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 technique was developed for visualizing virus particles electrophoretically transferred from infected leaves to nitrocellulose membranes. This technique was used to study the distribution of peanut mottle virus (PMV) particles in vivo. The immobilized transferable virus proteins from leaf tissue to nitrocellulose membranes were detected by an enzyme-linked immunobinding technique. The free binding groups on the nitrocellulose membrane were blocked with casein. The nitrocellulose membrane was then treated with PMV-specific antiserum. The virus-bound antibodies were located by protein A-peroxidase or protein A-alkaline phosphatase conjugate followed by the peroxidase substrate mixture, 4-chloro-1-naphthol and H2O2, in the first case or the alkaline phosphatase substrate mixture, 5-bromo-4-chloro-3-indolyl phosphate and Nitroblue tetrazolium in the second case. The locations of virus particles were detected by the corresponding reaction product. A mirror image was obtained on nitrocellulose membrane showing a pattern identical to that of necrotic lesions on leaf pieces but with a significantly larger size of local lesion copy, indicating the presence of virus particles in the apparently healthy tissue outside the necrotic lesion. The new technique is expected, because of its relative simplicity and sensitivity, to be useful for many other fields of biological research where the in vivo distribution of pathogens, antigenic molecules including drugs, or cells of specific antigenic molecules such as cancer or genetically engineered cells needs to be investigated.
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PMID:Mirror image in vivo electroblotting technique, a new technique for visualizing virus particles electrophoretically transferred from infected leaves to nitrocellulose membranes. 798 32

Two rotating bioreactors in tandem have been incorporated into a continuous-flow/stopped-flow sample/reagent processing setup for the determination of alkaline phosphatase (EC3.1.3.1) activity in serum samples. The strategy circumvents incompatibility of buffer systems as well as that of the immobilized enzymes utilized in the bioreactors (alkaline phosphatase and alcohol oxidase, EC 1.1.3.13). The determination is indirect in nature although recorded responses are directly related to the enzyme activity in the sample. It couples the following enzyme-catalyzed reactions: (1) hydrolysis of p-nitrophenyl dihydrogen phosphate catalyzed by alkaline phosphatase, (2) enzymatic reaction between unreacted p-nitrophenyl dihydrogen phosphate with methanol, and (3) conversion of the residual methanol to the corresponding aldehyde and H2O2, catalyzed by alcohol oxidase. The H2O2 is amperometrically determined at a stationary Pt-ring electrode (applied potential + 0.600 V vs a Ag/AgCl, 3.0 M NaCl reference).
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PMID:Continuous-flow/stopped-flow system incorporating two rotating bioreactors in tandem: application to the determination of alkaline phosphatase activity in serum. 801 34

For brightfield detection of two different DNA target sequences in one sample, we developed a double-target in situ hybridization (ISH) technique, using biotin- and digoxigenin-labeled chromosome-specific DNA probes. First, several immunochemical detection systems were optimized and compared for sensitivity and simultaneous applicability. Two non-interfering immunochemical systems were chosen for simultaneous detection of the DNA probe labels. This resulted in combination of an alkaline phosphatase (AP)-conjugated avidin-biotin system with a horseradish peroxidase (HRP)-conjugated antibody system for detection of biotin- and digoxigenin-labeled DNA probes, respectively. Development of AP with New Fuchsin-naphthol phosphate and HRP with diaminobenzidine-H2O2 resulted in stable, well-contrasting (red and black, respectively) color precipitates visible by conventional light microscopy. The double-target ISH technique was successfully applied on a wide variety of biological materials, such as metaphase spreads, cytospin, and Thin-prep samples of cytological specimens, frozen tissue sections, and formalin-fixed, paraffin-embedded tissue sections. In particular, on tissue sections, where quantitative interpretation of ISH data can be hampered by truncation of nuclei, the double-target ISH technique appeared to be a valuable tool for demonstration of chromosome aberrations and chromosome imbalances.
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PMID:Double-target in situ hybridization in brightfield microscopy. 802 26

