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 bacterial expression vector is described for investigation of protein-protein interactions. Important features of the vector include partition of the cI repressor of bacteriophage lambda into two functional domains separated by a multicloning site, and low level auto-regulated expression of human genes as C-terminal fusions to the DNA-binding domain of cI. Two different reporter systems have been employed; expression of either a suppressor tRNA or the alkaline phosphatase gene is dependent in both cases on the extent of repression of the major leftward promoter of lambda (lambdaP(L)). The cAMP-dependent protein kinase (PKA) has been used as a model protein complex because both homodimer and heterodimer interactions are known to occur and because cAMP acts as a modulator of these interactions. It has been shown that the product of the repressor gene with newly incorporated expressed polylinker restriction sites still functions as a repressor. Substitution of the dimerisation domain of the cI repressor with the regulatory subunit of PKA does not diminish the ability of a cI fusion protein to repress expression of the reporter gene from lambdaP(L), indicating that the regulatory subunit of PKA dimerises the fusion protein in the Escherichia coli cytoplasm. Substitution instead with the catalytic subunit of PKA destroys the repression ability of cI, which is partially restored by separate expression of the regulatory subunit within the same cell. Complete restoration is achieved using a host E. coli strain which has lost its ability to synthesise cAMP and again this can be reversed by the addition of exogenous cAMP to these cells. Human PKA has been reconstituted in the E. coli cytoplasm, where all subunit interactions appear functional and respond as expected to the allosteric modulator cAMP.
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PMID:A novel bacterial vector system for monitoring protein-protein interactions in the cAMP-dependent protein kinase complex. 903 6

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus as well as other moulds. This mycotoxin contaminates animal feed and human food and is nephrotoxic for all animal species studied so far. OTA is immunosuppressive, genotoxic, teratogenic and carcinogenic. Recently lipid peroxidation induced by OTA has been reported. OTA, a structural analogue of phenylalanine, inhibits protein synthesis by competition with phenylalanine in the phenylalanine-tRNA aminoacylation reaction, constituting the main mechanism of OTA-induced cytotoxicity. Since it seems impossible to avoid contamination of foodstuffs by toxigenic fungi, investigation is required for preventing the toxicity of OTA. An attempt to prevent its toxic effect, mainly the inhibition of protein synthesis, has been made using aspartame (L-aspartyl-L-phenylalanine methyl ester) a structural analogue of both OTA and phenylalanine. Protein synthesis was assayed in monkey kidney cells (Vero cells) treated by increasing concentrations of OTA (10-100 microM). After 24 h incubation, protein synthesis was inhibited by OTA in a concentration dependent manner (the 50% inhibitory concentration, IC50, was c. 14.5 microM). Aspartame (A19), at tenfold higher concentrations than OTA (100-1000 microM), was found to partially protect against the OTA-induced inhibition of protein synthesis in Vero cells, and more efficiently when added 24 h prior to the toxin (IC50 34 microM) than together (IC50 22 microM). As expected A19(250 microM) prevented the OTA-induced leakage of certain enzymes, including lactate dehydrogenase, gamma-glutamyl transferase, alkaline phosphatase, into the culture medium, and the concomitant decrease of their intracellular activity in OTA (25 microM)-treated cells. In order to investigate the effect of aspartame (A19) on OTA-protein binding as explanation of the above results, the mycotoxin time- and concentration-dependent binding to human samples was studied in static diffusion cells with two compartments separated by a dialysis membrane. When A19 (34 microM) was added to the upper compartment containing plasma before installing OTA (50, 250, 1240 microM) in the lower one. OTA binding was largely prevented (95-98%). When A19 (34 microM) was added to the lower compartment simultaneously with the toxin (50, 250, 1240 microM), for the lowest concentration of OTA, the same efficiency was shown in preventing OTA binding, but at the two high concentrations A19 seemed less efficient.
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PMID:Reduction of the ochratoxin A-induced cytotoxicity in Vero cells by aspartame. 913 7

The process of translation termination in eukaryotes has been monitored by different types of assays, each with its own merits. We have developed an in vivo system where the reporter protein is secreted from the cells in culture thus enabling continuous monitoring of translation termination activity by simple sampling of the cell culture media. Using this system, cell cultures can be challenged with various stimuli during growth and the cellular responses on the translational level can be investigated in vivo as well as in vitro. Sampling is rapid, easy, and non-destructive to the cells, which enables measurement of translational fidelity in real time during time-course experiments. In particular with this system it is possible to assess very low levels of stop codon suppression. The reporter enzyme, secreted alkaline phosphatase (SEAP), becomes tagged with the S-peptide when there is readthrough of a stop codon placed between the C-terminus of the SEAP and the S-peptide. The tagged SEAP is bound to a matrix and the bound SEAP activity is measured versus total SEAP activity in the medium as a reference. With this assay we have confirmed that eRF1 acts as an antisuppressor in cells transfected with a cognate suppressor tRNA as well as in control cells, where a small but significant level of readthrough (suppression) could be detected. We have also characterized suppression of the three stop codons individually, and especially UGA is prone for wobbling.
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PMID:A sensitive assay of translational fidelity (readthrough and termination) in eukaryotic cells. 1064 65

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [alpha-(32)P]GDP in the presence of T1 ribonuclease the 3'-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA(Met) (f); the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [alpha-(32)P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.
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PMID:Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase. 1079 69

