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)

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of a GMP residue from GTP to the 5' end of RNA to form a cap structure identified as G(5')pppN-, has been isolated from HeLa cell nuclei. The enzyme has been purified approximately 1000-fold and separated by column chromatography (using DEAE-cellulose, phosphocellulose, Cibacron blue-agarose, and GTP-agarose) from a variety of other activities, including RNA triphosphatase and mRNA (guanine-7)methyltransferase. The reaction product was identified by its resistance to Penicillium nuclease and alkaline phosphatase, sensitivity to venom phosphodiesterase, and electrophoretic and chromatographic mobilities relative to authentic standards. Optimal enzyme activity was obtained at pH 7.5 in the presence of Mn2+ or Mg2+, GTP, and an appropriate acceptor polyribonucleotide. The enzyme was inhibited by elevated concentrations of salt and by sulfhydryl-binding reagents but was unaffected by S-adenosylmethionine or S-adenosylhomocysteine. A molecular weight of 48,500 was estimated by sucrose gradient centrifugation of purified enzyme.
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PMID:Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei. 735 12

It is now possible to detect low copy numbers of messenger ribonucleic acid (mRNA) while retaining good histologic morphology for the determination of specific gene expression in diseased tissues. This technology will allow the pathologist to provide important prognostic information about tumors (expression of oncogenes and growth factors), to identify the subclones within the tumor which may be most likely to metastasize (expression of adhesion molecules and proteases) and to identify etiologic genetic aberrations (viral insertions). A technique for in-situ hybridization to mRNA has been developed for use in formalin fixed paraffin embedded tissues which is suitable for a hospital histology laboratory. Optimal conditions for the procedure were determined by using a biotinylated poly (d)T oligonucleotide probe. Results were dependent on the tissue type, fixation time, condition of the tissue prior to fixation, and degree of digestion before hybridization. The temperature and conditions of hybridization were optimized so that the poly d(T) control probe and the longer test probe could be run simultaneously. Streptavidin and avidin alkaline phosphatase detection systems were tested using levamisole to minimize background staining, and a biotin blocking agent to reduce reaction to renal tubular biotin. Increasing the temperature of stringency washes did not significantly improve the specificity but had a markedly detrimental effect on tissue morphology. The mRNA appears to remain stable within routinely fixed surgical material over long periods of time allowing for large retrospective studies. A review of c-erbB-2 expression in 16 human breast lesions was carried out comparing mRNA in-situ hybridization to immunoperoxidase and cytosolic methods. By direct localization of both message and antigen, it was possible to demonstrate focal positivity that cytosolic methods did not detect. Aberrant translation was noted in one case, and c-erbB-2 expression in non-malignant breast was detected in two cases.
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PMID:mRNA in-situ hybridization using biotinylated oligonucleotide probes: implications for the diagnostic laboratory. 752 37

Active oxygen species including hydrogen peroxide (H2O2) play a major role in ischemia-reperfusion injury. In the present study, changes in myocardial H2O2 content as well as its subcellular distribution were examined in rat hearts subjected to ischemia-reperfusion. Isolated perfused rat hearts were made globally ischemic for 20 or 30 minutes and were reperfused for different durations. H2O2 content in these hearts was studied biochemically and changes were correlated with the recovery of function. These hearts were also analyzed for subcellular distribution of H2O2. Optimal conditions of tissue processing as well as incubation medium were established for reacting cerium chloride with H2O2 to form cerium perhydroxide, an insoluble electron-dense product. The chemical composition of these deposits was confirmed by x-ray micro-analysis. Global ischemia caused complete contractile failure in minutes and after 30 minutes of ischemia, these was a > 250% increase in the myocardial H2O2 content. Depressed contractile function recovery in the early phase of reperfusion was accompanied by approximately a 600% increase in the myocardial H2O2 content. Brief pre-fixation with low concentrations of glutaraldehyde, inhibition of alkaline phosphatase, glutathione peroxidase, and catalase, post-fixation but no post-osmication, and no counterstaining yielded the best cytochemical definition of H2O2. In normal hearts, extremely small amounts of cerium hydroperoxide precipitates were located on the endothelial cells. X-ray microanalysis confirmed the presence of cerium in the reaction product. Ischemia resulted in a stronger reaction, particularly on the sarcolemma as well as abluminal side of the endothelial cells; and upon reperfusion, cerium precipitate reaction at these sites was more intense. In the reperfused hearts, the reaction product also appeared within mitochondria between the cristae as well as on the myofibrils, but Z-lines were devoid of any precipitate. The data support a significant increase in myocardial H2O2 during both the phase of ischemia and the first few minutes of reperfusion. A stronger reaction on the sarcolemma and abluminal side of endothelial cells may also indicate enhanced H2O2 accumulation as well as vulnerability of these sites to oxidative stress injury.
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PMID:Hydrogen peroxide changes in ischemic and reperfused heart. Cytochemistry and biochemical and X-ray microanalysis. 767 88

