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

---

Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid, cheap, and sensitive method has been developed for determining proteolytic activity of different classes of endoproteinases. The method is based on a solid-phase assay employing as substrate biotinylated gelatin adsorbed onto microtiter plates. Enzymatic activity is measured by incubating proteinase with the immobilized biotin-protein. Any remaining, undigested substrate bound to the microtiter plate is assayed with streptavidin-alkaline phosphatase. It was established that papain, pepsin, thermolysin, and trypsin all hydrolyzed the biotinylated substrate to varying degrees. Furthermore, the activity of these proteinases was blocked by their respective inhibitors. The assay presented is quick, highly reproducible, inexpensive, and useful for detecting all classes of endoproteolytic enzymes. By using different biotinylated proteins or peptides as substrates, and employing specific buffers and inhibitors, this assay may be utilized for detecting other and more specific endoproteinases.
...
PMID:An assay for detecting nanogram levels of proteolytic enzymes. 766 84

The authors report on their experience with an HPV non-radioactive in situ hybridization kit and describe the favorable results gained with the amended protocol, which are as follows: 1. The application of a decreased amount of both the probe and the chromogen substrate did not alter the quality of reactions. Therefore we were able to make 60 reactions instead of the originally suggested 21. 2. The proteolytic enzyme digestion time could be prolonged by changing proteinase-K for pepsin which intensifies the signal of hybridization. 3. By changing the order of hybrid detection and posthybridization washing, we succeeded in removing the excess amount of probe-ABC-AP-BAAV-ABC-AP conglomerates without losing the target sequence. 4. Using alkaline phosphatase or ABC-AP-BAAV-ABC-AP complex instead of peroxidase it was possible to demonstrate a very low number of gene copies, even if they were not detectable following the original instructions.
...
PMID:Non-isotopic in situ hybridization of human papilloma virus on histologic sections: an amended protocol. 784 39

Secretion of bicarbonate increases the pH at the duodenal mucosal surface, a process which contributes to the protection against acid/pepsin injury. Previously, we have shown that dopaminergic compounds stimulate the duodenal bicarbonate secretion in situ, in the anaesthetized rat, through an action on peripheral dopamine D1 receptors. In order to study the possible involvement of cyclic adenosine-3',5'-monophosphate (cAMP) as an intracellular mediator in enterocytes isolated from rat duodenum, cells were collected by a combination of enzyme treatment and calcium chelation. Two major cell fractions, one mainly from villi and the other mainly of crypt origin, were studied. In the villus cell fraction, the activity of alkaline phosphatase was 1.6 +/- 0.2 mumol mg protein-1 min-1 and that of sucrase 98.8 +/- 16.4 nmol mg protein-1 min-1. In the crypt fraction, activities were 0.7 +/- 0.1 and 28 +/- 10.5, respectively. Effects of dopamine, two selective dopamine receptor agonists and vasoactive intestinal peptide (VIP) on intracellular accumulation of cAMP were examined by radio-immunoassay (RIA). In the crypt cell fraction, VIP (10(-7) M) caused an increase in cAMP which was maximal after 5 min (78 +/- 28% above control, P < 0.01). In the villus cell fraction, maximal responses to VIP (60 +/- 24% above control, P < 0.05), did not occur until after 60 min of incubation. In contrast, there were no significant differences between villi and crypt enterocytes in respect to effects of dopamine, the dopamine D1-receptor agonist SKF-38393 and the D2-receptor agonist quinpirole.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Dopamine and vasoactive intestinal peptide stimulate cyclic adenosine-3',5'-monophosphate formation in isolated rat villus and crypt duodenocytes. 790 64

The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.
...
PMID:Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli. 790 63

