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 simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa x skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precipitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce the IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs gamma radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use.
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PMID:A simplified and rapid staining method for the HeLa x skin fibroblast human hybrid cell neoplastic transformation assay. 127 39

We explored the possibility of simultaneous application of histochemical and immunohistochemical staining techniques on the same paraffin-embedded human tissue section. Conventional histological stains (PAS, Alcian, Alcian-PAS, Van Gieson, Gomori silver impregnation, and Giemsa) were used in association with a battery of markers (keratins, leucocyte common antigen, S-100 protein, Factor VIII-related antigen) that are widely employed in diagnostic and experimental studies. We found that the best procedure was to perform immunostaining before the histochemical reaction, as this enables all the other possible combinations to be carried out. In addition, several detection systems, such as peroxidase-anti-peroxidase (PAP), alkaline phosphatase-anti-alkaline phosphatase (APAAP), and avidin-biotin complex (ABC), were tested and all gave consistent results. Some minor modifications of the histological staining methods were necessary, but the current immunohistochemical techniques could be used as established. Preliminary findings indicate that immunohistochemistry can be combined with histochemistry techniques by means of a relatively simple procedure whose only disadvantage is the time required to carry out the double staining.
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PMID:Simultaneous visualization of immunodetected antigens and tissue components revealed by non-enzymatic histochemical stains. 128 Jun 67

Chondroitin sulfate localization in mouse epiphyseal cartilage was studied using CS-56 monoclonal antibody immunospecific for the glycosaminoglycan portion of the molecule. For light and fluorescence microscopy, decalcified specimens were embedded in paraffin, Lowicryl, or were frozen and cryostat-sectioned, and the antigen-antibody reaction was demonstrated by treating sections with IgM-peroxidase, IgM-alkaline phosphatase, or IgM-fluorescein conjugates. For electron microscopy, decalcified and undecalcified specimens were embedded in Lowicryl; ultrathin sections from undecalcified specimens were decalcified by flotation on EDTA; sections from both types of specimens were treated with IgM-immunogold conjugate for demonstration of CS-56 reaction. Before immunoreaction, part of all decalcified sections were digested with Streptomyces or testicular hyaluronidase. Control sections were treated with either mouse and goat non-immune serum, or mouse monoclonal antiserum to human dendritic reticulum cells. Both light and electron microscopy show CS-56 reaction with cytoplasmic components of maturing and hypertrophic chondrocytes. Under the light microscope, immunoreaction was not visible in calcified matrix, and was visible in uncalcified matrix only after hyaluronidase digestion. Under the electron microscope, it was evident both in uncalcified and calcified matrix, although the latter showed few immunogold particles, usually placed on areas which appeared incompletely calcified. Gold particles were chiefly distributed at the periphery of calcification nodules and fully calcified matrix. These results show that CS-56, besides reacting with cytoplasm of maturing and hypertrophic chondrocytes, binds to crystal ghosts and other components of cartilage matrix, immunoreactivity decreasing as calcification increases. This suggests that chondroitin sulfate molecules are either degraded during calcification, or segregated into macromolecular complexes, or both degraded and segregated. The second possibility is supported by the increase of immunosensitivity induced by hyaluronidase digestion.
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PMID:Immunohistochemical investigation on the presence of chondroitin sulfate in calcification nodules of epiphyseal cartilage. 128 29

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.
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PMID:Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes. 131 62

We developed a method based on the use of various luminescent systems for identification of several nucleic acid sequences on the same dot blots. In a simultaneous assay, the target DNA molecules were hybridized with different DNA probes. These probes were labelled with biotin or digoxigenin or directly coupled to enzymes such as glucose-6-phosphate dehydrogenase, peroxidase or alkaline phosphatase. After hybridization, these labels were detected by luminescent reactions using an amplified camera. Rapid detection and specific identification of pathogenic agents such as human papillomaviruses (HPV) could be performed in a single step by this procedure. Polymerase chain reaction was carried out using general primers and virus types were identified on dot blots using short HPV 11, 16 and 18 specific oligonucleotides.
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PMID:A multiple luminescent procedure for the detection of different papillomaviruses on dot blots. 131 41

A comparison of the sensitivities of biotinylated and 32P-labelled human papillomavirus type 6b DNA probes was made. Slot blot hybridization results showed that the sensitivity of biotinylated probes was consistent with that of 32P-labelled, that is, 0.1 pg of pBR 322 plasmid containing 8 kbp HPV cDNA. In situ hybridization using 35S-labelled probes was applied to tissue from condylomata acuminata. After autoradiography, many silver grains were seen concentrated over the superficial koilocytic nuclei with some grains present in the cytoplasm. Biotinylated probes were visualized by 4 different means, i.e., streptavidin alkaline phosphatase, streptavidin biotinylated horseradish peroxidase, monoclonal anti-biotin antibody with 15 nm colloidal gold and streptavidin 5 nm colloidal gold. Strong reaction products were localized in the superficial nuclei while the cytoplasm of koilocytes showed weak hybridization signal. Pre-embedding methods were carried out for electron microscopic studies in which numerous granular diaminobenzidine (DAB) products were present in the nuclear chromatin while viral particles themselves had much fewer DAB products. This suggested that hybridization occurred more efficiently to yet unassembled viral genomes than to matured virions. Post-embedding methods using 15 nm colloidal gold were performed and showed singly scattered or clustered gold particles in superficial koilocytic nuclei.
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PMID:In situ hybridization at light and electron microscopic levels: identification of human papillomavirus nucleic acids. 132 21

