3 gene expression. The present findings suggest that exogenous nucleosides were selectively taken up by IEC-6 cells, increased the intracellular nucleotide pool, GTP and energy charge, and favoured cell morphological and functional changes during differentiation.
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PMID:Exogenous nucleosides accelerate differentiation of rat intestinal epithelial cells. 1791 45

Venoms of Heloderma horridum and Heloderma suspectum were analyzed for the possible presence of purine and pyrimidine nucleosides. Adenosine, cytidine, guanosine, hypoxanthine, inosine, and uridine were found in mug quantities. These amounts are much smaller than those seen in many elapid or viperine venoms, but greater and more varied than those found in crotaline venoms. While their contribution to the hypotension induced by Heloderma venoms may be minor, venom nucleosides nonetheless act in concert with kallikreins/hemorrhagins, alkaline phosphomonoesterase, 5'-nucleotidase, helodermin, helospectins, helothermine, and serotonin. The use of nucleosides as toxins is therefore a generalized squamate strategy, rather than the exclusive province of snakes. Both Heloderma venoms were found to be devoid of NADase and phosphodiesterase activities. Enzymes to release endogenous purines in the prey, are not significant components of Heloderma venoms.
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PMID:Nucleoside composition of Heloderma venoms. 1843 May 99


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