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)

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

Poly(A)-containing mRNAs labeled with [methyl-3H]methionine were isolated from nucleated erythroid cells obtained from the spleens of anemic mice. The RNAs were further separated into non-globin poly(A)-containing RNAs and highly purified globin mRNA by globin cDNA-cellulose affinity chromatography. DEAE-Sephadex column chromatography of the T2 ribonuclease digestion products of the cDNA-purified globin mRNA fraction yielded methylated resistant fragments with charges of -4.7 (Cap 1) and -5.3 (Cap 2). Digestion of the non-globin RNA fraction revealed a similar pattern with the addition of a methylated mononucleotide identified as 6-methyladenosine at -2 charges. Alkaline phosphatase treatment of the T2 resistant fragments reduced their charges by approximately 2, which is consistent with the removal of one terminal phosphate. Treatment of the globin T2 and alkaline phosphatase-resistant fragments withpenicillium P1 nuclease and alkaline phosphatase yielded a P1-resistant core structure in both fragments. In addition to the core, 2'-O-methylcytidine (Cm) was released from the more negatively charged globin fragment. The P1-resistant cores of the cap structures eluted from DEAE-Sephadex with the known standard m2G5'ppp5'Am and were found to be pyrophosphatase-sensitive establishing a 5'-5'-triphosphate linkage. The pyrophosphatase and alkaline phosphatase digestion products of the globin Cap 1 and Cap 2 core structures were analyzed by high voltage electrophoresis and paper chromatography and found to be 7-methyiguanosine (m7G) and the dimethylated nucleoside 6-methyl-2'-O-methyladenosine (N6mAm). A small amount of the singularly methylated adenosine, 2'-O-methyladenosine (Am) was also observed. The predominant sequences of the methylated nucleosides in the globin cap structures are therefore m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm.
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PMID:Methylated nucleosides in globin mRNA from mouse nucleated erythroid cells. 83 41

Poly(A)-containing messenger RNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has two oxidizable end groups per molecule of mol. wt. 220000. When the mRNA is subjected to stepwise degradation by beta-elimination, only one oxidizable end-group is found. This indicates that one of the 2',3' hydroxyl end-groups is linked through the normal 3'--5' phosphodiester bond, but that the other is linked in such a way that after stepwise degradation no new 2',3 hydroxyl group is revealed. This structure could be a 5'-linked 5'-phospho di- or tri-ester. On digestion with ribonuclease the isoniazid-labelled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3' end plus fragments that are not found after stepwise degradation. These fragments have a charge of --6 and --8 from pancreatic ribonuclease or --7 from ribonuclease T1 digestion. These charges are changed to --3.4 and --4.1 after pancreatic ribonuclease, ribonuclease T2 and alkaline phosphatase digestion. methyl-3H-labelled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labelled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3 H-labelled nucleotides with the electrophoretic mobility of pm7G and pAm. It is concluded that globin mRNA has the 5' sequences m7G(5')ppp'AmpYpGp ... and m7G(5')pppAmpApGpYp.
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PMID:The nature of the 5'-linked 5' nucleotide sequence at the 5' end of rabbit globin messenger ribonucleic acid. 94 25

Poly[15(IPGVG),(RGYSLG)], where RGYSLG is a protein kinase site, was synthesized. On raising the temperature of a 5 mg/ml solution, this polypeptide undergoes an inverse temperature transition at 18 degrees C in which it folds into a contracted state by optimizing intramolecular hydrophobic interactions. Averaging the data of five experiments, phosphorylation by means of a 3':5' cyclic AMP dependent protein kinase to the extent of one phosphate in 360 residues raises the temperature of the folding transition to 32 degrees C. The shift is completely reversed on dephosphorylation by alkaline phosphatase. Phosphorylation is hereby shown to be the most potent chemical perturbation known for shifting the temperature of an inverse temperature transition, which has been shown to be an efficient mechanism for achieving chemomechanical transduction (mechanochemical coupling).
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PMID:Phosphorylation and dephosphorylation modulation of an inverse temperature transition. 185 15

