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)

An oligonucleotide probe (23-mer) containing a single biotinylated deoxyuridine residue at the 3' terminus was prepared by a primer extention reaction using Escherichia coli DNA polymerase I (Klenow fragment). For efficient synthesis of the probe, it was necessary to add about 16-fold molar excess of the template oligonucleotide (pentadecanucleotide) to the primer oligonucleotide (nonadecanucleotide) in the reaction mixture and to continue the reaction for 2.5 hr at 4 degrees C. The probe was purified by polyacrylamide gel electrophoresis under denaturing conditions. The probe could be specifically and tightly bound with Avidin D (Vector Laboratories) in 1 M NaCl. It could be hybridized to a plasmid DNA containing a perfectly matched complementary sequence, but not to a DNA containing five consecutive noncomplementary bases. The hybridized biotinylated probe could be detected by Avidin D and biotinylated alkaline phosphatase, even when 1.8 ng of the plasmid DNA (0.5 fmoles) was used. A general approach to the enzymatic synthesis of oligonucleotides containing a single biotinylated deoxyuridine at the 3' end is described.
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PMID:Biotin-labeled oligonucleotides: enzymatic synthesis and use as hybridization probes. 674 43

Analogs of phosphophoryn, a calcium-binding phosphorylated protein found in mineralized dentin, were synthesized by solid phase peptide synthesis. The dentin phosphophoryn appears to contain some sequence blocks of (Asp-PhosphoSer)n. As this sequence is difficult to synthesize, polymers of (alpha-L-Glu-L-Ser) were prepared. The 30-peptide, (alpha-L-Glu-L-Ser)15, was phosphorylated by reaction with orthophosphoric acid in the presence of trichloroacetonitrile in anhydrous dimethylsulfoxide. Calcium ion binding studies were carried out with both the 30-peptide and phosphorylated 30-peptide. The conformation of the original 30-peptide, (Glu-Ser)15, was examined, in the presence and absence of calcium ion, by circular dichroism measurements. Purified bovine phosphophoryn, previously studied by the same techniques, was partially dephosphorylated by alkaline phosphatase, and its calcium ion binding properties were determined. Dephosphorylation to 31% of the initial phosphorus content reduced the number of high affinity sites to approximately 30% of the initial value. However, the stoichiometry of binding indicated that both phosphate and carboxylate groups participate in the high affinity binding and that the binding constant was decreased only slightly. Partial phosphorylation of the 30-mer raised the calcium binding constant, Ka, from 2.1 x 10(2) to 3.3 x 10(3) M-1 and increased the amount of binding from an electrostatic equivalent number of sites to a stoichiometric equivalent number. Concomitant with binding, there was a transition from random coil to beta-like structure. These data suggest that the repetitive (Asp-PhosphoSer)n regions in phosphophoryn and the (Glu-PhosphoSer)n sequence of the synthetic polymer have special conformations which favor the unidentate binding of calcium to the carboxyl groups and phosphate groups. and which enhance the binding affinities of the carboxyl groups in such sequences in a cooperative fashion.
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PMID:Cooperativity in calcium ion binding to repetitive, carboxylate-serylphosphate polypeptides and the relationship of this property to dentin mineralization. 678 Apr 83

A tendon is predominantly composed of collagen Type I. To determine the synthesis of collagen Type I after a rotator cuff tear, an in situ hybridization technique was applied to localize cells containing procollagen alpha 1 Type I in the proximal stump of five torn supraspinatus tendons obtained during surgery. Specimens were fixed in 10% buffered formalin, embedded in paraffin, and sectioned at 6 microns. A 22 mer oligonucleotide corresponding to a sequence coding a part of human procollagen alpha 1 Type I messenger RNA (mRNA) was used as a hybridization probe. The probe was 3'-end labeled with digoxigenin-11-dUTP, and the probe-mRNA hybrids were enzymatically visualized using conventional chromogens for alkaline phosphatase. The procollagen alpha 1 type I mRNA was clearly observed in the cells near the margin of the tear. However, they were not consistently found in the vicinity of the intratendinous extension of the tear and in cells of the subacromial bursa. It is concluded that this method should be used to study the characteristics of collagen synthesis in a torn rotator cuff tendon.
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PMID:Localization of mRNA of procollagen alpha 1 type I in torn supraspinatus tendons. In situ hybridization using digoxigenin labeled oligonucleotide probe. 802 Feb 12

An antisense phosphorothioate oligodeoxynucleotide 27-mer complementary to the rev gene mRNA of the human immunodeficiency virus (HIV-1) was administered to rats through intravenous injections and subcutaneous infusions in order to investigate the disposition of this compound. In addition, nonlethal toxic responses of the rat were evaluated. A biphasic plasma clearance with t1/2 alpha of 20-25 min and t1/2 beta of 27-41 hr was observed. Single doses ranging from 35 to 3257 micrograms were examined, and the plasma concentration and area under the plasma concentration-time curve (AUC) were found to be directly proportional to the dose. Continuous subcutaneous infusion of 50 mg over 28 days was also examined. The oligonucleotide is completely eliminated in the urine over 3 days. Electrophoretic analysis demonstrated that the excreted compound has the same mobility and UV-absorbance profile as the administered compound. Measurement of accumulation and distribution into tissues revealed unique tissue-specific rates and extent of oligonucleotide movement into and out of tissues. Results of the chronic infusion study suggest that uptake into tissue is not saturated, even after 28 days of infusion. Analysis of blood plasma from oligonucleotide-treated animals shows a possible transient elevation in levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), but not alkaline phosphatase (AP), gamma-glutamyltransferase (GT), and bilirubin. The data collectively support the potential utility of phosphorothioate oligonucleotides as therapeutic agents in vivo.
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PMID:Pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuous infusion. 806 15

