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)

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

Ubiquinol-1 in aerated aqueous solution inactivates several enzymes--alanine aminotransferase, alkaline phosphatase, Na+/K(+)-ATPase, creatine kinase and glutamine synthetase--but not isocitrate dehydrogenase and malate dehydrogenase. Ubiquinone-1 and/or H2O2 do not affect the activity of alkaline phosphatase and glutamine synthetase chosen as model enzymes. Dioxygen and transition metal ions, even if in trace amounts, are essential for the enzyme inactivation, which indeed does not occur under argon atmosphere or in the presence of metal chelators. Supplementation with redox-active metal ions (Fe3+ or Cu2+), moreover, potentiates alkaline phosphatase inactivation. Since catalase and peroxidase protect while superoxide dismutase does not, hydrogen peroxide rather than superoxide anion seems to be involved in the inactivation mechanism through which oxygen active species (hydroxyl radical or any other equivalent species) are produced via a modified Haber-Weiss cycle, triggered by metal-catalyzed oxidation of ubiquinol-1. The lack of efficiency of radical scavengers and the almost complete protection afforded by enzyme substrates and metal cofactors indicate a 'site-specific' radical attack as responsible for the oxidative damage.
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PMID:Enzyme inactivation by metal-catalyzed oxidation of coenzyme Q1. 135 46

Progesterone (P)-induced PRL secretion in estradiol (E)-primed monkeys is not due to direct pituitary stimulation, because lactotropes do not express progestin receptors (PR). However, the hypothalamus, particularly the tuberoinfundibular dopaminergic system (TIDA), plays a major role in the regulation of PRL secretion. To determine whether hypothalamic dopamine neurons are progestin target cells, the colocalization of PR and tyrosine hydroxylase (TH), a phenotypic marker of dopaminergic neurons, was examined with double immunocytochemistry. Two methods for visualizing the antigens were applied; the first was a dual peroxidase method, and the second was a peroxidase-alkaline phosphatase method. In addition, the question of whether E induces PR in dopamine neurons was explored. Spayed female monkeys were treated with empty Silastic capsules, E-filled capsules for a period of 28 days, or E capsules supplemented with P capsules for the last 14 days of E treatment. Only the E- plus P-treated monkeys exhibited an increase in serum PRL during the P treatment period. Frontal sections at the level of the optic chiasm and arcuate nucleus were examined for the colocalization of TH and PR. After E treatment, hypothalamic PR-positive cells increased in both intensity and number. Neurons expressing both TH and PR were detected in the rostral hypothalamus, lateral to the third ventricle (A11-rostral) and in a discrete subventricular population (A11-subvent). The lateral population continued caudally (A11-caudal). The A11-subvent population exhibited little steroid regulation. Of the remaining A11 TH neurons, approximately 20% exhibited PR in the spayed and E-treated groups. Addition of P doubled the percentage of PR-containing TH neurons in this group. Although very few TH-positive neurons in the ventral arcuate nucleus contained PR (A12-ventral), many double labeled neurons were observed in the dorsal arcuate region (A12-dorsal). Ventral arcuate TIDA neurons were not regulated by steroids, but E plus P increased PR expression in A12-dorsal. Double labeled cells were rarely seen in the zona incerta (A13) or the emerging ventral tegmental area (A10). In summary, P probably does not act directly on ventral arcuate TIDA neurons to stimulate PRL secretion. However, the frequency of PR-positive dopamine neurons in the A11-rostral, A11-caudal, and A12-dorsal groups increased with E and P treatment. Therefore, the contribution of the PR-positive periventricular dopamine neurons to progestin-stimulated PRL secretion may be important.
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PMID:Immunocytochemical colocalization of hypothalamic progestin receptors and tyrosine hydroxylase in steroid-treated monkeys. 135 39

In order to investigate the possible involvement of corticotropin-releasing factor (CRF) and somatostatin (SRIF) on thyrotropin-releasing hormone (TRH) neuronal cell activity in the rat hypothalamic paraventricular nucleus, we have proceeded to the simultaneous localization of CRF or SRIF and TRH. For this purpose, we used a dual immunostaining procedure that employed antibodies to CRF and SRIF and peroxidase-labeled goat anti-rabbit IgG as a first sequence, and antibodies to a cryptic fragment (Phe178-Glu199) of pro-TRH (to label TRH neurons) and alkaline phosphatase-labeled goat anti-rabbit IgG as the second sequence. A rich innervation of the paraventricular nucleus by immunoreactive CRF and SRIF fibers was observed. A large number of CRF and SRIF nerve endings were seen intimate anatomic proximity and often appeared to surround TRH-containing cell bodies. These results strongly suggest that TRH neurons might be regulated by both CRF and SRIF. These interactions might be the neuroanatomical basis for the already observed inhibitory effects of CRF and SRIF on TRH release.
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PMID:Neuroanatomical connections between corticotropin-releasing factor (CRF) and somatostatin (SRIF) nerve endings and thyrotropin-releasing hormone (TRH) neurons in the paraventricular nucleus of rat hypothalamus. 135 8

A simple to use, robust, quantitative, and extremely sensitive colorimetric assay for alkaline phosphatase (EC 3.1.3.1), designed to be used as a detection system in diagnostic assays employing antibodies or gene probes, is described. This technology is based on the novel principle of prosthetogenesis, according to which a purpose-designed substrate (a prosthetogen) for a primary analyte-linked enzyme label is hydrolyzed to produce a prosthetic group for a detector enzyme system. The prosthetogen employed here is a derivative of FAD which is phosphorylated at the 3'-position of the ribose ring (FADP), the label enzyme is alkaline phosphatase, and the detector is a D-amino-acid oxidase/horseradish peroxidase-coupled system. Essentially each turnover of every molecule of alkaline phosphatase produces a molecule of D-amino-acid oxidase for detection. Thus enormous amplification of the initial signal is achieved in short time periods because of the relatively high turnover number of alkaline phosphatase for FADP. The system can be formatted as a stable, preformed, freeze-dried preparation containing all analytical components, which is reconstituted simply by addition of buffer solution. This methodology can quantitate less than 0.1 amol of alkaline phosphatase in 30 min at 25 degrees C using microtiter plates.
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PMID:Amplified assay of alkaline phosphatase using flavin-adenine dinucleotide phosphate as substrate. 136 Jul 71


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