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)

Human placental cells were transformed with wild-type simian virus 40 (SV40) and temperature-sensitive SV40 mutants of the A and B classes. Four criteria for transformation were used: decreased generation time, increased saturation density, increased efficiency of growth on plastic, and ability to overgrow a nontransformed monolayer. Cell lines transformed by tsA mutants lost the transformed phenotype at the restrictive temperature (40 degrees); therefore, the A function of SV40 is required for the maintenance of the transformed phenotype activity, an inhibition of human chorionic gonadotropin synthesis, and an increase in thymidine kinase activity were seen when human placental cells transformed by wild-type or tsB mutants of SV40 were grown at 33 degrees or 40 degrees and when tsA transformants were grown at 33 degrees. When tsA transformants were grown at 40 degrees, alkaline phosphatase activity and human chorionic gonadotropin synthesis were greatly stimulated and thymidine kinase activity was greatly reduced, approximating their levels in the placenta.
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PMID:Human placental cells transformed by tsA mutants of simian virus 40: a model system for the study of placental functions. 20 98

Herpes simplex virus type 1 (HSV-1) encoded thymidine kinase converts 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd), a highly specific anti-herpes agent, into the 5'-diphosphate (AIdUDP) derivative in vitro. AIdUDP was identified by its acid lability, sensitivity to alkaline phosphatase hydrolysis, chromatographic behavior, and ratio of double isotope (125I, 32P) labeling. ATP, but not AMP, is a phosphate donor, and the direct transfer of the beta and gamma phosphate of ATP as pyrophosphate to AIdUrd was ruled out. The presence of a phosphoramidate bond was supported by the acid lability of AIdUDP which has a half life (t1/2) of 320 min at pH 3.0. At neutral pH, the hydrolysis products are AIdUrd and orthophosphate, with AIdUrd monophosphate being the probable hydrolytic intermediate at these pH values. However, at acidic pH, some pyrophosphate was detected in addition to AIdUrd and orthophosphate. AIdUrd competitively inhibited the phosphorylation of thymidine and deoxycytidine. Escherichia coli thymidine kinase, even though 100-fold higher in activity, was unable to phosphorylate AId-Urd under similar incubation conditions.
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PMID:Phosphorylation of 5-iodo-5'-amino-2',5',dideoxyuridine by herpes simplex virus type 1 encoded thymidine kinase. 22 42

Isoelectric focusing of plasmodial extracts of Physarum polycephalum demonstrated the presence of several multiple enzyme variants of thymidine kinase, which appear sequentially during the nuclear division cycle. Variants (A) + (A1) are the only enzyme variants found in the late G2-phase, whereas the variants (C) + (C1) are only present at the time of mitosis and S-phase (1, 2). Evidence is presented that multiple forms of thymidine kinase (A) + (A1) with high pI arise by dephosphorylation of a primary translation product with low pI (C and/or C1). The thymidine kinase fractions (A) + (A1) and (C) + (C1) + (c1) were separated and partially purified by DEAE-cellulose chromatography. The enzyme variants (C) + (C1) are converted in vitro by an endogenous enzymatic factor as well as by bacterial alkaline phosphatase into the variants (A) + (A1).
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PMID:Thymidine kinase enzyme variants in Physarum polycephalum. In vitro interconversion of the enzyme variants. 23 Jan 87

BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 x 10(-5) and 1 x 10(-5), respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and surveys on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 micro M hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.
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PMID:Alteration of human breast tumor cell membrane functions by chromosome-mediated gene transfer. 23 36

Functional adaptation of the villus brush border and crypt have been evaluated preceding and following jejunoileal bypass for morbid obesity. Before surgery, 26 of 101 patients who were at least 100% above their ideal weight were randomly included into the study group, and control tissue specimens were collected from the jejunum and ileum. When five patients required revision of the bypass, jejunal and ileal specimens were collected from the functional (included) and nonfunctional (excluded) segments. At 19.2 +/- 5.0 (SD) months following the bypass procedure, there was an increase in alkaline phosphatase, sucrase and thymidine kinase specific activities within the functional remnants; the included ileum demonstrated a greater degree of adaptation than the included jejunum. In the nonfunctional jejunum there was a decrease in alkaline phosphatase and thymidine kinase specific activities, whereas no statistical alteration in mucosal enzyme activities occurred within the nonfunctional ileum. Serum total protein concentrations and serum magnesium levels were also evaluated before bypass and at revision. Mean serum magnesium levels became decreased, whereas serum total protein concentrations were not altered.
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PMID:Functional adaptation of the intestinal mucosal enzymes after jejunoileal bypass for morbid obesity. 66 64

The localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, E.C. 1.1.1.34) in the villous and crypt cells of the small intestine was accomplished after separating these cells from the mucosal layer by sequential dissociation in a "dual-buffer" system. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villous cell, and thymidine kinase, specific to the crypt cell. Cells obtained were 95-100% viable, and no relative difference in lability was observed, as evidenced by the equal distribution of acid phosphatase. This method of cell separation was an improvement over the "scraping" technique which damaged cells severely and produced villous preparations that contained little or no reductase activity. The HMG-CoA reductase specific activity in whole cell homogenates of the ileal villi was 0.47 and of the crypts was 0.27 nmol/min per mg of protein, considerably higher values than have been reported earlier. Also in comparison to the crypts, the villi incorporated 1.5-fold more [(14)C]-acetate into sterols, a ratio similar to that describing the distribution of HMG-CoA reductase in the two cell populations. These results unequivocally establish that the villi have higher HMG-CoA reductase activity than the crypts and confirm an earlier report from this laboratory that the villi are a major site of sterol synthesis. The sterol bio-synthetic capacity of the small intestine was highest in the ileum and decreased towards the jejunum. The HMG-CoA reductase specific activity of the ileum averaged 0.30 and that of the jejunum 0.10 nmol/min per mg of protein; however, the cholesterol content of the ileum was slightly lower than the jejunum. These results are discussed to suggest the possibility that the sterol content of the ileum may largely be due to in situ synthesis.
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PMID:3-Hydroxy-3-methylglutaryl coenzyme A reductase in isolated villous and crypt cells of the rat ileum. 92 17

Recent studies have demonstrated the presence of estrogen receptor (ER) in both normal human osteoblast-like and osteoblast-like osteosarcoma cells. The number of ER in cultured osteoblastic cells is very low (200-500 sites/cell). This has complicated characterization of the biological role of estrogens in bone cells. To study the responsiveness of bone cells to estrogens, we established osteoblast-like cell lines expressing higher ER levels. ROS 17/2.8, an osteoblastic cell line, was stably transfected with the cDNA encoding for the mouse ER. After a selection period, positive clones were isolated and evaluated for the presence of ER by both Northern blot analysis and ligand binding assays. Using these techniques, we detected a significant increase in the level of both ER transcript and binding compared to that in wild-type cells. The levels of expressed ER protein were similar to those reported in normal human osteoblast-like cells in primary culture (approximately 2000 sites/cell). To test whether the exogenously inserted ER was responsive, both wild-type and ER stably transfected cells were transiently transfected with a reporter construct containing an estrogen-responsive element linked to a truncated thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Exposure of the cells to increased concentrations of estradiol induced a slight increase in CAT activity in wild-type cells (approximately 1.5-fold) at maximal stimulation; however, it provoked a clear concentration-dependent increase in CAT activity in the ER stably transfected cells, with a maximal stimulation of approximately 10-fold. This event was receptor mediated, since ICI 164,384, an ER antagonist, blocked the enhancement of estradiol-induced CAT activity, and it was specific, since other steroid hormones did not stimulate CAT activity. Finally, we evaluated the ability of ER to modulate an endogenous estrogen-responsive gene by measuring the activity of the enzyme alkaline phosphatase. In addition, diethylstilbestrol, a synthetic estrogen agonist, increased the activity of both the CAT reporter gene and the endogenous alkaline phosphatase enzyme. In summary, we have established osteoblast-like cells expressing high levels of an exogenously inserted ER, which has characteristics similar to those of the endogenous ER in terms of its Kd. Finally, the exogenous ER regulates both exogenously inserted construct (VITERECAT) and endogenous properties of the cells (enzymatic activity and proliferation).
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PMID:Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor. 157 85

