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)

A dot blot hybridization assay, using a biotinylated cDNA probe, was able to detect feline infectious peritonitis virus (FIPV) RNA in Felis catus whole fetus (fcwf-4) cells infected with the FIPV isolates DF2, 79-1146, UCD1, and UCD2. The probe cross-hybridized in the dot blot assay with nucleic acid of a closely related feline coronavirus, feline enteric coronavirus (FEVC)-79-1683. To construct the probe, a 2.5 kilobase cDNA, prepared from FIPV-DF2 genomic RNA, was molecularly cloned. The recombinant cDNA clone was digested with the restriction endonuclease Rsa I, and an 870 basepair Rsa I fragment was isolated from vector DNA by agarose electrophoresis and glass-milk purification. This fragment was complementary to the 3' three fourths of the nucleocapsid gene. The hybridization probe was prepared by random primed labeling in the presence of biotin-11-dUTP. Using an avidin-alkaline phosphatase conjugate and chemiluminescent substrate detection system, virus could be detected in as few as 3000 infected cells. In an in vivo study, the probe was used to detect FIPV RNA in peripheral blood mononuclear leukocytes (PBML) isolated at various post-infection days (PID) from cats experimentally infected with the FIP-producing coronavirus isolate FIPV-79-1146 or FIPV-DF2. Viral RNA could be detected in as few as 12,000 PBML isolated from cats at PID 7 and in 50,000 PBML at PID 22. There was no consistent pattern, however, between hybridization results and prognosis or severity of disease at the time of sampling. Despite some cross-hybridization with FECV RNA, this probe should be useful for diagnosis of FIP, because cats infected with FECV most likely do not become viremic.
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PMID:Detection of feline infectious peritonitis virus infection in cell cultures and peripheral blood mononuclear leukocytes of experimentally infected cats using a biotinylated cDNA probe. 838 40

A tripartite fusion construct encoding the amino-terminal half of EcoRI endonuclease followed by amino acids 217-299 of the filamentous bacteriophage gene I protein (pI) attached to the enzymatic portion of alkaline phosphatase results in the production of two proteins. The larger protein, pIf, is the complete tripartite fusion protein while the smaller protein, pIf*, results from internal initiation of translation at pI methionine 241. Both pIf and pIf* span the Escherichia coli inner membrane via a 20-amino-acid hydrophobic stretch of pI with their amino termini in the cytoplasm and their carboxyl-terminal alkaline phosphatase domains in the periplasm. The alkaline phosphatase moiety of approximately 70% of pIf is released into the periplasm by in vivo proteolysis, but only about 10% of pIf* is cleaved. Neither DegP, OmpT, nor protease III are responsible for the cleavage in vivo, and leader peptidase is unable to cleave the fusion protein in vitro. Deletion and substitution analyses demonstrate that the degree of periplasmic cleavage depends on the sequence of the cytoplasmic domain of the fusion proteins. Possible mechanisms for this transmembrane-directed cleavage event are compared to proposed models for signal transduction.
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PMID:The membrane domain of a bacteriophage assembly protein. Transmembrane-directed proteolysis of a membrane-spanning fusion protein. 844 11

The gene I protein (pI) of the f1 filamentous bacteriophage is a non-capsid protein that is required for the assembly of the bacteriophage. It spans the Escherichia coli inner membrane once with its amino terminus in the cytoplasm and its carboxyl-terminal portion in the periplasm. The presence of moderate amounts of this protein in the membrane results in rapid inhibition of cell growth, probably from a loss of membrane potential. Previous observations defined a 55-amino-acid sequence within pI required for its membrane insertion which includes a 20-residue hydrophobic stretch preceded by a 13-residue positively charged amphiphilic helix. To define the minimal sequence required for membrane translocation and for growth inhibition, a deletion analysis was performed on a tripartite fusion construct containing the 55-residue pI sequence flanked upstream by the amino-terminal portion of EcoRI endonuclease and downstream by the enzymatic portion of alkaline phosphatase. Only the 20-residue hydrophobic stretch immediately preceded by 1 arginine residue is required for membrane insertion of the fusion proteins. This region also sufficed to inhibit cell growth provided it contained protein domains exposed in both the cytoplasm and periplasm. It was not possible to separate the domains required for membrane insertion and cell growth inhibition. No requirement for the positively charged amphiphilic helix was detected either for membrane insertion or growth inhibition, suggesting that it plays a role in phage assembly and not membrane insertion.
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PMID:The membrane domain of a bacteriophage assembly protein. Membrane insertion and growth inhibition. 844 12

