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)

In situ hybridization techniques have rapidly become widely used by the molecular biologist for the localization of specific nucleic acid sequences in individual cells or tissues. We describe the demonstration of Sox gene mRNA in chick tissue that has been embedded in the plastic methyl methacrylate to permit the preparation of sections for high-resolution light microscopy. Polymerization of the plastic was induced by using either N,N-dimethylaniline or N,N-3,5-tetramethylaniline. The in situ hybridization technique used was non-isotopic and used a digoxigenin-labelled probe detected with an antibody bound to alkaline phosphatase, which was then localized using X-phosphate-Nitro BT as a substrate-chromogen mix. Various pretreatments of the tissue sections were investigated, including the use of proteinase K, and heat-mediated techniques using a microwave oven and a pressure cooker. The best results were produced using pressure cooking on tissue in which the plastic had been chemically polymerized with N,N-3,5-tetramethylaniline. For the demonstration of Sox 11, this combination had a critical influence on the staining results, but for Sox 21 all protocols used produced good staining.
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PMID:Non-isotopic in situ hybridization to detect chick Sox gene mRNA in plastic-embedded tissue. 934 59

One hundred paraffin-embedded cervical biopsy specimens were tested for the presence of human papilloma virus (HPV) by in situ hybridization (ISH), and by direct and indirect in situ PCR (IS-PCR) in order to evaluate the efficiency of the different in situ methods in detecting HPV infection. ISH was performed using either commercial DNA probes or a cocktail of 5'-digoxigenin labeled oligoprimers. The same were used for ISH during indirect IS-PCR. To enhance the sensitivity of ISH several polymers, i.e., polyvinyl alcohol (PVA), polyethylene glycol, and polyvinylpyrrolidone were added to the alkaline phosphatase nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) reaction. Furthermore, tyramide signal amplification (TSA) was tried for signal amplification. Those samples treated with PVA during the NBT/BCIP reaction did not show any signal amplification whereas those treated with TSA exhibited a dramatic increase in sensitivity with usually acceptable signal to noise ratios. Our results show that, regarding sensitivity, ISH with subsequent signal amplification by TSA can be used as an almost equivalent alternative to the more cumbersome IS-PCR on routinely processed tissue specimens. When considering reproducibility, it is superior to IS-PCR.
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PMID:Comparison of in-situ hybridization, direct and indirect in-situ PCR as well as tyramide signal amplification for the detection of HPV. 1009 May 69

GM-CSF is a hematopoietic growth factor. In vitro it stimulates the proliferation of myeloid progenitors and formation of granulocyte and macrophage colonies. It was found that GM-CSF in vitro is also stimulated the function of mature granulocytes, but we have no information about such influence in vivo. The purpose of this investigation was the evaluation in vivo of the GM-CSF effect on phagocytosis, bactericidal activity, and lysosome enzyme activities in granulocytes. GM-CSF was injected into mice subcutaneously during 5 consecutive days in the dose of 1 microgram/kg/d. The examination of the percent of cell phagocytizing bacteria (Staphylococcus aureus), NBT test, bactericidal activity and activation of acid phosphatase, alkaline phosphatase, peroxidase and esterase was performed every day and an evident increase of the tested parameters was found. These results prove in vivo activation of granulocytes by GM-CSF.
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PMID:[Phagocytosis and killing of Staphylococcus aureus by granulocytes in mice after granulocyte-macrophage colony stimulating factor (GM-CSF) injection]. 1022 30

Acute interdigital phlegmon (AIP) is a commonly occurring anaerobic bacterial infection in cattle. This study examined in vitro the interaction of bovine polymorphonuclear granulocytic neutrophils (PMN) from blood with bacterial species involved in AIP. Polymorphonuclear neutrophils were purified from whole bovine blood, exposed to one of the three putative etiologic agents of AIP and comparatively assessed for phagocytosis using light microscopy. Fusobacterium necrophorum and Prevotella intermedia were effectively phagocytosed by PMN, but Porphyromonas levii was phagocytosed significantly less effectively by PMN. The effect of high titre anti-P. levii bovine serum on antibody-mediated phagocytosis by PMN was also evaluated. High titre serum increased the efficiency of phagocytosis of P. levii by bovine PMN. This was independent of heat labile complement factors. Antibodies specific for P. levii were assessed for protease activity capable of cleaving bovine immunoglobulins (IgG, IgG1, IgG2, and IgM). Partially purified supernatant from broth cultures of P. levii were incubated with biotinylated immunoglobulins (Igs). Samples were taken from times 0 to 72 h and examined using SDS-PAGE followed by Western blot analysis. Streptavidin-alkaline phosphatase and NBT-BCIP were used to visualize the Igs for heavy and light chains as well as lower molecular weight fragments of these glycoproteins. Porphyromonas levii produced an immunoglobulin protease which readily cleaved bovine IgG into fragments, but did not act against IgM. Specifically, the enzyme may be a significant virulence factor as it may act to neutralize the antibodies demonstrated necessary for effective PMN-mediated phagocytosis.
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PMID:Bovine polymorphonuclear neutrophil-mediated phagocytosis and an immunoglobulin G2 protease produced by Porphyromonas levii. 1036 68