Polyadenylated [poly(A)+] RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4 degrees C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37 degrees C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T1 after 3' end labeling of the poly(A)+ RNA with RNA ligase. These radioactively labeled poly(A) oligonucleotides were shown by electrophoresis through DNA sequencing gels to average 10 bases in length, with major components of 5, 9, 10, 11, and 12 bases. The lengths of these poly(A) sequences are in agreement with estimates obtained from RNase A and RNase T1 digestions of [3H]adenine-labeled poly(A)+ RNA molecules. Poly(A)+ RNA molecules from M. vannielii were labeled at their 5' termini with T4 polynucleotide kinase after dephosphorylation with calf intestine alkaline phosphatase. Pretreatment of the RNA molecules with tobacco acid pyrophosphatase did not increase the amount of phosphate incorporated into poly(A)+ RNA molecules by polynucleotide kinase, indicating that the poly(A)+ RNA molecules did not have modified bases (caps) at their 5' termini. The relatively short poly(A) tracts, the lack of 5' cap structures, and the instability of the poly(A)+ RNA molecules isolated from M. vannielii indicate that these archaebacterial poly(A)+ RNAs more closely resemble eubacterial mRNAs than eucaryotic mRNAs.
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PMID:Polyadenylated, noncapped RNA from the archaebacterium Methanococcus vannielii. 258 34

Poly-A RNA extracted from the rat liver was translated in a cell-free wheat germ system and a rabbit reticulocyte lysate. The subunit of tryptophan pyrrolase precipitated by specific antiserum after synthesis in vitro has the same molecular weight as the corresponding subunit derived from the rat liver. With specific antiserum prepared against tyrosine aminotransferase, however, a radioactive protein from both the in vitro assays was precipitated with an about 5% higher molecular weight than the tyrosine aminotransferase subunit precipitated from rat liver. The immunological evidence and the comparison of the specific peptide patterns prepared by cyanogen bromide treatment showed that the in vitro product corresponds to tyrosine aminotransferase. Various concentrations of potassium or spermidine used in the wheat germ translation system did not alter the size of the enzyme subunit synthesized. The run of the tyrosine aminotransferase purified form the rat liver in the SDS-polyacrylamide gel electrophoresis was not influenced by treatment with Escherichia coli alkaline phosphatase. The possibility is discussed that the larger enzyme synthesized in vitro represents a precursor molecule which is cleaved proteolytically in vivo.
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PMID:Translation of poly-A RNA from rat liver in vitro. Evidence for a high molecular weight subunit of tyrosine aminotransferase. 615 19

Poly(ADP-ribose) is routinely detected by the use of radioactive polymers formed from labeled substrates. In this report a simple and time-saving method for the biotinylation and the detection of poly(ADP-ribose) on blots is described. The polymer modified by light-induced reaction with photobiotin was colorimetrically detected and quantified, using streptavidine-alkaline phosphatase conjugates. The separation of poly(ADP-ribose) chains on polyacrylamide gels was not affected by the biotinylation of the polymers. When biotinylated poly(ADP-ribose) was used to detect the poly(ADP-ribose) binding capability of proteins in ligand blots, the results were comparable to those obtained with poly([32P]ADP-ribose). Experiments with histones and rat liver nuclear proteins demonstrate that in studies on poly(ADP-ribose)-protein interaction, this method is applicable to the detection of poly(ADP-ribose) binding proteins.
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PMID:The use of biotinylated poly(ADP-ribose) for studies on poly(ADP-ribose)-protein interaction. 797 87

A synthetic porous three-dimensional structure that can mimic the architecture of actual tissues, provide sustained release of nutrients or growth factors, and serve as a template for cell seeding would be an ideal substrate for tissue engineering. Poly(l-lactic acid) (PLLA) foams were fabricated for this purpose, based on the principle of phase separation from homogeneous naphthalene solutions. Complex shapes could be readily fabricated, and resulting foams had relatively uniform, open cells throughout the matrix. Densities and total pore-surface areas were in the range of 0.05-0.1 g/cm3 and 0.8-1.3 m2/g, respectively. The loss tangent of these foams ranged from 0.07 to 0.128, as measured by thermomechanical analysis. Naphthalene residue in the resulting foams went below 0.2 wt% after extensive vacuum sublimation. Feasibility of incorporating drugs or nutrients into such a highly porous structure was demonstrated by the dispersion of two model compounds, bromothymol blue (BTB) and sulforhodamine B (SD), in the matrix. Sustained release of BTB from the foam with a porosity as high as 87% was observed for more than 2 months. Alkaline phosphatase, as a model protein to be incorporated, lost approximately 30% of its bioactivity during the fabrication. As a cell-culture substrate, the PLLA foams performed as well as the flat PLLA surface in supporting the growth of rat osteosarcoma cells (ROS 17/2.8) and in maintaining their functions such as alkaline phosphatase activity and osteocalcin synthesis. UMR-106 cells cultured in the foam also expressed a higher degree of mineralization than those cultured on the flat PLLA substrate.
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PMID:Poly(L-lactic acid) foams with cell seeding and controlled-release capacity. 884 55