Studies assessing mechanisms of proximal tubular cell (PTC) physiology and pathophysiology increasingly utilize cell culture systems to avoid the complexity of whole organ/whole animal experiments. However, no well-differentiated PTC line derived from adult human kidney currently exists. Therefore, the goal of this research was to establish such a line by transduction with human papilloma virus (HPV 16) E6/E7 genes. A primary PTC culture from normal adult human renal cortex was exposed to a recombinant retrovirus containing the HPV 16 E6/E7 genes, resulting in a cell line designated HK-2 (human kidney-2) which has grown continuously in serum free media for more than one year. HK-2 cell growth is epidermal growth factor dependent and the cells retain a phenotype indicative of well-differentiated PTCs (positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3 beta 1 integrin, fibronectin; negative for factor VIII-related antigen, 6.19 antigen and CALLA endopeptidase). Furthermore, HK-2 cells retain functional characteristics of proximal tubular epithelium (Na+ dependent/phlorizin sensitive sugar transport; adenylate cyclase responsiveness to parathyroid, but not to antidiuretic, hormone). The E6/E7 genes are present in the HK-2 genome, as determined by PCR. To assess its potential usefulness as a tool for studying injury and repair, HK-2 cells were exposed to a toxic concentration of H2O2 +/- iron chelation (deferoxamine) or hydroxyl radical scavenger (Na benzoate) therapy. Only the former blocked H2O2 cytotoxicity, reproducing results previously obtained with freshly isolated rat proximal tubular segments. In conclusion, an immortalized adult human PTC line has been established by transduction with HPV 16 E6/E7 genes. It appears to be well-differentiated on the basis of its histochemical, immune cytochemical, and functional characteristics, and it can reproduce experimental results obtained with freshly isolated PTCs. Thus, this new PTC line could have substantial research application.
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PMID:HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. 812 21

Ki-67 nuclear antigen is expressed in upper epithelial levels of intraepithelial neoplasia of the cervix and vulva, variably in condyloma, and in basal and parabasal cells of normal squamous mucosa in histologic preparations. The application of antibodies to Ki-67 as a marker of squamous intraepithelial lesions in cervical smears was explored using either air-dried, acetone-fixed cervical smears obtained from 106 consenting patients or a single slide from archival two-slide cases of squamous intraepithelial lesions MIB-1 monoclonal antibody to Ki-67 was tested using two immunocytochemical techniques. In one set of smears, avidin-biotin peroxidase was used for detection and diaminobenzidine with H2O2 as the chromogen. Some specimens were incubated with 0.3% H2O2 and phosphate buffered saline for blockade of endogenous peroxidase. Alternatively, other air-dried smears were stained using alkaline phosphatase antialkaline phosphatase for detection and new fuchsin as the chromogen. Nuclear staining in squamous intraepithelial lesions was identified in air-dried smears using all of the above methods. Slides stained with avidin-biotin peroxidase and blocked with 0.3% H2O2 and phosphate buffered saline showed less background staining from neutrophils and erythrocytes compared with those without blocking. Slides stained using alkaline phosphatase antialkaline phosphatase showed excessive cytoplasmic staining of endocervical cells, making intraepithelial difficult. No nuclear staining of squamous intraepithelial lesions was observed in destained archival smears. Air-dried smears blocked with 0.3% H2O2 and phosphate buffered saline, incubated with MIB-1, and stained using avidin-biotin peroxidase gave the best results for identification of Ki-67 expression in squamous intraepithelial lesions.
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PMID:Protocol for immunocytochemical detection of SIL in cervical smears using MIB-1 antibody to Ki-67 [corrected]. 872 81

