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)

A new method for the determination of lipopolysaccharide (LPS) admixtures in protein solutions has been developed. The method includes the periodate oxidation of LPS, biotinylation with biotin hydraside, immobilization on a nitrocellulose membrane and the development of biotinylated LPS in the streptavidin--alkaline phosphatase system. Proteins are previously removed from the solution by treatment with hot phenol. Development with the use of 5-bromoinodyl phosphate and nitrotetrazolium blue makes it possible to detect about 30 pg of LPS immobilized on the nitrocellulose membrane.
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PMID:[The determination of lipopolysaccharide admixtures in protein preparations]. 1085 36

An immunoelectrochemical method coupled with immunomagnetic separation was developed for rapid detection of Salmonella Typhimurium in chicken carcass wash water. Samples of chicken carcass wash water were inoculated with Salmonella Typhimurium at different cell numbers. Possible nonspecified inhibitors in the wash water were minimized by filtration and centrifugation. An approximately 9.4% loss of Salmonella cells was found after filtration (P < 0.01). The samples were mixed with anti-Salmonella-coated magnetic beads (ASCMB) and alkaline phosphatase-labeled anti-Salmonella (APLAS) to form ASCMB-Salmonella-APLAS conjugates. The conjugates were separated from the solution using a magnetic separator and then incubated with phenylphosphate substrate to produce phenol. The number of Salmonella was determined by measuring the phenol concentration using an amperometric tyrosinase carbon paste electrode in a flow injection analysis system. Under optimized parameters (1 mM MgCl2, 0.2 microg/ml APLAS, and 1 mM phenylphosphate in pH 7.0 Tris buffer solution), Salmonella Typhimurium in chicken carcass wash water could be identified and enumerated within 2.5 h with a detection limit of 5 x 10(3) CFU/ml. A linear relationship on a log-log scale was found between Salmonella cell number and the peak current ratio for Salmonella concentrations ranging from 10(3) to 10(7) CFU/ml (R2 = 0.963). The peak currents of multibacteria samples, containing Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, were not significantly different from Salmonella-only samples (P > 0.01).
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PMID:Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method. 1094 78

The alkaline phosphatase (ALP) activity test has been used since 1935 to assess the effectiveness of pasteurization. Different analytical methods exist for detecting ALP in milk. Unfortunately, there is little information about ALP activity in ewe's milk. The aim of this study was to assess and compare the official European method (spectrophotometric method) and the Fluorophos method (fluorometric method) regarding their use in ewe's milk. Bulk ewe's milk samples were taken from a flock and from three different dairies. A portion of the original sample was pasteurized at 63 degrees C for 30 min in a circulating bath; another portion was heated to and kept at 95 degrees C for about 2 min, and 0.1% (vol/vol) of raw milk was added. The samples obtained were analyzed in duplicate using the spectrophotometric and fluorometric methods. The relation between ALP activity determined by the two methods was characterized by the following equation: Y = 1.34 + 0.0039X (where Y = ALP in microg of phenol per ml of milk and X = ALP in mU/liter; R2 = 91.5%). Precision parameters (repeatability [r], standard deviation of repeatability [s(r)], and relative standard deviation of repeatability [RSDr]) for both methods were calculated. The values of RSDr for the Fluorophos method were 4.30 for pasteurized milk and 2.96 for 0.1% raw milk, close to the value indicated by Rocco in whole cow's milk (RSDr = 4.4). The repeatability for the official method (r = 2.16) was close to that indicated for whole cow's milk (r = 2).
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PMID:Evaluation of spectrophotometric and fluorometric methods for alkaline phosphatase activity determination in ewe's milk. 1098 2

The electrooxidation of a biotin pyrrole has allowed the formation of biotinylated polypyrrole films. Gravimetric measurements based on a quartz crystal microbalance demonstrate the efficient coupling of avidin, biotinylated polyphenol oxidase (PPO-B) and avidin-labeled alkaline phosphatase (AP-A) with the underlying biotinylated polymer film. The estimated mass increase corresponds to the anchoring of 1.6-1.8 equivalent layer of proteins. A step-by-step construction of bienzyme multilayers composed of PPO-B and AP-A was carried out on the electrode surface modified by the biotinylated polypyrrole film through avidin-biotin bridges. A spatially controlled distribution of the two enzymes was performed by the formation of one AP-A layer on 1, 5, and 10 PPO-B layers. The resulting bienzyme electrodes were applied to the determination of phenyl phosphate on the basis of amperometric detection of enzymically generated o-quinone at -0.2 V. Their analytical performances were analyzed in relation to the design of the enzyme architectures and in comparison with the amperometric behavior of the monoenzymatic electrodes (PPO-B electrode and AP-A electrode). It appears that at the 10-layer-PPO-B polypyrrole electrode only 4% of phenol is transformed, whereas 42-69% of phenyl phosphate is enzymatically consumed and detected at the AP-A polypyrrole electrode, depending on the enzyme activity. For the bienzymatic AP-A/PPO-B polypyrrole electrodes, the activity of each immobilized enzyme clearly affects the biosensor performance, the main limiting factor being the very low efficiency of PPO-B at pH 8.8.
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PMID:Elaboration and characterization of spatially controlled assemblies of complementary polyphenol oxidase-alkaline phosphatase activities on electrodes. 1146 32

