Phenotypic change caused by transcriptional bypass of uracil in nondividing cells.
(9/1786)
Cytosine deamination to uracil occurs frequently in cellular DNA. In vitro, RNA polymerase efficiently inserts adenine opposite to uracil, resulting in G to A base substitutions. In vivo, uracil could potentially alter transcriptional fidelity, resulting in production of mutant proteins. This study demonstrates that in nondividing Escherichia coli cells, a DNA template base replaced with uracil in a stop codon in the firefly luciferase gene results in conversion of inactive to active luciferase. The level of transcriptional base substitution is dependent on the capacity to repair uracil. These results provide evidence for a DNA damage-dependent, transcription-driven pathway for generating mutant proteins in nondividing cells. (+info)
A comparison of electron-capture GLC, electrolytic-conductivity GLC and UV-absorption HPLC for the analysis of some herbicides in foods.
(10/1786)
A comparison of gas chromatography with electron-capture or electrolytic-conductivity (nitrogen mode) detection, and high-pressure liquid chromatography (HPLC) with UV-absorption detection (254 nm) was carried out for the analysis of several herbicides in foods. Linuron, propanil, terbacil, benzoylprop-ethyl, and the fungicide DCNA in samples of cabbage, corn, potato, and wheat spiked at 2 and 0.2 ppm were examined. The pesticides were extracted with acetone, partitioned into petroleum ether-methylene chloride, and cleaned up on a 2% deactivated Florisil column before direct chromatographic analysis. Electron-capture gas-liquid chromatography (GLC) was most suitable for DCNA and benzoylprop-ethyl while UV-absorption HPLC was best for terbacil analysis. Linuron and propanil gave similar results for both electron-capture GLC and HPLC. Electrolytic-conductivity GLC could detect all pesticides at the 0.2 ppm level and exhibited the least number of extraneous peaks in the chromatograms. (+info)
The effect of a thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors.
(11/1786)
Thymidine phosphorylase (TP) is an enzyme involved in the reversible conversion of thymidine to thymine and is identical to an angiogenic factor, platelet-derived endothelial cell growth factor. TP is expressed at higher levels in a wide variety of solid tumors than in the adjacent nonneoplastic tissues. Patients with TP-positive colon and esophageal tumors have a poorer prognosis than those with TP-negative tumors. We have recently synthesized a new TP inhibitor (TPI), 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride. We investigated the effect of TPI on angiogenesis in KB cells transfected with platelet-derived endothelial cell growth factor cDNA, KB/TP, and a mock transfectant, KB/CV, using the mouse dorsal air sac assay model. We found that KB/TP cells had a higher angiogenic ability than KB/CV cells and that TPI completely suppressed angiogenesis by KB/TP. Furthermore, at a dose of 50 mg/kg/day, TPI considerably decreased the growth rate of KB/TP cells xenografted into nude mice. Microvessel density in KB/TP tumors was higher than that in KB/CV tumors, and TPI did not significantly change the density in either of the tumors. The apoptotic index in KB/TP tumors was significantly lower than that in KB/CV tumors, and TPI significantly increased the apoptotic index in KB/TP tumors but not in KB/CV tumors. These findings, taken together with previous reports, suggest that the expression of TP plays an important role in tumor growth and that TPI suppresses tumor growth by increasing the proportion of apoptotic cells and probably inhibiting angiogenesis. (+info)
Identification of the binding site for the extrahelical target base in N6-adenine DNA methyltransferases by photo-cross-linking with duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position.
(12/1786)
DNA methyltransferases flip their target bases out of the DNA double helix for catalysis. Base flipping of C5-cytosine DNA methyltransferases was directly observed in the protein-DNA cocrystal structures of M.HhaI and M.HaeIII. Indirect structural evidence for base flipping of N6-adenine and N4-cytosine DNA methyltransferases was obtained by modeling DNA into the three-dimensional structures of M.TaqI and M.PvuII in complex with the cofactor. In addition, biochemical evidence of base flipping was reported for different N6-adenine DNA methyltransferases. As no protein-DNA cocrystal structure for the related N6-adenine and N4-cytosine DNA methyltransferases is available, we used light-induced photochemical cross-linking to identify the binding site of the extrahelical target bases. The N6-adenine DNA methyltransferases M.TaqI and M.CviBIII, which both methylate adenine within the double-stranded 5'-TCGA-3' DNA sequence, were photo-cross-linked to duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position in 50-60% and almost quantitative yield, respectively. Proteolytic fragmentation of the M. CviBIII-DNA complex followed by Edman degradation and electrospray ionization mass spectrometry indicates photo-cross-linking to tyrosine 122. In addition, the mutant methyltransferases M. TaqI/Y108A and M.TaqI/F196A were photo-cross-linked with 6-fold and 2-fold reduced efficiency, respectively, which suggests that tyrosine 108 is the primary site of modification in M.TaqI. Our results indicate a close proximity between the extrahelical target base and tyrosine 122 in M.CviBIII or tyrosine 108 in M.TaqI. As both residues belong to the conserved motif IV ((N/D/S)(P/I)P(Y/F/W)) found in all N6-adenine and N4-cytosine DNA as well as in N6-adenine RNA methyltransferases, a similar spatial relationship between the target bases and the aromatic amino acid residue within motif IV is expected for all these methyltransferases. (+info)
The uptake and metabolism of uridine by the slime mould Physarum polycephalum.