Embryonic mouse pre-metatarsals were removed from embryos at 13 days of gestation and cultured in a defined, serum-free medium for up to 15 days. By histological analysis, we observe that the cultured pre-metatarsal tissue undergoes a similar developmental profile as pre-metatarsals growing normally in vivo. The initial mesenchyme condensation regions undergo differentiation and morphogenesis to form distinct rods made up of cartilage tissue. A marker of this differentiation step is the synthesis of type II collagen. Metabolic labelling, pepsin digestion, SDS-PAGE, and autoradiography were used to demonstrate this protein when cartilage tissue is present in the cultures. After additional culture time, terminal chondrocyte differentiation and morphogenesis take place in specific regions of the cartilage rods to form bands of hypertrophied chondrocytes. One marker of this differentiation step is the synthesis of the enzyme alkaline phosphatase. We have measured the activity of this enzyme throughout the culture period and see a substantial increase at the time of terminal chondrocyte differentiation. Another feature of hypertrophied chondrocytes is that the matrix around the cells becomes calcified. Calcified matrix in our cultured pre-metatarsals was visualized by staining with alizarin red. By supplementing the defined culture medium with ITS, we observed that terminal chondrocyte differentiation took place in a shorter culture time. Supplementation of the medium with serum results in a similar acceleration of terminal differentiation, and, with additional culture time, an osteoid-like matrix forms around the central region of the rods.
...
PMID:Embryonic mouse pre-metatarsal development in organ culture. 843 20

Cultured chick embryo tibial hypertrophic chondrocytes released matrix vesicles and Type X collagen into the culture medium. When the culture medium was filtered through a 0.1 micron nitrocellulose filter, both the matrix vesicles, measured as alkaline phosphatase, and the Type X collagen were retained quantitatively. None of the other collagen types in the culture medium was retained on the filter. Dissolution of the matrix vesicles on the filter in detergent solutions resulted in quantitative solubilization of the Type X collagen also. These results suggested that the Type X collagen was intimately associated with the matrix vesicles. However, when membrane filters that were composed of materials other than nitrocellulose, and that had a range of pore sizes, were used to filter the culture medium, the ratios of total matrix vesicles to total Type X collagen retained on the filters ranged from 53 (polysulfone membranes) to 0.3 (nitrocellulose-cellulose acetate membranes). Thus it is concluded that the quantitative retention of matrix vesicles and Type X collagen on 0.1 micron nitrocellulose filters was due to true filtration of the matrix vesicles and to selective adsorption of the Type X collagen. Removal of the noncollagenous extensions from the Type X collagen by brief pepsin or trypsin treatment converted the Type X collagen to its 45 kDa collagenous domain, which was no longer retained on nitrocellulose filters, suggesting that the adsorption of the Type X collagen on the filters was through one or both of its noncollagenous extensions. When the culture medium was subjected to ultracentrifugation to pellet the matrix vesicles, 98% of the membrane-associated alkaline phosphatase (matrix vesicles) was pelleted but only 15-20% of the Type X collagen was recovered in the pellet. These results indicate that matrix vesicles and Type X collagen are not the associated products of hypertrophic chondrocyte.
...
PMID:Type X collagen does not bind to matrix vesicles. 850 64

We describe a new application of a bright-field microscopic procedure for rapid enzyme cytochemical detection of repeated DNA sequences in metaphase preparations and frozen tissue sections. Various chromosome-specific oligonucleotide primers were used in up to three sequential primed in situ (PRINS) labeling reactions together with Taq DNA polymerase and biotin, digoxigenin and/or fluorescein isothiocyanate (FITC)-modified nucleotides. DNA target sequences were localized simultaneously by the precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color) and horseradish peroxidase-teramethylbenzidine (PO-TMB, green color) reaction in hematoxylin counterstained metaphases and interphase nuclei using a standard bright-field microscope. In addition, a protocol is reported for the application of PRINS to frozen tissue sections from normal colon and bladder epithelium. Methanol/acetic acid fixation in combination with a pepsin digestion before performing the PRINS reaction proved to be critical steps in the total procedure that permits access of the PRINS reactants, while preserving the morphology of the nuclei in the tissue. Quantification of PRINS signals showed the majority of epithelial cells with the expected two chromosome copies. The described procedures can be considered valuable tools for application in molecular cytogenetics, cell biology and pathology.
...
PMID:Rapid bright-field detection of oligonucleotide primed in situ (PRINS)-labeled DNA in chromosome preparations and frozen tissue sections. 882 52