A rapid and sensitive detection process for Escherichia coli O157:H7 was developed using alkaline phosphatase (APase)-labeled anti-E. coli O157 antibodies to tag the targeted bacteria. Immunomagnetic beads or antibody-labeled streptavidin-coated magnetic beads were then used to capture the APase-tagged E. coli. Immunomagnetically captured bacteria were washed and distributed into microplates or optical cuvettes. The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed. Less than 1000 cfu/ml of E. coli O157:H7 could be detected. This approach was applied to detect the bacteria artificially spiked in beef hamburgers. Less than 1 cfu/g of E. coli O157:H7 produced a significant response after cultural enrichment for 4-6 h at 37 degrees C.
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PMID:Detection of immunomagnetically captured Escherichia coli O157:H7 by antibody-conjugated alkaline phosphatase. 1157 17

Sea bass (Dicentrarchus labrax) were injected intraperitoneally once (single dose) or three times (fractionated dose) with phenol or OH-phenols (hydroquinone, resorcinol, and pyrocatechol). On the basis of the lethal doses, OH-phenols were more toxic than phenol, and pyrocatechol was the most powerful compound. Hematological, metabolic and antioxidant blood parameters were measured 3 days after the end of the treatment. Metabolic variations as specific effects on erythrocytes were revealed and differences between single and fractionated doses were observed. OH-phenols-treated fish showed disorders in the metabolic toxicity indicators as hypoglycemia, low blood urea nitrogen level (BUN) and decrease of alkaline phosphatase activity (ALP). In addition, quantitative structure-activity relationships were developed using the n-octanol:water partition coefficient (log K(ow)). Positive correlations were found with ALP, plasma glucose and hemoglobin.
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PMID:In vivo effects of phenolic compounds on blood parameters of a marine fish (Dicentrarchus labrax). 1179 Mar 55

Renal injury-induced by phenol injection activates renal sympathetic afferent pathways, increases norepinephrine release from the posterior hypothalamus, activates renal efferent pathways, and provokes a rapid and persistent hypertension. This study aimed to determine whether phenol injury provoked a redistribution of proximal Na(+) transporters from internal stores to the apical cell surface mediated by sympathetic activation, a response that could contribute to generation or maintenance of hypertension. Anesthetized rats were cannulated for arterial blood pressure tracing and saline infusion and then 50 microl 10% phenol or saline was injected into one renal cortex (n = 7 each). Fifty minutes after injection, kidneys were removed and renal cortex membranes from injected kidneys were fractionated on sorbitol gradients and pooled into three windows (WI-WIII) that contained enriched apical brush border (WI); mixed apical, intermicrovillar cleft and dense apical tubules (WII); and intracellular membranes (WIII). Na(+) transporter distributions were determined by immunoblot and expressed as percentage of total in gradient. Acute phenol injury increased blood pressure 20-30 mmHg and led to redistribution of Na(+)/H(+) exchanger type 3 (NHE3) out of WIII (from 22.79 +/- 4.75 to 10.79 +/- 2.01% of total) to WI (13.07 +/- 1.97 to 27.15 +/- 4.08%), Na(+)-P(i) cotransporter 2 out of WII (68.72 +/- 1.95 to 59.76 +/- 2.21%) into WI (9.5 +/- 1.62 to 18.7 +/- 1.45%), and a similar realignment of dipeptidyl-peptidase IV immunoreactivity and alkaline phosphatase activity to WI. Renal denervation before phenol injection prevented the NHE3 redistribution. By confocal microscopy, NHE3 localized to the brush border after phenol injection. The results indicate that phenol injury provokes redistribution of Na(+) transporters from intermicrovillar cleft/intracellular membrane pools to apical membranes associated with sympathetic nervous system activation, which may contribute to phenol injury-induced hypertension.
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PMID:Responses of proximal tubule sodium transporters to acute injury-induced hypertension. 1238 25

