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)

Diaphyseal bone from normal Sprague-Dawley rats was delipidated in chloroform-methanol and demineralized in 0.6 N HCl at 4 degrees C. The bones were then implanted for 7-28 days into rats made rachitic by a low-phosphate, vitamin D-deficient diet (VDP-) for 3 weeks. Bones from VDP- and normal rats were also implanted into normal hosts. When normal rats were used as the host environment, a consistent sequence of cartilage induction and bone formation was observed. Demineralized rachitic bone (RB) implanted into normal host rats resulted in cartilage and bone induction similar to that seen for normal bone (NB) implants. Transmission electron microscopy of RB in normal hosts revealed morphologically normal chondrocytes and cartilage matrix with normal mineralization. In contrast, implantation of NB in VDP- hosts resulted in delayed chondrogenesis and lack of calcification. Furthermore, similar results were observed when RB was implanted into VDP- hosts. Treatment of VDP- hosts with either 1 alpha-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 did not accelerate the sequential appearance of precartilage or cartilage. However, 24,25-(OH)2D3 administered alone or in combination with 1 alpha-OHD3 significantly increased the amount of calcified cartilage observed at 2 weeks postimplantation compared to implants from either untreated VDP-hosts or those treated only with 1 alpha-OHD3. New bone formation was observed at 4 weeks postimplantation in all vitamin D-treated groups as determined by von Kossa staining or direct electron microscope examination. There was no apparent difference in the quantitative or qualitative bone formed within the various vitamin D-treated groups. Serum calcium and phosphorus levels were lower and alkaline phosphatase levels were higher in VDP- hosts compared with normal animals or those treated with vitamin D metabolites. The results of this study show a reduction in the capacity of progenitor cells in VDP- rat hosts to respond to osteoinductive factor(s). This impaired response appears to be corrected by vitamin D metabolites.
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PMID:Enhancement of osteoinduction by vitamin D metabolites in rachitic host rats. 144 1

Smokeless tobacco contains a nonnicotine inhibitor of posttranslational modification of collagen (hydroxylation of [3H]proline) by cultured chick embryo tibias and osteoblasts. This study was undertaken to determine whether a methanol extract of smokeless tobacco (STE) containing the inhibitor has similar effects on collagen-producing cells and tissues other than bone. Its effects on DNA synthesis and cell proliferation (incorporation of [3H]thymidine) were also determined. Frontal bone, aorta, and cartilage were incubated for 2 days in medium containing STE. Glycolysis (lactate production) was stimulated by 80% in cartilage, but was not affected in the other tissues; medium alkaline phosphatase activity was unaffected. In frontal bone and cartilage, [3H] hydroxyproline content was decreased 88% and 57%, respectively, and [3H]proline content was decreased 68% and 37%, respectively; neither was affected in the aorta. Confluent cultures of collagen-producing mouse fibroblasts or primary osteoblasts obtained from chick embryo calvarias were incubated for 2 days in medium containing increasing concentrations of STE. Glycolysis and DNA synthesis were not affected. Cell proliferation was unaffected in fibroblasts, but was inhibited (34%) at the highest STE concentration in osteoblasts. AIPase activity was not detectable in fibroblast medium, but was decreased up to 72% in osteoblast medium. Inhibition of collagen synthesis by STE was concentration related in both cell types. At the highest concentration, [3H] hydroxyproline and [3H]proline contents in the cell layers were decreased to the following respective values: fibroblasts 56% and 45% and osteoblasts 50% and 29%, respectively. When incubation with STE was discontinued for 1 day, recovery did not occur. These findings suggest that inhibition of collagen synthesis by STE is not specific for bone, that collagen-producing cells are directly affected, and that recovery is not immediate. This inhibitor could contribute to the periodontal disease often seen in users of smokeless tobacco. Its identification and removal would produce a safer product.
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PMID:Inhibition of cell metabolism by a smokeless tobacco extract: tissue and species specificity. 174 13

Problems encountered in attempts to purify mevalonate-5-diphosphate decarboxylase from rat liver are addressed. These are the quantitative, facile separation of [14C]isopentenol in the radiochemical assay (2) the instability of the enzyme activity and (3) the very low activity in rat liver. The assay was modified by using Sep Pac C18 filters to bind and release [14C]isopentenol. Authentic isopentenol was quantitated by absorbance at 210 nm wavelength and the extinction coefficient estimated to be epsilon m = 3.26 X 10(3). Recovery of authentic isopentenol from aqueous solution after binding and elution into methanol was quantitative from 10-100 nmols. Recovery of [14C]ispentenol from assay mixtures using 2-[14C]mevalonate-5-diphosphate and alkaline phosphatase to hydrolyze phosphate was quantitative using Sep Pac filter but not using petroleum ether extraction. Enzyme activity was stabilized by phenylmethylsulfonyl fluoride, aprotinin and leupeptin and was stable at -73 degrees C for 3 months. Activity of the decarboxylase was increased by 5-fold after feeding young rats 2.5% cholestyramine for ten days to four weeks.
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PMID:Modification of the radiochemical assay of rat liver mevalonate-5-diphosphate decarboxylase and induction and stabilization of the activity. 176 97