The immortalized rat calvarial bone cell line RCT-1 responds to treatment with retinoic acid (RA) by increased expression of osteoblast phenotype-related features, including the induction of liver/bone/kidney alkaline phosphatase (ALP) activity. ALP mRNA could not be demonstrated in unstimulated cells, but was first detected in cells treated for 6 h with 1 microM RA. Cycloheximide failed to block the RA induction of ALP mRNA, indicating that de novo protein synthesis was not a requirement for the RA effect and that the ALP gene may be a direct target for RA action. This was confirmed by nuclear run-on assays, which demonstrated a 2.5-fold increase in the abundance of ALP transcripts after 6 h of RA treatment. To determine whether the RA responsiveness was mediated by a specific segment of the ALP promoter, RCT-1 cells were transfected with a series of plasmids containing deletions of the 5'-flanking sequence of the human ALP gene fused to the chloramphenicol acetyl transferase (CAT) gene. CAT activity was measured in cells cultured in the presence of RA or vehicle. All but the smallest construct, which contained 44 basepairs up-stream of the initiation of transcription, were found to mediate a 2- to 3-fold increase in the expression of CAT activity in response to RA. Furthermore, when the region -108 to -45 of the human ALP gene was inserted into the expression vector pBLcat2, in a position immediately up-stream of the herpes simplex virus thymidine kinase promoter, the construct was found to mediate a 2-fold enhancement of CAT activity in response to RA. In gel retardation assays, a major band was present corresponding to the formation of a complex between the 32P-labeled probe containing the -108 to -45 sequence and proteins present in nuclear extracts of RCT-1 cells stimulated for 3 h with RA. These data suggest that the sequence of 64 basepairs (-108 to -45) 5' to the transcription start site is involved in the RA inducibility of the human ALP gene.
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PMID:Retinoic acid stimulates transcriptional activity from the alkaline phosphatase promoter in the immortalized rat calvarial cell line, RCT-1. 158 26

The aim of the present study was to characterize rat mucosal colonic cells harvested from the crypt continuum during differentiation and dimethylhydrazine-induced neoplasia. The collection of colonocytes was performed using a modified nonenzymatic isolation procedure based on Ca2+ chelation and gentle mechanical dissociation. Light and electron microscopy histomorphological examinations, [3H]thymidine incorporation studies, and activity gradients of alkaline phosphatase, thymidine kinase, and cytoskeleton-associated protein tyrosine kinase indicated that distinct cell populations were harvested from the various crypt regions in a temporal sequence mirroring their zonal and functional distribution in situ. After dimethylhydrazine administration, marked protein tyrosine kinase activity was noted in colonic cells harvested from upper crypt zones. The misplaced and sustained kinase activity preceded the actual polyp or tumor formation. This observation is consistent with the expansion of colonic proliferative compartments beyond allowable boundaries during the preneoplastic period. Companion studies in human colonic epithelial specimens corroborate the findings observed in normal and transformed murine colonocytes. It is believed that the characterization and manipulation of colonocytes using our in vitro model will provide important clues to the molecular events underlying the differentiation program and carcinogenic process in the colonic cell.
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PMID:Isolation and characterization of normal and neoplastic colonic epithelial cell populations. 199 90

Male Wistar rats were fed for four weeks on defined diets containing no fiber additions, 10% levels of insoluble fiber derivatives (cellulose or alfalfa), or 5% levels of viscous fiber derivatives (pectin, guar gum, or metamucil). After an overnight fast, the pancreas was assayed for protein, amylase, lipase, trypsin, and chymotrypsin. Homogenates of small intestinal mucosa were analyzed for protein, alkaline phosphatase, invertase and thymidine kinase. There were, with few exceptions, no dietary effects on the exocrine pancreatic enzymes. The specific activities of the villus marker enzymes (invertase and alkaline phosphatase) tended to be higher in the proximal (but not middle or distal) intestines of the fiber-fed groups, while total activities were the same in all groups. In contrast, the activity of the crypt marker, thymidine kinase, was highest in the distal intestinal segments, and even higher in animals given the alfalfa, guar gum or metamucil-supplemented diets.
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PMID:Dietary fiber and intestinal adaptation: effects on intestinal and pancreatic digestive enzyme activities. 240 60


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