We describe a high-resolution, fluorescence-based method for localizing endogenous alkaline phosphatase in tissues and cultured cells. This method utilizes ELF (Enzyme-Labeled Fluorescence)-97 phosphate, which yields an intensely fluorescent yellow-green precipitate at the site of enzymatic activity. We compared zebrafish intestine, ovary, and kidney cryosections stained for endogenous alkaline phosphatase using four histochemical techniques: ELF-97 phosphate, Gomori method, BCIP/NBT, and naphthol AS-MX phosphate coupled with Fast Blue BB (colored) and Fast Red TR (fluorescent) diazonium salts. Each method localized endogenous alkaline phosphatase to the same specific sample regions. However, we found that sections labeled using ELF-97 phosphate exhibited significantly better resolution than the other samples. The enzymatic product remained highly localized to the site of enzymatic activity, whereas signals generated using the other methods diffused. We found that the ELF-97 precipitate was more photostable than the Fast Red TR azo dye adduct. Using ELF-97 phosphate in cultured cells, we detected an intracellular activity that was only weakly labeled with the other methods, but co-localized with an antibody against alkaline phosphatase, suggesting that the ELF-97 phosphate provided greater sensitivity. Finally, we found that detecting endogenous alkaline phosphatase with ELF-97 phosphate was compatible with the use of antibodies and lectins. (J Histochem Cytochem 47:1443-1455, 1999)
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PMID:A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity. 1054 17

A multiplex PCR was employed to amplify unique conserved sequences of DNA from the pathogens Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae from cerebrospinal fluid samples of patients suffering from acute pyogenic meningitis. The accurate identification of the PCR amplified product was achieved by hybridizing dot-blots of the PCR products to probes which were specific, biotinylated internal sequences of the amplified target DNA. Detection of the hybrids was done in a colour reaction using streptavidin-alkaline phosphatase conjugate and BCIP/NBT substrates. The entire protocol took only 7 h for the correct identification of the pathogen present in clinical samples of cerebrospinal fluid. The sensitivity and specificity were >95%.
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PMID:Rapid diagnosis of acute pyogenic meningitis by a combined PCR dot-blot assay. 1079 66

Stem Cell Factor (SCF) can stimulate the growth and development of primitive multipotential and unipotential hematopoietic stem cells, either alone or in combination with other cytokines such as Granulocyte-Macrophage Colony Stimulating factor (GM-CSF) and Granulocyte-CSF (G-CSF). It was found that these cytokines can also stimulate the function of granulocytes but there is no information concerning SCF influence on the function of these mature cells. SCF was injected into mice subcutaneously during 5 consecutive days in a dose of 1 microgram/kg/d. An examination of the percentage of phagocytic granulocytes and NBT test was performed. The activity of acid phosphatase (AcP), alkaline phosphatase (AP), peroxidase (MPO) and esterase were determined by cytochemistry methods. On the basis of obtained results we can conclude that SCF evidently increases all tested parameters connected with the metabolism of phagocytosis.
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PMID:[Phagocytosis and oxidative metabolism of granulocytes in mice after stem cell factor (SCF) injection]. 1086 43

The investigations covered a group of 20 patients with uterine myomas aged 38 to 57 years. The control group consisted of 20 healthy women. The following indices were used in the assessment of non-specific immunity: 1) determination of the total leukocyte number and neutrophil number in peripheral blood, 2) the investigation of NBT reduction by neutrophils, 3) the evaluation of myeloperoxidase activity in neutrophils. 4) the assessment of alkaline phosphatase activity in neutrophils, 5) the evaluation of the neutrophil adherence ability, 6) determination of phagocytic activity of neutrophils, 7) determination of lysozyme activity in serum, 8) the evaluation of concentration of immune complexes in serum. In the group of patients with myomas the following indices were found as a statistically significant: a) the increase of leukocyte and neutrophil number b) the increase of the index of stimulated reduction of NBT, c) the decrease of myeloperoxidase activity, d) the increase of activity of alkaline phosphatase in neutrophils, e) the increase of the index of adherence, f) the decrease of the phagocytic activity.
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PMID:[Immunological disorders in patients with uterine myomas]. 1098 81

This report demonstrates the stability of NBT substrate after multiple exposures to alkaline phosphatase. Perhaps more important than the ability to reuse substrates, the report provides some insight into the mechanisms by which tetrazoliums are reduced and evidence for the formation of an intermediary product, i.e., a half-formazan that is reduced more rapidly. (J Histochem Cytochem 49:1189-1190, 2001)
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PMID:Stability of nitroblue tetrazolium-based alkaline phosphatase substrates. 1151 89

Electrospray (ES) deposition has been applied to fabricate protein microarrays for immunochemical assay. Protein antigens were deposited as arrays of dry spots on a surface of aluminized plastic. Deposition was performed from water solutions containing a 10-fold (w/w of dry protein) excess of sucrose. Upon contact with humid air, the spots turn into microdroplets of sucrose/protein solution from which proteins were either adsorbed or covalently linked to clean or modified aluminum surfaces. It was found that covalent binding of antigens via aldehyde groups of oxidized branched dextran followed by reduction of the Schiff bonds gives the highest sensitivity and the lowest background in microarray-based ELISA, as compared to other tested methods of antigen immobilization. The minimum concentration of a primary mouse antibody detected in indirect ELISA with such antigen microarrays was approximately 0.3-1.0 ng/mL for ELF-97 or BCIP/NBT substrates of alkaline phosphatase.
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PMID:Immobilization of proteins in immunochemical microarrays fabricated by electrospray deposition. 1179 78


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