Direct label alkaline phosphatase (AP) conjugated oligonucleotide probes (AP-DNA) were prepared to assess their utility for allele-specific detection of single base substitutions. Oligonucleotide conjugates were designed to detect point mutations in the genes for lipoprotein lipase (LPL) and coagulation factor-V (FV). Genomic DNA samples, including ones known to harbor point mutations in the genes for LPL and FV, were prepared from whole blood and subjected to polymerase chain reaction (PCR). PCR products were analyzed by Southern hybridization with the allele-specific AP-DNA probes and restriction endonuclease analysis. Thermal profiles for hybridization indicate optimal allele-specific selectivity was achieved with temperatures ranging from 45 degrees C to 55 degrees C at a total Na divided by concentration of 150 mM. Under these conditions the base changes studied were easily discriminated with allele specific hybridization signals in excess of 200:1 as estimated by scanning densitometry. Complete concordance was observed between hybridization and restriction analyses for 175 LPL and 201 FV clinical and reference samples. The total time for analysis of the PCR products was less than 2 h with a dot blot hybridization protocol.
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PMID:Allele-specific hybridization of lipoprotein lipase and factor-V Leiden missense mutations with direct label alkaline phosphatase-conjugated oligonucleotide probes. 893 92

Porphyromonas gingivalis has been isolated from periodontitis lesions in subjects from many geographical locations. The purpose of this investigation was to determine whether similar ribotypes of P. gingivalis could be detected among strains isolated in different countries. A total of 198 isolates of P. gingivalis were obtained from 52 periodontitis patients in Boston (130 isolates), Bergen, Norway (17 isolates), Khartoum, Sudan (26 isolates), and Bucharest, Romania (25 isolates). DNA was isolated from each strain, cut separately by the restriction endonucleases KpnI and PstI. The resulting preparations were subjected to electrophoresis in a 0.8% agarose gel using a Tris-acetate EDTA buffer. Uncut lambda and a 1000-bp fragment of 16S rRNA were included as internal standards in each lane. In addition, a HindIII digest of lambda was present in a separate lane in each run. The DNA fragments were transferred to a nylon membrane by downward capillary transfer. 16S rRNA bands were detected using a 1000-kb digoxigenin-labelled probe generated by a polymerase chain reaction. At the same time, a digoxigenin-labelled probe to lambda was employed to detect the internal and molecular weight standards. The bands were detected using antibody to digoxigenin conjugated to alkaline phosphatase and chemiluminescence. The positions of the bands relative to the internal standards were determined and normalized to correct for run-to-run variations, and the molecular weight of each band was determined by comparison with standards within each gel. The resulting data for the 2 enzymes were combined and subjected to cluster analysis using an average unweighted linkage sort. In some instances, isolates that appeared to be of identical ribotype using one endonuclease gave different ribotypes using the other. Strains of P. gingivalis within a subject were usually identical, except for 3 patients who harbored 2 different ribotypes/individual. All subsequent analyses employed a single ribotype strain for each subject. A total of 32 ribotypes were observed for isolates from distant countries. A total of 11.5% of the patients had isolates exhibiting the same ribotype: ribotype 7a. Identical ribotypes of P. gingivalis can be recovered from subgingival plaque samples of periodontitis patients in different countries.
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PMID:Detection of identical ribotypes of Porphyromonas gingivalis in patients residing in the United States, Sudan, Romania and Norway. 922 34

The homogenate from Caco-2 cells of day 13 or 15 after subculturing had high omega-hydroxylation activity of docosahexaenoic acid (22:6(n-3)) or arachidonic acid (20:4(n-6)). Activity, maximal at pH 8.0, was inhibited in the presence of CO or metyrapone and in the absence of NADPH. Omega-hydroxylation activity of lauric acid in the homogenate was not detected. Apparent Michaelis constant (Km) values for 22:6(n-3) and 20:4(n-6) were found to be 4 and 7 microM. Omega-hydroxylation activity considerably increased with growth up to day 13 and then decreased until day 20 even though alkaline phosphatase (ALP) and leucine-aminopeptidase (LAP) activity increased with growth to day 20. Metyrapone in cultures caused omega-hydroxylation, ALP and LAP activity to decrease, while sodium butyrate dose-dependently increased that of omega-hydroxylation, ALP and an endogenous endonuclease and decreased lactate dehydrogenase (LDH) activity in culture medium. The omega-hydroxylation system thus appears quite likely to be associated with cytochrome P450, differentiation and/or apoptosis rather than cytotoxic cell death of Caco-2 cells. Substrate specificity, however, differed from that of human cytochrome P450 4A11.
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PMID:Docosahexaenoic/arachidonic acid omega-hydroxylation system and differentiation in the human colonic adenocarcinoma cell line, Caco-2. 946 91

To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E. coli exonuclease III.
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PMID:Characterization of Bacillus subtilis ExoA protein: a multifunctional DNA-repair enzyme similar to the Escherichia coli exonuclease III. 1054 Jul 38