Two types of nonradioactive DNA detection systems were optimized for use with nylon membranes in Southern transfers. A luminol substrate system (consisting of an enhanced chemiluminescent reaction utilizing luminol enzyme substrate) was used with peroxidase-labeled probe DNA, and a dioxetane-based substrate was used with alkaline phosphatase/antibody and digoxigenin-labeled probe DNA. Chemiluminescence was detected by autoradiography. Methods for reprobing the membranes were also optimized for both systems; blots could be reprobed at least ten times. Results showed that excellent sensitivity and low background can be achieved on both amphoteric and positively charged nylon membranes, using either detection system.
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PMID:Chemiluminescent detection of DNA on nylon membranes. 132 8

The brush border of normal small-intestine epithelial cells is rich in enzymes that are involved in the digestive process. Such molecules can be used as markers to analyze cell lineages and differentiation properties of colorectal cancers. Monoclonal antibodies detecting dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase, alkaline phosphatase, maltase-glucoamylase and lactase have been used to analyze the phenotype of colorectal cancers, adjacent mucosa and histologically normal distant mucosa. The avidin-biotin peroxidase complex method was used. Expression of dipeptidyl peptidase-IV, aminopeptidase N, sucrase-isomaltase and alkaline phosphatase was common in non-neoplastic mucosa adjacent to, and distant from, the tumor; in contrast, endopeptidase F, maltase-glucoamylase and lactase were rarely expressed in normal distant mucosa and more frequently expressed in mucosa adjacent to the tumor. Dipeptidyl peptidase-IV, aminopeptidase N, endopeptidase F, sucrase-isomaltase and alkaline phosphatase were frequently expressed in colorectal cancers, whereas maltase-glucoamylase and lactase were rarely expressed. Two general patterns of antibody reactivity were observed: diffuse cytoplasmic and apical; apical reactivity was generally associated with more differentiated tumors. A logistic predictive regression model indicated that enzyme expression in colorectal cancers followed a coordinate pattern, but was unrelated to the location of the tumor, Dukes stage or differentiation grade. In conclusion, expression of brush-border-associated enzymes occurs frequently in colorectal cancers and is regulated in a co-ordinated manner. These markers can be used for the phenotypic sub-classification of colorectal cancers.
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PMID:Intestinal brush-border-associated enzymes: co-ordinated expression in colorectal cancer. 134 6

In situ hybridization experiments, using oligodeoxyribonucleotides specific for the two major expressed human tyrosine hydroxylase mRNAs, were performed on human brain sections at the level of the mesencephalon. The specificity of the probes was ascertained by Northern blot experiments carried out with independently in vitro synthesized human tyrosine hydroxylase mRNAs. For in situ hybridization experiments, oligodeoxyribonucleotides were labelled with nucleotides tagged with digoxigenin or biotin molecules. The hybridized oligonucleotides were detected by antibodies coupled with peroxidase and alkaline phosphatase enzymes, which yield, with appropriate substrates, brown and purple products, respectively. The simultaneous detection of the two mRNAs with digoxigeninated and biotinylated probes revealed that these two mRNAs are co-expressed in single cells. The purple product obtained with alkaline phosphatase exhibits a discrete distribution within the dopaminergic cells suggesting these mRNAs are associated with sub-cellular structures. Finally, a heterogeneity in the intensity of the labelling of reactive cells with both probes was visualized as well as the expression of the two mRNA species in neurites.
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PMID:Co-expression of tyrosine hydroxylase messenger RNA 1 and 2 in human ventral mesencephalon revealed by digoxigenin- and biotin-labelled oligodeoxyribonucleotides. 135 96

Adherence of Pseudomonas aeruginosa to the cornea is a requisite step in the pathogenesis of bacteria-induced corneal disease. P. aeruginosa is capable of attaching to host epithelial cells by its pili, but there is little information regarding the epithelial receptors of this adhesin in the cornea. Using nitro-cellulose blotting of polyacrylamide gels of solubilized adult mouse corneal epithelium, four major proteins (molecular weights: 38, 42, 57, and 66 kD) and several minor proteins were identified that bound purified pili from strain PAK and its hyperpiliated mutant PAK/PR1. These proteins were identified by immunoblotting either with pilus-specific monoclonal antibodies, XLR-3 and PK 3B, or using peptide PAK 128-144 (OX). The glycosylated nature of the proteins was determined using similar gel electrophoresis of corneal epithelial proteins, blotting onto nitrocellulose, and staining the blots with lectins conjugated to either horseradish peroxidase or alkaline phosphatase. All four major pilus-binding proteins were stained with concanavalin A lectin (mannose and glucose) and either wheat germ agglutinin lectin (WGA, specific for sialic acid and N-acetylglucosamine) or succinylated WGA lectin (only N-acetylglucosamine). Staining for peanut agglutinin lectin (galactose beta(1-3) N-acetylgalactosamine) was seen for the 42-, 57-, and 66-kD proteins. The importance of the carbohydrate portions of these corneal proteins in pili binding was confirmed by preincubation of corneal epithelial blots with periodate or pili with sialic acid, both of which abolished the pili binding. These studies indicate that corneal epithelial pilus-binding proteins are glycoproteins in nature and that sialic acid may be a constituent of these pilus-specific receptors in the adult mouse corneal epithelium.
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PMID:Corneal epithelial glycoproteins exhibit Pseudomonas aeruginosa pilus binding activity. 135 76


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