A mouse embryo culture model was used to determine whether embryonic prostaglandin H synthase (PHS)-catalyzed bioactivation and resultant oxidative damage to embryonic protein and DNA may constitute a molecular mechanism mediating phenytoin and benzo[a]pyrene teratogenesis. Embryos were explanted from CD-1 mouse dams on gestational day 9.5 (vaginal plug = day 1) and incubated for either 4 h (biochemistry) or 24 h (embryotoxicity) at 37 degrees C in medium containing either phenytoin (20 micrograms/ml, 80 microM), benzo[a]pyrene (10 microM), or their respective vehicles. As previously observed with phenytoin (Mol. Pharmacol.48: 112-120, 1995), embryos incubated with benzo[a]pyrene showed decreases in anterior neuropore closure, turning, yolk sac diameter, and somite development (p < .05). Addition of the antioxidative enzyme superoxide dismutase (SOD) substantially enhanced embryonic SOD activity (p < .05) and completely inhibited benzo[a]pyrene embryotoxicity (p < .05). Substantial PHS was detected in day 9.5 embryos using SDS/PAGE, anti-PHS antibody, and alkaline phosphatase-conjugated donkey anti-goat IgG. Embryonic protein oxidation was detected by the reaction of 0.5 mM 2,4-dinitrophenylhydrazine with protein carbonyl groups. This method was first validated by using a known hydroxyl radical-generating system consisting of vanadyl sulfate and H2O2, with bovine serum albumin or embryonic protein as the target. Embryonic proteins were characterized by SDS/PAGE, anti-dinitrophenyl antisera, and peroxidase-labeled goat anti-donkey IgG. Using enhanced chemiluminescence, the number and content of oxidized protein bands detected between 25 and 200 kDa were substantially increased by both phenytoin and benzo[a]pyrene. Addition of the reducing agent dithiothreitol, or SOD or catalase, decreased protein oxidation in phenytoin-exposed embryos. Both phenytoin (Mol. Pharmacol.48: 112-120, 1995) and benzo[a]pyrene enhanced embryonic DNA oxidation, determined by the formation of 8-hydroxy-2'-deoxyguanosine, as measured by high-performance liquid chromatography (HPLC) (p < .05). Phenytoin also enhanced the oxidation of embryonic glutathione (GSH) to its GSSG disulfide, as measured by HPLC (p < .05). These results provide direct evidence that, in the absence of maternal or placental processes, embryonic PHS-catalyzed bioactivation and reactive oxygen species-mediated oxidation of embryonic protein, thiols, and DNA may constitute a molecular mechanism mediating phenytoin and benzo[a]pyrene teratogenesis.
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PMID:Evidence for embryonic prostaglandin H synthase-catalyzed bioactivation and reactive oxygen species-mediated oxidation of cellular macromolecules in phenytoin and benzo[a]pyrene teratogenesis. 901 24

Polymorphonuclear cells kill microorganisms by the stock of antibiotic proteins and peptides stored in their lysosomal granules and have the ability to produce reactive oxygen intermediates (ROI) such as H2O2, O2-, and HOCl. Since the components involved in the microbicidal functions of buffalo (Bos bubalis) polymorphonuclear cells (PMN) have not been characterized, an assessment was made of the levels of various enzymes, the extent of extracellular release of these enzymes, and also their ability to produce H2O2/O2- upon activation with opsonized zymosan (OZ) or lipopolysaccharide (LPS). Using GPC-HPLC, OZ was shown to be a more potent secretagogue than LPS, causing a significantly greater release of low-molecular-weight components. Varying levels of the enzymes (myeloperoxidase, lactate dehydrogenase, acid and alkaline phosphatases, beta-galactosidase, beta-D-glucuronidase, elastase and lysozyme) were recorded in the buffalo PMN and both the activators (OZ and LPS) caused significant release of all the enzymes except alkaline phosphatase. Both the activators also caused a significant increase in H2O2/O2- production by the PMN. However, OZ caused a more pronounced activation than LPS. The studies revealed the presence of oxygen-dependent and oxygen-independent microbicidal systems with buffalo PMN, which responded more effectively to zymosan activation.
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PMID:The effect of activation of granulocytes on enzyme release and hydrogen peroxide and superoxide production in buffaloes. 915 8