Optimal conditions for detection of calcitonin mRNA were examined by in situ hybridization (ISH) method using a non-radioactive probe in the rat thyroid gland. An oligonucleotide complementary to rat calcitonin mRNA was synthesized with a DNA synthesizer, labelled at 3'-end by using digoxigenin-11-deoxyuridine triphosphate (dUTP) and terminal transferase (Boehringer Mannheim), and used as a probe. Hybrid chains formed by probe and calcitonin mRNA were visualized by anti-digoxigenin-alkaline phosphatase conjugate, nitroblue tetrazolium (NBT) and X-phosphate (Boehringer Mannheim). To determine the optimal conditions for ISH, relations between tissue fixation and proteinase K treatment, and between hybridization temperature and time were mainly surveyed in the present study. As for the relation between tissue fixation and proteinase K treatment, good results were obtained in sections fixed by immersion in 10% formalin at 4 degrees C for 2 hr, and digested with 1 microgram/milliliter proteinase K at 37 degrees C for 20 min. Under this condition, the most intense signals were obtained after hybridization at 37 degrees C overnight. Alternative application of ISH and immunostaining to each of the adjacent sections revealed a small number of cells which were immunonegative but displayed hybridization signals.
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PMID:[Optimal conditions for detection of calcitonin mRNA by in situ hybridization (ISH) method using a non-radioactive probe in the rat thyroid gland]. 780 91

Ischemic tissue necrosis (ITN) has been described as a complication of hyperparathyroidism (HPT) in patients with end-stage renal disease (ESRD) and is associated with a mortality rate of up to 80%. Early recognition of ITN is important but difficult. Optimal treatment is controversial. Based on an analysis of the English literature and a recent clinical experience, a mathematical expression to aid in the identification of high-risk patients (2 x [CaPO(4) - 5] x alkaline phosphatase x PTH ratio) was developed. The values for this expression were calculated in 3 recently reported cases and our case (n = 4). The values were compared with those of a group of 54 hyperparathyroid chronic hemodialysis patients (controls); the mean values were significantly different (p < 0.001). The expression, consisting of 4 easily measured laboratory values, appears to differentiate patients with this complication of ITN from patients with only severe HPT. Ten new additional cases were evaluated using the equation; the sensitivity of the equation was 80% and the specificity 92%, positive predictive value was 66% and the negative predictive value 96%. Long-term validation of this equation is required but it appears to be discriminatory and, thus, promising, given the potentially lethal consequences of ITN.
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PMID:Mathematical formulation to help identify the patient at risk of ischemic tissue necrosis--a potentially lethal complication of chronic renal failure. 814 Nov 79

The expression of alkaline phosphatase (APase) activity by purified B cells from lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice was determined. Optimal APase activity was expressed after costimulation with interleukin-5 and dextran sulfate (DXS), whereas LPS, which is highly effective on B lymphocytes from normal mice, was unable to induce enzyme expression, even in the presence of DXS. The simultaneous determination by flow cytometry of both cellular APase, by using a fluorescent azo dye technique, and DNA content showed that APase was highly expressed by about one-tenth of cells in G1 phase, whereas it was present in more than 50% of cells in S and G2/M phases. The enzyme, as visualized by confocal microscopy after cell sorting on the basis of DNA content, was found to be localized mainly in vesicular structures distributed throughout the cytoplasm in G1 cells. It was distributed in patches and essentially localized at the cell periphery in S cells, whereas clear capping of activity was observed in G2/M cells.
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PMID:Expression and visualization during cell cycle progression of alkaline phosphatase in B lymphocytes from C3H/HeJ mice. 818 40

An alkaline phosphatase was purified from conidia of a Neurospora crassa wild type strain. The M(r) of the purified native enzyme was estimated as ca 145,000 and 110,000 by gel filtration, in the presence and absence of magnesium ions, respectively. A single polypeptide band of M(r) 36,000 was detected by SDS-PAGE, suggesting that the native enzyme was a tetramer of apparently identical subunits. Conidial alkaline phosphatase was an acidic protein (pl = 4.0 +/- 0.1), with 40% carbohydrate content. Optimal pH was affected by substrate concentration and magnesium ions. Low concentrations of calcium ions (0.1 mM) had slight stimulatory effects, but in excess (5 mM) caused protein aggregates with decreased activity. The enzyme specificity against different substrates was compared with those reported for constitutive or Pi-repressible alkaline phosphatases produced by N. crassa. The results suggested that the conidial alkaline phosphatase represented a different class among other such enzymes synthesized by this organism.
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PMID:Conidial alkaline phosphatase from Neurospora crassa. 858 77