With the aim to determine whether bone metabolism in young women using low-dose oral contraception is influenced by vitamin D receptor (VDR) genotype, we designed the prospective clinical study of 41 healthy women aged 20-27 years. Twenty-one women of the study group were prescribed an oral contraceptive (30 microg ethynyl estradiol and 150 microg levonorgestrel) and 20 women of the control group a nonhormonal contraceptive or none. Biochemical markers of bone metabolism (bone-specific alkaline phosphatase, osteocalcin, deoxypyridinoline) and VDR genotype, using BsmI endonuclease, were determined. After 3 months in the study group, the BB genotype subgroup showed significantly decreased osteocalcin (p = 0.010), in the Bb genotype subgroup bone-specific alkaline phosphatase (p = 0.043) and osteocalcin (p = 0.006) decreased, and in the bb genotype subgroup no changes were observed. In the control group, there were no significant changes in markers of bone metabolism regarding VDR genotype. In conclusion, our study shows that in young women VDR gene polymorphism could influence bone metabolism during low-dose oral contraceptive use.
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PMID:Vitamin D receptor gene polymorphism and bone metabolism during low-dose oral contraceptive use in young women. 1252 55

Gene transfer to the major salivary glands is an attractive method for the systemic delivery of therapeutic proteins. To date, nonviral gene transfer to these glands has resulted in inadequate systemic protein concentrations. We believe that identification of the barriers responsible for this inefficient transfection will enable the development of enhanced nonviral gene transfer in salivary glands and other tissues. One potential barrier is the degradation of plasmid DNA by endonucleases. To test this hypothesis, we coadministered two endonuclease inhibitors ((zinc and aurintricarboxylic acid (ATA)) with plasmid DNA, containing the secreted alkaline phosphatase gene (SEAP), to the submandibular glands of rats. The effect of zinc and ATA on SEAP expression, tissue accumulation of plasmid DNA, and plasmid DNA stability was then characterized. We observed that mixtures containing zinc/DNA, ATA/DNA, and zinc/ATA/DNA significantly enhanced both systemic transgene expression and the amount of plasmid DNA associated with treated tissues. The relative endonuclease inhibitory activity of zinc, ATA, and zinc/ATA correlated with the observed effects on transfection efficacy. The use of zinc/ATA enhanced the efficacy of salivary gland transfection by at least 1000-fold versus DNA alone. Importantly, this improved performance resulted in robust systemic secretion of an exogenous protein (SEAP), thus demonstrating the potential this nonviral gene transfer technology has as a method to treat systemic protein deficiencies.
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PMID:Enhanced systemic transgene expression after nonviral salivary gland transfection using a novel endonuclease inhibitor/DNA formulation. 1462 68

Cerebral ischemia and reperfusion induces rapid accumulation of oxidative DNA lesions in the brain, which, if not repaired promptly, may trigger cell death. The base-excision repair (BER) pathway is the main mechanism employed by neurons to repair various types of oxidative DNA damage. Recent studies have suggested that the cellular activity of BER is highly regulated (up- or down-regulated) after ischemic brain injury, and this regulation may contribute to the outcome of cell injury. The mechanism through which cellular BER is regulated in response to neuronal injury is currently poorly understood. In the present study, we have examined BER regulation in the rat model of focal ischemic brain injury induced by 2 hr of middle cerebral artery occlusion and 0-72 hr of reperfusion. As determined using cerebral nuclear extracts, focal ischemia resulted in a marked reduction in BER activities, including the overall BER activity, AP endonuclease activity and DNA polymerase-beta activity, indicating functional impairment of the BER pathway. BER reduction occurred as early as 0.5 hr after the onset of reperfusion. Thereafter, BER activity failed to recover, and there were persistent accumulations of apurinic/apyrimidinic abasic sites and DNA single-strand breaks in ischemic tissues. The reduction in BER during the early reperfusion phase (less than 6 hr) was not accompanied by any alterations in the levels of essential BER enzymes in brain extracts. However, increased serine- and threonine-specific phosphorylation was detected for both AP endonuclease and DNA polymerase-beta after ischemia, with the time course of serine phosphorylation closely correlated to that of changes in BER activity. Furthermore, dephosphorylation of nuclear extracts with alkaline phosphatase largely restored AP endonuclease and DNA polymerase-beta activities. Taking advantage of the neuroprotective effect of mild hypothermia (33 degrees C), which was induced in the brain during the first 2 hr of reperfusion, we found that the post-ischemic suppression of BER activity is a reversible event. Hypothermic treatment diminished the serine-specific phosphorylation of AP endonuclease and DNA polymerase-beta, promoted BER activities, and attenuated the levels of oxidative DNA lesions after ischemia. These results suggest that the functional impairment of the BER pathway after severe focal cerebral ischemia is due to the loss-of-function post-translational modifications of repair enzymes. Further investigations elucidating the precise mechanism underlying the post-translational regulation of BER enzymes may lead to novel therapeutic strategies for cerebral ischemia.
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PMID:Impaired DNA repair via the base-excision repair pathway after focal ischemic brain injury: a protein phosphorylation-dependent mechanism reversed by hypothermic neuroprotection. 1712 26


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