Enzyme-based hybridization assays for the simultaneous electrochemical measurements of two DNA targets are described. Two encoding enzymes, alkaline phosphatase and beta-galactosidase, are used to differentiate the signals of two DNA targets in connection to chronopotentiometric measurements of their electroactive phenol and alpha-naphthol products. These products yield well-defined and resolved peaks at +0.31 V (alpha-naphthol) and +0.63 V (phenol) at the graphite working electrode (vs. Ag/AgCl reference). The position and size of these peaks reflect the identity and level of the corresponding target. The dual target detection capability is coupled to the amplification feature of enzyme tags (to yield fmol detection limits) and with an efficient magnetic removal of non-hybridized nucleic acids. Proper attention is given to the choice of the substrates (for attaining well resolved peaks), to the activity of the enzymes (for obtaining similar sensitivities), and to the selection of the enzymes (for minimizing cross interferences). The new bioassay is illustrated for the simultaneous detection of two DNA sequences related to the BCRA1 breast-cancer gene in a single sample in connection to magnetic beads bearing the corresponding oligonucleotide probes. Prospects for electrochemical coding of multiple DNA targets are discussed.
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PMID:Dual enzyme electrochemical coding for detecting DNA hybridization. 1243 May 95

Prevotella intermedia, a Gram-negative obligate anaerobic black-pigmented oral bacterium, belongs to a small group of microorganisms that is closely associated with the initiation of periodontal diseases. Lipopolysaccharide (LPS), an outer membrane component, is one of the main virulence factors of this bacterium. The aim of this study was to examine the effects of Prev. intermedia lipopolysaccharide, extracted by the hot-phenol-water method, on differentiation (alkaline phosphatase activity) and mineralisation (calcium incorporation) of fetal mouse calvarial cells in vitro and to determine the release of the important osteolytic factors nitric oxide, interleukin-6 (IL-6) and matrix metalloproteinases by these cells after treatment with different concentrations of Prev. intermedia lipopolysaccharide (0.2-25 microg/ml). By gelatin zymography, we also characterized the matrix metalloproteinases released by these osteoblasts. Treatment with Prev. intermedia lipopolysaccharide dose-dependently inhibited bone formation by reducing alkaline phosphatase activity and calcium incorporation and induced the release of nitric oxide, IL-6 and the latent proforms of MMP-2 and MMP-9 by fetal mouse osteoblasts in organoid culture. These results indicate that the lipopolysaccharide from Prev. intermedia not only participates in periodontal tissue destruction and alveolar bone resorption, but also inhibits bone formation.
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PMID:Effects of lipopolysaccharide extracted from Prevotella intermedia on bone formation and on the release of osteolytic mediators by fetal mouse osteoblasts in vitro. 1245 May 17

Renal cortical phenol injection provokes acute sympathetic nervous system-dependent hypertension and a shift of proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3) and Na(+)-P(i) cotransporter type 2 (NaPi2) to apical microvilli. This study aimed to determine whether proximal tubule (PT) Na(+) transporter redistribution persists chronically and whether the pool sizes of renal Na(+) transporters are altered. At 5 wk after a 50-microl 10% phenol injection, blood pressure is elevated: 154 +/- 8 vs. 113 +/- 11 mmHg after saline injection. Cortical membranes were fractionated into three "windows" enriched in apical brush border (WI), mixed apical and intermicrovillar cleft (WII), and intracellular membranes (WIII). NHE3 relative distribution in these windows, assessed by immunoblots and expressed as %total, remained shifted to apical from intracellular membranes (WI: 25.3 +/- 3 in phenol vs.12.7 +/- 3% in saline and WIII: 9.1 +/- 1.3 in phenol vs. 18.9 +/- 3% in saline). NaPi2 and dipeptidyl-peptidase IV also remained shifted to WI, and alkaline phosphatase activity increased 100.9 +/- 29.7 (WI) and 51.4 +/- 17.5% (WII) in phenol-injected membranes. Na(+) transporter total abundance [NHE3, NaPi2, thiazide-sensitive Na-Cl cotransporter, bumetanide-sensitive Na-K-2Cl cotransporter, Na-K-ATPase alpha(1)- and beta(1)-subunits, and epithelial Na(+) channel (ENaC) alpha- and beta-subunits] was profiled by immunoblotting. Only cortical NHE3 abundance was altered, decreasing to 0.56 +/- 0.06. The results demonstrate that phenol injury provokes a persistant shift of PT NHE3 and NaPi2 to the apical microvilli, along with a 44% decrease in total NHE3, evidence for an escape mechanism that would counteract the redistribution of a larger fraction of NHE3 to the apical surface by normalizing the total amount of NHE3 in apical membranes.
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PMID:Chronic renal injury-induced hypertension alters renal NHE3 distribution and abundance. 1255 35


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