An ELISA-based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC-5 cells as per the standard micro-Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID50 of virus grown on MRC-5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC-5 cells in phosphate buffered saline, ph 7.2. The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC-5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37 degrees C for 1-2 h and was unaffected by complement. At 4 degrees C, CMV was inactivated in 1-2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization virus read microscopically correlated with ELISA absorbance of < 0.1. CPE produced by approximately 1 TCID50 of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 (P < 0.001) for 257 human plasma samples and 0.85 (P < 0.001) for 50 immune globulin preparations.
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PMID:An enzyme immunoassay based micro-neutralization test for titration of antibodies to human cytomegalovirus (CMV) and its correlation with direct ELISA measuring CMV IgG antibodies. 873

Human bone marrow contains a population of cells capable of differentiating along multiple mesenchymal cell lineages. Recently, techniques for the purification and culture-expansion of these human marrow-derived Mesenchymal Stem Cells (MSCs) have been developed. The goals of the current study were to establish a reproducible system for the in vitro osteogenic differentiation of human MSCs, and to characterize the effect of changes in the microenvironment upon the process. MSCs derived from 2nd or 3rd passage were cultured for 16 days in various base media containing 1 to 1000 nM dexamethasone (Dex), 0.01 to 4 mM L-ascorbic acid-2-phosphate (AsAP) or 0.25 mM ascorbic acid, and 1 to 10 mM beta-glycerophosphate (beta GP). Optimal osteogenic differentiation, as determined by osteoblastic morphology, expression of alkaline phosphatase (APase), reactivity with anti-osteogenic cell surface monoclonal antibodies, modulation of osteocalcin mRNA production, and the formation of a mineralized extracellular matrix containing hydroxyapatite was achieved with DMEM base medium plus 100 nM Dex, 0.05 mM AsAP, and 10 mM beta GP. The formation of a continuously interconnected network of APase-positive cells and mineralized matrix supports the characterization of this progenitor population as homogeneous. While higher initial seeding densities did not affect cell number of APase activity, significantly more mineral was deposited in these cultures, suggesting that events which occur early in the differentiation process are linked to end-stage phenotypic expression. Furthermore, cultures allowed to concentrate their soluble products in the media produced more mineralized matrix, thereby implying a role for autocrine or paracrine factors synthesized by human MSCs undergoing osteoblastic lineage progression. This culture system is responsive to subtle manipulations including the basal nutrient medium, dose of physiologic supplements, cell seeding density, and volume of tissue culture medium. Cultured human MSCs provide a useful model for evaluating the multiple factors responsible for the step-wise progression of cells from undifferentiated precursors to secretory osteoblasts, and eventually terminally differentiated osteocytes.
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PMID:Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. 902 89

We have developed a serum-free medium for the growth and differentiation of periodontal ligament-derived cells (PLC). In addition, the expression of both fibroblast growth factor (FGF) and FGF receptor (FGFR) in the PLC was investigated by immunohistochemical examination, heparin affinity chromatography (HAC), and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Optimal growth of the cells was achieved in Iscove's modified Dulbecco's medium supplemented with insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, sodium selenite, and oleic acid in type-I collagen-coated dishes. Both FGF-1 and FGF-2 stimulated cell growth and inhibited differentiation as measured by inhibition of alkaline phosphatase activity of the cells. An immunohistochemical analysis of FGF-1 and FGF-2 revealed that immunoreactive FGF-1 and FGF-2 were detected predominantly in the cytoplasm of growing cells. In addition, perinuclear FGF-1 staining and nuclear FGF-2 staining were observed in the same growing cells. In contrast, a faint diffuse staining of FGF-1 and FGF-2 was detected in cytoplasm of the confluent differentiated cells. The 2.15 M NaCl eluate from HAC of the cell extracts exhibited growth-promoting activities for the PLC, and it also stimulated the growth of human umbilical vein-derived endothelial cells and inhibited binding of [125I]-FGF to its receptors, indicating the cells produced FGFs or FGF-like growth factors. RT-PCR analysis revealed that the cells expressed FGFR-1 mRNA but not mRNAs for FGFR-2, FGFR-3 and FGFR-4 mRNA. These results suggest that the FGF-FGFR-1 system plays an important role in the growth and differentiation of periodontal ligament-derived cells.
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PMID:Growth and differentiation of periodontal ligament-derived cells in serum-free defined culture. 915 47


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