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

---

Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Following hybridization of membrane-bound target-DNA with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CIAP]. This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deep-blue coloured, water-insoluble precipitate directly adhering to the membrane. The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot- and Southern-blots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations.
...
PMID:Non-radioactive labeling and detection of nucleic acids. I. A novel DNA labeling and detection system based on digoxigenin: anti-digoxigenin ELISA principle (digoxigenin system). 207 99

Sequences of hepatitis B virus DNA were detected specifically in the sera of infected individuals by a non-radioactive riboprobe nucleic acid hybridization assay. The nucleic acids contained in serum specimens were prepared by an overnight proteinase K-/SDS-digest, phenol/chloroform-extraction and ethanol-precipitation, and immobilized on nylon membranes by the dot-blot technique. Digoxigenin-labelled 1.4 kb RNA-probes were generated by the phage sp 6 RNA-polymerase from a linearized HBV DNA transcription template containing the appropriate promoter. Hybridized probes were detected by alkaline phosphatase-conjugated sheep anti-digoxigenin Fab-fragments, and 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium salt (NBT) as chromogenic substrates for the subsequent ELISA procedure. With a detection limit of 0.5-1.0 pg HBV DNA and a high specificity, the results were comparable to those obtained by the standard assay employing radioactively labeled DNA-probes.
...
PMID:A nonradioactive riboprobe assay for the detection of hepatitis B virus DNA in human sera. 208

In an ongoing study of brain microvasculature in humans at autopsy, we had the opportunity to analyze the overall scheme of this vascular supply. The native endothelial membrane enzyme, alkaline phosphatase, is used to precipitate black lead sulfide salt in the vessel wall, rendering the brain microvasculature visible by both light microscopy and microradiography. There are six distinct patterns of intraparenchymal afferent blood supply to the supratentorial brain: short arterioles from a single source (e.g., those in the cortex); short- to intermediate-length arterioles, single source (anterior two-thirds of the corpus callosum); short- to intermediate length arterioles and arteries, dual source (subcortical U fibers); intermediate-length arterioles and arteries, triple source (extreme/external capsule and claustrum); long arteries and arterioles, single source (centrum semiovale); and large, long muscular arteries, single source (thalamus and basal ganglia). The nature of this arrangement offers some protection to certain regions of the cerebrum from circulatory challenges such as hypotension, while leaving other areas vulnerable. The distal arterioles supplying two of these protected regions, the U-fiber area and the extreme/external capsule and claustrum area, also exhibit the feature of interdigitation, which can offer additional collateral potential from one arteriolar territory to the next. Aging, hypertension, diabetes mellitus, and atherosclerosis can have a significant impact on brain microcirculation. The way in which vascular patterns dictate the distribution of these effects is discussed. The ability to stain the cerebral microvessels and demonstrate the finer points of their patterns in sections and microradiographs has enabled us to resolve some long-standing questions about vascular connections and directions.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Features of the cerebral vascular pattern that predict vulnerability to perfusion or oxygenation deficiency: an anatomic study. 211 4

A degenerative oligodeoxyribonucleotide probe deduced from the first 19 amino acids of the mature alkaline phosphatase IV (APase IV) protein was used to clone a DNA fragment internal to the coding region of the phoAIV gene of Bacillus subtilis. An insertional mutation was constructed in the phoAIV locus using the integrative plasmid, pJM103, containing the cloned DNA fragment. The strain with the interrupted phoAIV gene showed no detectable APase IV product on Western-blot analysis. The impact of the phoAIV interruption on total APase production in B. subtilis 168 was analyzed under both phosphate starvation and sporulation culturing conditions. The mutation in phoAIV reduced total APase-specific activity by 75% in phosphate-starved cells, and resulted in the elimination of a salt-extractable membrane APase, as well as the secreted APase IV. Analysis of this membrane APase indicated that it is a phoAIV gene product which is localized within the membrane fraction of the lysed cell and not secreted. There was no effect on the production of sporulation APase. The phoAIV::pJM103 insertion was mapped and determined to be located at approx. 73 degrees on the B. subtilis 360 degrees chromosome.
...
PMID:The Bacillus subtilis phoAIV gene: effects of in vitro inactivation on total alkaline phosphatase production. 212 17