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3'-OH residue of a variety of nucleosides, nucleoside 5'-phosphates and dinucleotides of the type nucleoside(5')oligophospho(5')nucleoside is described here for the first time. Using micromolar concentrations of [alpha-32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5')diphospho(5')adenosine (Ap2A), adenosine (5')triphospho(5')adenosine (Ap3A), adenosine(5')tetraphospho(5')adenosine (Ap4A), adenosine(5')pentaphospho(5')adenosine (Ap5A), guanosine(5')diphospho(5') guanosine (Gp2G), guanosine(5')triphospho(5')guanosine (Gp3G), guanosine(5')tetraphospho(5')guanosine (Gp4G), and guanosine(5')pentaphospho(5')guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase. The presence of 1 mM GMP inhibited competitively the polyadenylylation of tRNA. We hypothesize that the type of methods used to measure polyadenylation of RNA is the reason why this novel property of E. coli poly(A) polymerase has not been observed previously.
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PMID:Poly(A) polymerase from Escherichia coli adenylylates the 3'-hydroxyl residue of nucleosides, nucleoside 5'-phosphates and nucleoside(5')oligophospho(5')nucleosides (NpnN). 1142 92

We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.
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PMID:Assessment of transcriptional gene activity in situ by application of HOPE-fixed, paraffin-embedded tissues. 1192 70

An improved synthesis of 3-beta-D-ribofuranosylwybutine (2) has been achieved by the Wittig reaction between 4,6-dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]-purine-7-carbaldehyde (8) and the phosphorane derived from (R)-2-[(methoxycarbonyl)amino]-3-(triphenylphosphonio)propanoate (9), followed successively by methylation, hydrogenation, and deprotection. On the other hand, the minor nucleoside wybutosine of yeast tRNA(Phe) was isolated on a scale of 80 microg by partial digestion of unfractionated tRNA (1 g) with nuclease P1, followed successively by reversed-phase column chromatography, complete digestion with nuclease P1/alkaline phosphatase, and reversed-phase HPLC. Comparison of this nucleoside with 2 has unambiguously established that the structure of wybutosine is (alphaS)-alpha-[(methoxycarbonyl)-amino]-4,6-dimethyl-9-oxo-3-beta-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester (2).
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PMID:Practical synthesis of wybutosine, the hypermodified nucleoside of yeast phenylalanine transfer ribonucleic acid. 1196 3

Large-scale isolation of the minor nucleoside wyosine of torula yeast tRNA(Phe) was accomplished by a combination of enzymatic digestion and reversed-phase chromatography: the wyosine-containing nucleotide fraction, which was obtained by partial digestion of unfractionated tRNA (1 g) with nuclease P1, was concentrated by reversed-phase column chromatography followed by complete digestion with nuclease P1/alkaline phosphatase. The nucleoside mixture thus obtained was purified by reversed-phase HPLC, providing wyosine (70 microg). Comparison of this nucleoside with a chemically synthesized authentic sample has unambiguously established that the structure of wyosine is 4,6-dimethyl-3-beta-D-ribofuranosyl-3,4-dihydro-9H-imidazo[1,2-a]purin-9-one (2).
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PMID:Structure of wyosine, the condensed tricyclic nucleoside of torula yeast phenylalanine transfer ribonucleic acid. 1196 8

A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient. No identification could be reached using phenotypic techniques. Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library. Sequencing of the 16S rRNA gene revealed its identity as Moraxella atlantae, a species isolated only rarely and published only once as causing infection. In retrospect, the phenotypic characteristics fit the identification as M. atlantae (formerly known as CDC group M-3). Comparative 16S rRNA sequence analysis indicates that M. atlantae, M. lincolnii, and M. osloensis might constitute three separate genera within the MORAXELLACEAE: After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed.
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PMID:Bacteremia due to Moraxella atlantae in a cancer patient. 1208 12

The first synthesis of (alphaS,betaS)-beta-hydroxy-alpha-[(methoxycarbonyl)amino]-4,6-dimethyl-9-oxo-3-beta-D-ribofuranosyl-4,9-dihydro-3H-imidazo[1,2-a]purine-7-butanoic acid methyl ester [(alphaS,betaS)-11] has been achieved by OsO(4) oxidation of [S-(E)]-4-[4,6-dimethyl-9-oxo-3-[2,3,5-tris-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-4,9-dihydro-3H-imidazo[1,2-a]purin-7-yl]-2-[(methoxycarbonyl)amino]-3-butenoic acid methyl ester (13) followed by successive gamma-deoxygenation through the cyclocarbonates, separation from the (alphaS,betaR)-isomer by means of flash chromatography, and deprotection. On the other hand, the minor nucleoside of rat liver tRNA(Phe) was isolated on a scale of 100 microg by partial digestion of unfractionated tRNA (1 g) with nuclease P(1), followed by reverse-phase column chromatography, complete digestion with nuclease P(1)/alkaline phosphatase, and reverse-phase HPLC. Comparison of this nucleoside with the synthetic one has unambiguously established its structure to be (alphaS,betaS)-11.
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PMID:Synthesis and structure of the hypermodified nucleoside of rat liver phenylalanine transfer ribonucleic Acid. 1237 57


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