The amount and distribution of hyaluronan in a PTFE polymer used to support an artificial cornea implanted in the rabbit cornea were determined. The findings were used to describe the polymer-corneal stroma interface and the reason for the translucence and wettability of this originally opaque and hydrophobic biomaterial. PTFE disks (6 mm in diameter, 0.2 mm thick, 50 microns in pore size) were implanted after a free-hand intralamellar dissection. The corneas were removed 15 days, 1 month, and 3 months after implantation. The hyaluronan content of pepsin-solubilized corneal stromal extracts and its distribution (7 microns cryostat sections) were investigated using an alkaline phosphatase-linked hyaluronectin assay that specifically detects nanogram amounts of hyaluronan. A PTFE polymer implant caused large, transient increases in hyaluronan density in the implanted stroma. The presence of amphiphilic hyaluronan in the polymer 15 days post implantation probably produced translucence and wettability of this opaque, hydrophobic implant despite the absence of cells. The hyaluronan density in the PTFE polymer increased considerably during the first month and then decreased to stabilize at a moderate level by the third month. These changes in hyaluronan density parallel the invasion of the polymer by inflammatory cells during the first month and the subsequent replacement of these cells by fibroblasts. The PTFE polymer is a good interface that is compatible with the native corneal stroma, and our results indicate that hyaluronan, because of its amphiphilic character, plays a major role in the polymer wettability and translucence and in the production of typical corneal extracellular matrix within the pores of the polymer.
...
PMID:Quantification and localization of hyaluronan in a PTFE polymer implanted in the corneal stroma. 957 77

The activities of digestive enzymes and alkaline phosphatase from the hepatopancreas of Macrobrochium nipponense were determined under different environmental factors (calcium concentrations 20 mg.L-1, 35 mg.L-1, 60 mg.L-1, 80 mg.L-1, 150 mg.L-1; salinity 7@1000, 14@1000, pH 7.6, 8.8, 9.8). The results showed that higher Ca2+ concentration could enhance the pepsin activity, but inhibit the trysin-like activity in hepatopancreas of M. nipponense. The activities of pepsin, trysin-like, alkaline phosphatase in hepatopancreas of M. nipponense were higher under salinity of 14@1000 than under salinity of 7@1000 and 20@1000. It showed that the activities of digestive enzymes and alkaline phosphatase of shrimp increased gradually with increasing pH value from 7.6 to 9.8.
...
PMID:[Effect of different environmental factors on the activities of digestive enzymes and alkaline phosphatase of Macrobrochium nipponense]. 1256 Nov 82

1. The effects of whole grains of wheat on the digestive tract of broiler chickens was studied. A complete pelleted feed was compared with free choice feeding of whole wheat and a pelleted protein concentrate, given from 7 to 29 d of age. 2. Pepsin activity in proventriculus tissue was lower in whole wheat-fed birds than in complete diet-fed birds. The weight (g/kg body weight) of the gizzard was higher in whole wheat-fed birds and its contents had a lower pH. 3. In the intestine, there were no differences in deoxyribonucleic acid (DNA) concentration, protein/DNA, ribonucleic acid (RNA)/DNA, RNA/protein ratios or alkaline phosphatase activity expressed per tissue weight. The weight (g/kg body weight) of the duodenum was lower in whole wheat-fed birds and its contents had a higher pH. Also the activities of alkaline phosphatase and leucine aminopeptidase in the duodenum, and maltase in the ileum, expressed per unit of bird weight, were lower in whole wheat-fed birds. 4. These results suggest that whole grain feeding increases the chemical (pepsin in proventriculus) and physical (gizzard muscle) functionality of the upper part of the digestive tract but decreases the digestive capacity of the intestine. Higher gizzard functionality may play a positive role in the control of bacterial populations. The lower digestive enzyme activities in the intestine may be detrimental in situations of mucosal deterioration caused by intestinal disease.
...
PMID:Differences in the digestive tract characteristics of broiler chickens fed on complete pelleted diet or on whole wheat added to pelleted protein concentrate. 1282 14


<< Previous 1 2 3 4 5 6 Next >>

---