We detected in urine by HPLC two enzyme fractions of alkaline phosphatase (AP, EC 3.1.3.1), soluble and particulate, analogous to those in serum. The second fraction was eluted with high-salt-content eluent at the same elution time as high-molecular-mass, or particulate, AP in serum. AP characterization in urine from a patient with acute rejection crisis showed a greater sensitivity of the particulate form to treatment with L-phenylalanine, which suggests a higher content of intestinal-type AP in the particulate form. The soluble fraction showed a more liver-type AP behavior. Changes in the chromatograms after the sample was treated with 1-butanol and Triton X-100 support a membrane origin of the particulate AP. Urinalyses from patients with acute renal disease showed increased activity of soluble and particulate AP, with a relatively greater increase of particulate AP.
...
PMID:Particulate (high-molecular-mass) and soluble alkaline phosphatase in urine determined by high-performance liquid chromatography. 224 72

Effects of dephosphorylation on interactions of neurofilaments (NFs) with microtubules (MTs) were studied by the cosedimentation method. Centrifugation conditions were chosen so that MTs pelleted but NFs did not. While NFs isolated from bovine spinal cords did not cosediment with MTs polymerized in the presence of taxol, NFs dephosphorylated with Escherichia coli alkaline phosphatase began to coprecipitate with MTs. The dephosphorylated NFs bound to MTs but not to the unpolymerized tubulin dimer. The binding was not observed in the presence of high salt or with MTs containing microtubule-associated proteins. The cosedimentation experiments using purified NF subunit proteins showed that the dephosphorylation-induced binding of NFs to MTs was mediated by the largest subunit of NF (NF-H). Negative staining electron microscopy confirmed bindings of the dephosphorylated NFs and NF-H to MTs. Densitometric measurement of the bound and unbound NF-H after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the binding of the dephosphorylated NF-H to MT was saturable and gave the following binding parameters. Approximately 1 mol of NF-H bound per 10 mol of tubulin dimer with a high affinity site (Kd = 3.8 x 10(-8) M) and per 16 mol of tubulin dimer with a low affinity site (Kd = 1.1 x 10(-7) M).
...
PMID:Dephosphorylation-induced interactions of neurofilaments with microtubules. 225 37

A method for the microassay in vitro of lipoprotein: proteoglycan interactions is described. The wells of a plastic 96-well microtitration plate are coated with low density lipoprotein. A limiting quantity of biotin-conjugated proteoglycan is allowed to bind to each coated well, and the amount of the latter retained in wells is estimated spectrophotometrically through subsequent binding of alkaline phosphatase-conjugated avidin. Many of the incubation parameters (e.g., time, pH, salt concentration, divalent cations), which influence the extent of binding of biotin-conjugated proteoglycan, have been studied and optimized. The effect upon binding of introducing different levels of proteoglycans or lipoproteins at the interaction step can be measured readily. Thus, the orders of increasing relative binding affinities were found to be high density lipoprotein less than Lipoprotein (a) less than low density lipoprotein; rat chondrosarcoma proteoglycan less than bovine nasal cartilage proteoglycan less than human aorta proteoglycan; chondroitin 4-sulfate less than chondroitin 6-sulfate less than dermatan sulfate for lipoproteins, proteoglycans, and glycosaminoglycans, respectively.
...
PMID:A competitive assay of lipoprotein: proteoglycan interaction using a 96-well microtitration plate. 232 81