A transposon was constructed allowing the rapid restriction mapping of plasmids. This transposon, Tn 5Map, contains a cleavage site for the I-SceI endonuclease which recognizes an 18-mer. After in vivo transposition of Tn5Map into the plasmid of interest, the plasmid is isolated and linearized with I-SceI. Splinkers labelled with digoxygenin and complementary to the left and right end of the linearized molecule are added and ligated. After partial digestion of the splinkered molecules with the restriction enzyme of interest, separation of the cleavage products in an agarose gel, and Southern transfer, the labelled fragments are visualized by the addition of the chemiluminescent substrate AMPPD and alkaline phosphatase. The restriction map can be directly read from the bottom to the top of the gel.
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PMID:Tn 5Map, a transposon for the rapid mapping of restriction sites in plasmids. 813 29

A technique of nested polymerase chain reaction (PCR) and hybridization with non-radioactive probes was developed for the detection and identification of HBV DNA and HBs-subtypes in very small volumes of human sera. Four oligonucleotide primers (20 mer) complementary to DNA sequences in the S region of HBV and probes (18 or 20 mer) conjugated to alkaline phosphatase were used for the present PCR assay. The results of the PCR assay coincide with those of enzyme immunoassay (EIA) in 14 HBe-positive and 59 HBe-negative samples with 98.6% of specificity. The HBV subtypes adr and adw were identified using an 18-mer DNA probe in 30 samples with an accuracy of 100%. Further, the DNA subtypes were clearly demonstrated in 3 samples where HBs-antigen was undetectable. These results indicate that amplification of HBV DNAs by PCR and their detection with non-radioactive probes is a reliable tool for diagnosis of HBV infection in clinical laboratories.
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PMID:[Detection and identification of HBV DNA and DNA subtypes of HBs-antigen by the use of the polymerase chain reaction and non-radioactive probes]. 836 May 15

The O6-alkylguanine-DNA alkyltransferase (ATase) is known to overcome the effects of promutagenic, precarcinogenic O6-alkylguanine induced in DNA by exposure to environmental, chemotherapeutic and dietary alkylating agents. Within an organ, the cell type-specific responses to these agents may be attributed, in part, to varying expression of critical DNA repair genes, like ATase. In order to determine the cell-specific expression of the human ATase gene, in situ hybridization was used to map the cellular distribution of ATase mRNA in tissue sections of normal human fetal and adult livers. Tissue sections were hybridized with a digoxigenin-labeled 39 base oligomer, antisense to ATase cDNA. Following immunodetection, using an alkaline phosphatase-conjugated anti-digoxigenin antibody, the ATase-specific mRNA levels were visualized in parallel with liver cell type identification. The specificity of the antisense probe and hybridization to human ATase mRNA was demonstrated by: (i) staining of Mer+ and not Mer- cells by the antisense probe; (ii) faint staining of liver sections when the antisense probe was not used during hybridization; (iii) no hybridization of liver sections by the sense probe; (iv) no staining of sections preincubated with RNase before hybridization; and (v) the retention of cell type-specific staining patterns in tissue sections incubated with DNase prior to hybridization with the antisense probe. The staining patterns appeared similar in adjacent sections of tissues obtained from the same liver and in sections obtained from either adult or fetal livers of different individuals. The expression of the ATase mRNA, as noted by stain intensity, appeared highest in all of the bile ductal cells. There was a heterogenous expression in hepatocytes, which varied from moderate to high stain. Staining in Kupffer cells also appeared to be high. Sinusoidal cells, endothelial cells of the hepatic artery and cells of the connective tissue showed weak hybridization, indicating low levels of ATase mRNA. These data explain, in part, the basis for a differential response of various cell types within the liver to the mutagenic and carcinogenic effects of alkylating agents.
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PMID:Cell type-specific expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human liver tissues as revealed by in situ hybridization. 847 40

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.
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PMID:Phosphorylation of human and bovine prothymosin alpha in vivo. 848 35

An alkaline phosphatase-conjugated 30-mer oligonucleotide probe was developed to detect the cholera toxin gene (ctx) in Vibrio cholerae O1. For rapid identification, V. cholerae O1 was grown on selective agar (thiosulfate-citrate-bile salts agar) or in alkaline peptone water and organisms were transferred directly to nylon membranes. Lysis of cells, denaturation of DNA, neutralization, and hybridization were carried out on the membrane. These procedures required only 3 h for completion. The results of the hybridization test with 88 clinical and 20 environmental isolates agreed almost exactly with the results of the immunological tests (anti-cholera toxin antibody-sensitized latex agglutination tests). The specificity of the probe was also tested with strains of enterotoxigenic Escherichia coli, V. cholerae non-O1, and Vibrio mimicus.
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PMID:Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene. 850 Dec 33

An alkaline phosphatase-labeled 30-mer oligonucleotide probe was designed to detect the gene for pilus colonization factor antigen III (CFA/III) of the human type of enterotoxigenic Escherichia coli (ETEC). The CFA/III probe was used to identify CFA/III-producing ETEC among 303 Escherichia coli obtained from subjects with traveler's diarrhea. Six isolates positive for the CFA/III gene were found. This result was confirmed immunologically by using a specific monoclonal antibody developed against CFA/III. These six isolates, isolated from travelers returning from India, Pakistan and China, were all positive for the gene of heat-labile enterotoxin and possessed an identical serotype (025:H-).
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PMID:Use of a new oligonucleotide probe for detection of colonization factor antigen III gene in enterotoxigenic Escherichia coli. 856 93


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