1. The presence of high-Mr and low-Mr acid phosphatases [orthophosphoric-monoester phosphohydrolase, (acid optimum), EC 3.1.3.2] in the skeletal muscle of frog Rana esculenta was reported. 2. The subcellular localization and some characteristics of both enzymes were also described. 3. The low-Mr AcPase was purified to homogeneity. The enzyme did not absorb on Concanavalin A-Sepharose 4B indicating that this was not a glycoprotein. 4. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr 13.7 +/- 0.8 kDa in the presence of sodium dodecyl sulphate. 5. The Mr of the native enzyme was 14.0 +/- 1.1 kDa as determined by gel filtration on a Sephadex G-100 column. The isoelectric point was 6.02. 6. The enzyme was strongly inhibited by 1 mM Ag+, Hg2+, Sn2+ and Cu2+ while other cations both at 10(-2) and 10(-3) M showed little or no effect. 7. The enzyme was insensitive to NaF and tartrate but was strongly deactivated by formaldehyde, PMB, Iodoacetamide and Triton X-100. Phosphate was a competitive inhibitor (k1 = 0.83 mM). 8. The best substrate for the enzyme was p-nitrophenylphosphate but phenylphosphate, flavin mononucleotide and o-P-tyrosine were also hydrolyzed, though at different rates. 9. The enzyme activity was enhanced in the presence of methanol, ethanol, acetone and glycerol indicating a phosphotransferase activity.
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PMID:Acid phosphatases in the frog (Rana esculenta) skeletal muscle. Purification and some properties of the low molecular weight enzyme. 178 53

Ochratoxin A, produced by a number of fungal species, has been found in many milieu, including porcine sera and coffee beans. It was therefore analysed by enzyme-linked immunosorbent assay (ELISA) in porcine sera, coffee products and fungal cultures, using monoclonal antibodies, a monoclonal antibody-linked immunoaffinity column (IAC) and high-performance liquid chromatography (HPLC). The chloroform extracts of acidified porcine sera were assayed directly by ELISA, with alkaline phosphatase and horseradish peroxidase as marker enzymes, at detection limits of 0.1 and 0.01 ng/ml, respectively. The presence of ochratoxin A in ELISA was confirmed by HPLC. The average contents in the five different lots tested were: 0.4 ng/ml in lot A (19 samples), 0.36 ng/ml in lot B (104 samples), 5.20 ng/ml in lot C (17 samples), 1.24 ng/ml in lot D (23 samples) and 0.22 ng/ml in lot E (24 samples). ELISA of methanol extracts of rice cultures showed the presence of more than 0.1 ng/g in 3 of 15 isolates of Aspergillus, in 16 of 67 isolates of Penicillum and in 7 of 17 isolates of Eupenicillum; none was found in an isolate of Emericella. IAC-HPLC analysis revealed that P. foetidus, which is similar to A. niger and is used for the production of a Japanese alcoholic drink (shou-chuu), also produced ochratoxin A. Use of IAC-HPLC to analyse coffee beans and instant coffee power resulted in the sharp resolution of ochratoxin A without complicated clean-up steps. The IAP-HPLC technique could thus be used for mass surveys of ochratoxin A residues in biological specimens.
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PMID:Use of monoclonal antibodies, enzyme-linked immunosorbent assay and immunoaffinity column chromatography to determine ochratoxin A in porcine sera, coffee products and toxin-producing fungi. 182 Mar 56

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.
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PMID:The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes. 184 39