Tissue cholestasis is a histologic feature in some patients with alcoholic liver disease, but its significance is unknown. We studied prospectively the clinical, laboratory, and histologic findings of 306 chronic male alcoholics in whom liver tissue was available. Tissue cholestasis permitted identification of two groups: group I, absent or mild cholestasis (239 patients), and group II, moderate to severe cholestasis (67 patients). Statistical evaluation was performed by Student's t test and regression analyses. In patients with tissue cholestasis, 97% had elevated serum cholylglycine levels, while only 61% had significant jaundice (serum bilirubin greater than 5 mg/dl). In patients without tissue cholestasis, 66% had elevated serum cholylglycine and 13.5% jaundice. Highly significant statistical correlations (P less than 0.0001) were found between cholestasis and malnutrition, prothrombin time, AST, alkaline phosphatase, bilirubin, Maddrey's discriminant function, serum cholylglycine level, albumin, and histologic severity score. In group I, 54% survived 60 months versus 22% in group II (P less than 0.0001). Highly significant statistical correlations (P less than 0.0001) were noted between serum cholylglycine levels and the parameters enumerated earlier, but not with survival. We conclude that tissue cholestasis is a highly significant prognostic indicator of outcome in alcoholic hepatitis and is more consistently associated with bile salt retention than jaundice.
...
PMID:Prognostic significance of cholestatic alcoholic hepatitis. VA Cooperative Study Group #119. 236 44

Acid phosphatase, alkaline phosphatase and 5'-nucleotidase activities were analyzed cytophotometrically in cryostat sections of rat liver up to 8 weeks after ligation and transsection of the common bile duct. Ligation resulted in cholestasis and induced alterations in both localization and activity of the enzyme investigated. The cellular distribution but not the activity of acid phosphatase changed in liver parenchyma. In control liver, the final reaction product was localized as discrete granules in the bile canalicular region of hepatocytes. The final reaction product was precipitated more diffusely within the cytoplasm after induction of cholestasis, most probably due to increased fragility of lysosomal membranes. In control liver, alkaline phosphatase activity was low and localized in the bile canalicular plasma membranes only. The total parenchymal activity increased threefold after the induction of cholestasis and is considered to be a compensatory mechanism in order to enhance the excretion of bile salts from hepatocytes. 5'-Nucleotidase was present at the bile canalicular and sinusoidal surfaces of plasma membranes of hepatocytes in control liver; total activity in pericentral areas was significantly higher than in periportal areas. Induction of cholestasis resulted in higher total activity and redistribution of the activity over all three surfaces of the plasma membranes, whereas heterogeneity over the different zones of the acinus disappeared. The appearance of the enzyme at lateral plasma membranes is suggested to be related to the formation of new sites for bile salt transport out of the hepatocytes. With respect to all three enzymes studied, alterations of liver parenchymal cells due to a disturbed bile transport were already established during the first week of cholestasis.
...
PMID:Quantitative changes in acid phosphatase, alkaline phosphatase and 5'-nucleotidase activity in rat liver after experimentally induced cholestasis. 238 57

Primary human hepatocytes were used to study bile salt hepatotoxicity and the hepatoprotective potential of ursodeoxycholate in vitro. Hepatocytes were obtained by collagenase perfusion of healthy human liver tissue and were treated with glycochenodeoxycholate for 24 hr 1 day after plating. Clear signs of cytotoxicity were observed at concentrations of about 100 mumol/L glycochenodeoxycholate. Toxicity was determined by release of alkaline phosphatase, gamma-glutamyl transferase, AST, ALT or lactate dehydrogenase into the culture medium, by measuring DNA synthesis of the cultured liver cells and by testing the viability of the hepatocytes using trypan-blue dye exclusion. Addition of ursodeoxycholate, which by itself proved to be of little toxicity, significantly reduced the hepatotoxic effects of glycochenodeoxycholate: 72% +/- 6% of the cells survived treatment with 500 mumol/L glycochenodeoxycholate alone, but addition of 100 mumol/L ursodeoxycholate increased the survival rate to 87% +/- 4% (p less than 0.05). Moreover, all enzymes tested were secreted at a significantly lower level when ursodeoxycholate was present. Similarly, the cellular DNA synthesis was maintained at significantly higher levels as a result of ursodeoxycholate treatment. We conclude that (a) primary human hepatocytes are a suitable model for studying hepatotoxicity of bile salts in vitro, (b) ursodeoxycholate reduces hepatotoxicity of other bile salts and (c) ursodeoxycholate can act hepatoprotectively by itself (i.e., alteration of the metabolism of other bile salts is not necessarily required).
...
PMID:Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. 240 54


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---