This study investigates the efficacy of using hydrogen peroxide as adjuvant therapy after extended local curettage for benign giant cell tumors of bone. Hydrogen peroxide is used clinically as a chemical adjuvant for removal of residual tumor cells, presumably by effervescent cleansing with minimal damage to surrounding soft tissue and bone cells. This investigation examined the effects of hydrogen peroxide on giant cell tumor cells and osteoblasts grown in culture. Fresh fragments of histologically confirmed giant cell tumor tissue (six patients) and trabecular bone (one patient) were excised. Cells obtained from the fragments were grown in culture. Confluent cell cultures were exposed to saline (control) or hydrogen peroxide (0.1-1000 mm) for 2 minutes, and incubation was continued for 12, 24, or 48 hours without hydrogen peroxide. Protein content, deoxyribonucleic acid content, tartrate resistant acid phosphatase activity, and alkaline phosphatase activity were measured in the cell layers. The medium from the final 12 hours of each incubation period was used to evaluate lactate production. Cell lysis or death occurred after exposing giant cell tumor cells and osteoblasts to 100 mm and 30 mm hydrogen peroxide, respectively, concentrations substantially lower than the 3% (880 mm) hydrogen peroxide commonly used clinically. These results support the theory of using a minimal concentration of hydrogen peroxide as a chemical adjuvant in the surgical treatment of giant cell tumors of bone.
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PMID:Hydrogen peroxide inhibits giant cell tumor and osteoblast metabolism in vitro. 952 Aug 98

The promutagenic base 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in DNA is known to be formed from oxygen radical attack on 2'-deoxyguanosine (dG) as a result of oxidative stress. Formation of 8-OH-dG from dG during workup is strongly dependent on temperature and transition metals and is mediated by oxygen radicals. The 8-OH-dG formation at temperatures between 0 and 140 degrees C for 1.5 h in an "ultrapure" solution followed a third-order equation. Fe2+ in the nM range mediated the formation of 8-OH-dG from dG without addition of H2O2. Fe3+, Cu+, and Cu2+ were shown to have weaker oxidative effects in comparison to Fe2+. The pH (5.0-9.0) had a very limited effect on 8-OH-dG formation. Acid phosphatase, which contains iron at its active site, caused the formation of 8-OH-dG, whereas alkaline phosphatase did not. Phenol was not found to be oxidative. Fe2+-catalyzed formation of 8-OH-dG was completely blocked by the nitroxide 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), whereas DMSO, mannitol, and DMPO had a significantly weaker protecting effect. Catalase cleaved the dG molecule and was not suitable for use. A simple, fast, and inexpensive method for 8-OH-dG workup and analysis was developed, and the background level seen in liver from 13-week-old male Sprague-Dawley rat was 0.23 +/- 0.020 8-OH-dG/10(5) dG, which is up to 200 times lower than reported values from some other methods and up to 26 times lower when compared to other reports using HPLC-EC methods. In summary, the TEMPO method reduces oxidation of dG to 8-OH-dG during workup by (1) using chemicals low in transition metals, (2) using a cold workup procedure, (3) limiting the incubation time, and (4) using the nitroxide TEMPO in all steps.
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PMID:Reduction of oxidation during the preparation of DNA and analysis of 8-hydroxy-2'-deoxyguanosine. 970 49

The enzyme-linked immunospot (ELISPOT) assay is an efficient technique for the enumeration of single cells secreting antibodies and cytokines. For simultaneous differentiation of individual cells producing interleukin-2 (IL-2) and/or interleukin-4 (IL-4) at a single cell level, a dual color ELISPOT assay has been developed. In the present system, the red spots corresponding to IL-2-secreting cells (T helper type 1, Th1 cells) were developed with a horseradish peroxidase and the amino ethyl carbazole (AEC)/H2O2. The light blue spots corresponding to IL-4-secreting cells (T helper type 2, Th2 cells) were developed with an alkaline phosphatase and the Vector blue. The mixed colored (indigo) spots corresponding to both kinds of cytokine-secreting cells (T helper type 0, Th0 cells) were developed with both substrates. With this system, we could detect the IL-2- and/or IL-4-secreting cells simultaneously in crude spleen cell preparation or purified CD4 fraction. This procedure provides a useful tool for quantitatively analyzing micro-levels of dynamic immune responses.
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PMID:Development of a dual color enzyme-linked immunospot assay for simultaneous detection of murine T helper type 1- and T helper type 2-cells. 971 57


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