The evaluation of contaminating breast cancer cells in hematopoietic grafts is of considerable importance for monitoring the efficiency of purging procedures. We report a comparison of three systems for the in vitro detection and enumeration of metastatic breast cancer cells. Breast cancer cells from established cell lines were mixed with Daudi cells at dilutions ranging from 1:10 to 1:1,000,000, and a predetermined number were fixed in defined areas on microscope slides coated with one of the following attachment factors: (i) Cell-Tak Cell and Tissue Adhesive, (ii) 0.1% solution of Poly-L-Lysine, or (iii) Cel-Line HTC Super Cured slides. We employed a specificity-proven pancytokeratin antibody (A45-B/B3) and the alkaline phosphatase-antialkaline phosphatase (APAAP) staining technique. In multiple experiments, one breast cancer cell in 1,000,000 Daudi cells could reliably be detected in the Cell-Tak and Cel-Line systems and 1 in 100,000, with the Poly-L-Lysine system. The observed number of seeded cells showed a highly significant correlation with the number of cells seeded (p < 0.0001 in all cases). Finally, we used the Cell-Tak method to evaluate clinical material from various sources: from patients with primary carcinomas of the breast, prechemotherapy, and during various chemotherapeutic regimens, as well as from patients with metastatic disease. The system consistently detected tumor cells in bone marrow samples from these patients. All peripheral blood samples from patients with metastatic disease tested positive at incidences ranging from 5 to 19/10(6) peripheral blood mononuclear cells. This is a simple and reliable technique that allows rapid screening of large cell numbers with high resolution of positive cells.
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PMID:Immunocytochemical detection of breast cancer cells: a comparison of three attachment factors. 911 15

Poly(L-lactide) (PLLA) products are molded by heat extrusion. These treatments may change chemical properties and biological response of the PLLA to cells. In this study, the effect of heat treatment of PLLA on osteoblast proliferation and differentiation was examined in vitro. Osteoblast-like MC3T3-E1 cells were cultured for 2 weeks on the PLLA subjected to various heating temperature and time combinations. The protein, DNA, and hydroxyproline (HYP) contents and alkaline phosphatase (ALP) activity of cells cultured on the untreated (non-heated) PLLA with a weight average molecular weight (Mw) of 1,000,000 (high Mw PLLA) were not significantly different from those of cells cultured on glass. The activation of osteoblast differentiation by the high Mw PLLA was weak. In contrast, increases in ALP activity and HYP content were found for cells cultured on the PLLA heated at a high temperature of 200 or 250 degrees C. Heat treatment of high Mw PLLA increased differentiation of MC3T3-E1 cells cultured upon it. Significant degradation of PLLA (decrease in molecular weight and increase in molecular weight distribution) were observed following heat treatment. The Mw of PLLA decreased from 1,000,000 to below 20,000, and 14.4 microg of L-lactic acid was released from 10 mg of PLLA by heating at 250 degrees C. Therefore, the effect of low Mw chemicals, which were expected to be the degradation products of high Mw PLLA after heat treatment, on MC3T3-E1 cell activities was examined. Increases in the protein, DNA and HYP amounts and ALP activity for cells cultured with L-lactide or L-lactic acid were observed at 100 microg/ml, but not at 10 microg/ml. When the cells were cultured on the low Mw PLLA (Mw 20,000), their biological parameters also increased. Twelve micrograms of L-lactic acid released from 10 mg of the low Mw PLLA during 2 weeks incubation. The concentration of L-lactic acid in the incubation solution of low Mw PLLA or heat-treated PLLA was too small to cause cell activation. These results suggested that increases in osteoblast differentiation on the heat-treated PLLA was not to due to soluble degradation chemicals, such as L-lactic acid, rather than the remaining low Mw PLLA.
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PMID:Effect of heat treatment of poly(L-lactide) on the response of osteoblast-like MC3T3-E1 cells. 1081 7


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