(13/1786)
1. Uridine is taken up by microplasmodia of Physarum polycephalum via a saturatable transport system with an apparent Km of 29 muM. An intracellular concentration significantly higher than that in the growth medium is attained, suggesting that the uptake is an active process. Both deoxyribonucleosides and ribonucleosides are competitive inhibitors of the uptake of uridine. 2. In contrast, the rate of entry of uridine into surface plasmodia is a linear function of the concentration of the nucleoside in the growth medium, and the uptake is not inhibited by other nucleosides. 3. As well as serving as a source of pyrimidine nucleotides for the synthesis of nucleic acids, uridine is also catabolised by P. polycephalum. Uracil accumulates in the growth medium and there is also significant conversion of C-2 of the pyrimidine ring to CO2. The proportion of uridine subject to catabolism in surface plasmodia is less than that observed for microplasmodia. (+info)
Specific binding of human MSH2.MSH6 mismatch-repair protein heterodimers to DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases.
(14/1786)
Previous studies have demonstrated recognition of DNA-containing UV light photoproducts by bacterial (Feng, W.-Y., Lee, E., and Hays, J. B. (1991) Genetics 129, 1007-1020) and human (Mu, D., Tursun, M., Duckett, D. R., Drummond, J. T., Modrich, P., and Sancar, A. (1997) Mol. Cell. Biol. 17, 760-769) long-patch mismatch-repair systems. Mismatch repair directed specifically against incorrect bases inserted during semi-conservative DNA replication might efficiently antagonize UV mutagenesis. To test this hypothesis, DNA 51-mers containing site-specific T-T cis-syn-cyclobutane pyrimidine-dimers or T-T pyrimidine-(6-4')pyrimidinone photoproducts, with all four possible bases opposite the respective 3'-thymines in the photoproducts, were analyzed for the ability to compete with radiolabeled (T/G)-mismatched DNA for binding by highly purified human MSH2.MSH6 heterodimer protein (hMutSalpha). Both (cyclobutane-dimer)/AG and ((6-4)photoproduct)/AG mismatches competed about as well as non-photoproduct T/T mismatches. The two respective pairs of photoproduct/(A(T or C)) mismatches also showed higher hMutSalpha affinity than photoproduct/AA "matches"; the apparent affinity of hMutSalpha for the ((6-4)photoproduct)/AA-"matched" substrate was actually less than that for TT/AA homoduplexes. Surprisingly, although hMutSalpha affinities for both non-photoproduct UU/GG double mismatches and for (uracil-cyclobutane-dimer)/AG single mismatches were high, affinity for the (uracil-cyclobutane-dimer)/GG mismatch was quite low. Equilibrium binding of hMutSalpha to DNA containing (photoproduct/base) mismatches and to (T/G)-mismatched DNA was reduced similarly by ATP (in the absence of magnesium). (+info)
Anti-Toxoplasma gondii activities and structure-activity relationships of novel fluoroquinolones related to trovafloxacin.
(15/1786)
Eleven novel fluoroquinolones closely related to trovafloxacin were evaluated for their in vitro activity against Toxoplasma gondii, and their structure-activity relationships were examined. The 50% inhibitory concentration (IC50) of trovafloxacin against T. gondii was 2.93 microM; the IC50 of the 11 analogs ranged from 0.53 to 14. 09 microM. Six analogs had IC50s lower than that of trovafloxacin. Examination of the structure-activity relationships of the compounds revealed that addition of a -CH3 at C-5 of the 1,8-naphthyridone ring, at C-2 of the azabicyclohexane ring, or on the -NH2 at the 6 position of the azabicyclohexane ring resulted in a four- to sixfold increase in activity. Moreover, replacement of 2,4-difluorophenyl by cyclopropyl at N-1 of the 1,8-naphthyridone ring increased activity twofold, and moving the -NH2 one atom further away from the azabicyclohexane ring decreased activity. There was no difference between the naphthyridone and quinolone analogs. These results indicate that structure-activity studies of compounds related to drugs active against T. gondii may be useful in producing compounds with more potent activities against the parasite. (+info)
Post-replicative base excision repair in replication foci.
(16/1786)
Base excision repair (BER) is initiated by a DNA glycosylase and is completed by alternative routes, one of which requires proliferating cell nuclear antigen (PCNA) and other proteins also involved in DNA replication. We report that the major nuclear uracil-DNA glycosylase (UNG2) increases in S phase, during which it co-localizes with incorporated BrdUrd in replication foci. Uracil is rapidly removed from replicatively incorporated dUMP residues in isolated nuclei. Neutralizing antibodies to UNG2 inhibit this removal, indicating that UNG2 is the major uracil-DNA glycosylase responsible. PCNA and replication protein A (RPA) co-localize with UNG2 in replication foci, and a direct molecular interaction of UNG2 with PCNA (one binding site) and RPA (two binding sites) was demonstrated using two-hybrid assays, a peptide SPOT assay and enzyme-linked immunosorbent assays. These results demonstrate rapid post-replicative removal of incorporated uracil by UNG2 and indicate the formation of a BER complex that contains UNG2, RPA and PCNA close to the replication fork. (+info)