Cryostat-sections of biopsies from HIV-infected patients or HIV/SIV-infected experimental animals pose a biohazard risk to laboratory workers. The objective of this study was to select a procedure that appropriately fixes cryo-sections and reduces the risk of HIV-1 infectivity. This inactivation procedure should preserve antigen binding capacity of host-produced antibodies and the antigenic structure of epitopes present in these tissues, while retaining sufficient morphologic detail. We tested the effect of seven different established fixation-inactivation procedures for HIV-1 on the detection of specific antibodies and membrane markers, compared to acetone fixation as a reference. Frozen sections of spleens from mice immunized with trinitrophenyl (TNP)-Ficoll were incubated with TNP-alkaline phosphatase to detect specific antibody-forming cells and follicular immune complexes containing TNP-specific antibodies. In addition, sections were stained with monoclonal antibodies directed against IgM (187-1), T-cells (anti Thy-1), and marginal metallophilic macrophages (MOMA-1). Five procedures proved useful as they gave results similar to regular acetone fixation. In contrast, two procedures with a methanol-containing fixative obscured both antigen binding sites and membrane antigens. Subsequently, these five selected procedures were tested on glass slide preparations of HIV-1 infected cell lines, expressing HIV-1 determinants defined by monoclonal antibodies. Finally, the procedures were tested on sections of an HIV-1 infected human lymph node, for detection of HIV-specific B-cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fixation of cryo-sections under HIV-1 inactivating conditions: integrity of antigen binding sites and cell surface antigens. 191 74

Increasing interest in receptor-regulated phospholipase C and phospholipase D hydrolysis of cellular phosphatidylcholine motivates the development of a sensitive and simple assay for the water-soluble hydrolytic products of these reactions, phosphocholine and choline respectively. Choline was partially purified from the methanol/water upper phase of a Bligh & Dyer extract by ion-pair extraction using sodium tetraphenylboron, and the mass of choline was determined by a radioenzymic assay using choline kinase and [32P]ATP. After removal of choline from the upper phase, the mass of residual phosphocholine was determined by converting it into choline by using alkaline phosphatase, followed by radioactive phosphorylation. In addition to excellent sensitivity (5 pmol for choline and 10 pmol for phosphocholine), these assays demonstrated little mutual interference (phosphocholine----choline = 0%; choline----phosphocholine = 5%), were extremely reproducible (average S.E.M. of 3.5% for choline and 2.9% for phosphocholine), and were simple to perform with instrumentation typically available in most laboratories. In addition, the ability to apply the extraction technique to the upper phase of Bligh & Dyer extracts permitted simple analysis not only of choline and phosphocholine, but also of phosphatidylcholine and lipid products of phospholipase C and phospholipase D activity (1,2-diacylglycerol and phosphatidic acid respectively) from the same cell or tissue sample.
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PMID:Isolation and enzymic assay of choline and phosphocholine present in cell extracts with picomole sensitivity. 211 61

In situ hybridization with non radioactive probes is more and more used to detect viral infections, especially human papillomavirus (HPV) infections. The quality of the reaction depends on several factors, such as sample preparation (including fixation and pretreatments). Their importance was evaluated on a model with cell lines including CaSki cells (harboring about 600 copies of HPV 16 DNA per cell) and Hela cells (containing 10-50 copies of HPV 18 DNA per cell). These cell lines were chosen in order to evaluate cytological and histological difficulties. Several parameters were studied: preparation of samples, fixation and hybridization duration. DNAs of biotinylated probes of HPV types 16 and 18 and cells were simultaneously denatured 10 min at 95 degrees C. Hybridization was carried out at 37 degrees C for various periods of time; it was followed by a 3-step reaction for detection of biotinylated DNA-DNA hybrids with immunoenzymatic staining using streptavidin-alkaline phosphatase complex. Typical intranuclear granulations were seen either in cell deposits fixed with acetone, methanol-acetic acid, paraformaldehyde, formaline, Bouin's, Baker's or Carnoy's fixatives; or in cytocentrifuged cells fixed with formaline, Bouin's or Baker's fixatives. The detergent pretreatment was unnecessary. On the contrary, the protease pretreatment was required with formaline, Bouin's or Baker's fixatives. In order to detect constantly HPV 16 in CaSki cells and HPV 18 in HeLa cells, hybridization should be performed for more than 4 h. The sensitivity of the technique could therefore be evaluated to few copies of HPV DNA per cell. This technique is reliable, sensitive and rapid; et can be applied to biopsy specimens fixed with Bouin's or Baker's fixatives and paraffin-embedded; it allows routine detection of HPV infections.
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PMID:[Human papillomavirus typing by molecular hybridization in situ with biotinylated probes. Optimization of sample preparation and hybridization time]. 216

Myeloma plasma cells were double stained using peroxidase and alkaline phosphatase labelled monoclonal anti-BrdU and anti-intracytoplasmic immunoglobulins. Samples were methanol fixed; DNA was denatured with formamide. The results allowed easy identification of plasma cells, their cytological examination and the calculation of percentage of plasma cells in S phase. Good correlation was found with the labelling index obtained with tritiated thymidine.
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PMID:Determination of plasma cell labelling index with bromodeoxyuridine using a double immunoenzymatic technique. 219 27


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