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Journal of Bacteriology Apr 1973Acriflavine uptake and resistance were investigated in red, sensitive Serratia marcescens cells and in orange, resistant mutant cells and their respective spheroplasts....
Acriflavine uptake and resistance were investigated in red, sensitive Serratia marcescens cells and in orange, resistant mutant cells and their respective spheroplasts. Acriflavine-sensitive cells bound more acriflavine than acriflavine-resistant cells. Spheroplasts from sensitive and resistant cells were both resistant to and bound similar amounts of acriflavine. Sensitive cells were resistant to acriflavine in medium supplemented with 0.01 M MgSO(4) and 0.5 M sucrose. In the presence of 0.01 M MgSO(4) and 0.5 M sucrose, acriflavine binding by sensitive cells was reduced to the level of binding by resistant cells. Inhibition of metabolism by carbon starvation, chloramphenicol, As(2)O(3), nitrosoguanidine, and bromouracil did not affect the uptake of acriflavine by sensitive and resistant cells. Rapid temperature changes did not alter the acriflavine-binding capacity of the cells, and no temperature dependence of acriflavine uptake or release was observed at 0 and 30 C. Acriflavine uptake by both sensitive and resistant cells increased with increase in pH from 5.7 to 8.0. The logarithm of acriflavine uptake was a linear function of the logarithm of the acriflavine concentration in the binding medium.
Topics: Acridines; Arsenic; Bromouracil; Chloramphenicol; Culture Media; Drug Resistance, Microbial; Glucose; Hydrogen-Ion Concentration; Mutation; Nitrosoguanidines; Oxides; Serratia marcescens; Spheroplasts; Temperature
PubMed: 4572726
DOI: 10.1128/jb.114.1.59-64.1973 -
Proceedings of the National Academy of... Feb 1958
PubMed: 16590151
DOI: 10.1073/pnas.44.2.112 -
Cell Cycle (Georgetown, Tex.) Feb 2017Transcriptional timing is inherently influenced by gene length, thus providing a mechanism for temporal regulation of gene expression. While gene size has been shown to...
Transcriptional timing is inherently influenced by gene length, thus providing a mechanism for temporal regulation of gene expression. While gene size has been shown to be important for the expression timing of specific genes during early development, whether it plays a role in the timing of other global gene expression programs has not been extensively explored. Here, we investigate the role of gene length during the early transcriptional response of human fibroblasts to serum stimulation. Using the nascent sequencing techniques Bru-seq and BruUV-seq, we identified immediate genome-wide transcriptional changes following serum stimulation that were linked to rapid activation of enhancer elements. We identified 873 significantly induced and 209 significantly repressed genes. Variations in gene size allowed for a large group of genes to be simultaneously activated but produce full-length RNAs at different times. The median length of the group of serum-induced genes was significantly larger than the median length of all expressed genes, housekeeping genes, and serum-repressed genes. These gene length relationships were also observed in corresponding mouse orthologs, suggesting that relative gene size is evolutionarily conserved. The sizes of transcription factor and microRNA genes immediately induced after serum stimulation varied dramatically, setting up a cascade mechanism for temporal expression arising from a single activation event. The retention and expansion of large intronic sequences during evolution have likely played important roles in fine-tuning the temporal expression of target genes in various cellular response programs.
Topics: Animals; Bromouracil; Conserved Sequence; Enhancer Elements, Genetic; Evolution, Molecular; Fibroblasts; Gene Expression Regulation; Genes; Humans; Male; Mice; MicroRNAs; Models, Biological; Oligonucleotide Array Sequence Analysis; Serum; Serum Response Factor; Time Factors; Transcription Factors; Transcription, Genetic; Uridine
PubMed: 28055303
DOI: 10.1080/15384101.2016.1234550 -
Journal of Nuclear Medicine : Official... Jul 2006Noninvasive imaging of a reporter gene is a new and promising technique to quantify transgene expression after gene therapy. This study was performed to demonstrate...
UNLABELLED
Noninvasive imaging of a reporter gene is a new and promising technique to quantify transgene expression after gene therapy. This study was performed to demonstrate visualization of lentiviral-marked cells by PET.
METHODS
We transduced nonhuman primate CD34+ hematopoietic cells with a lentiviral vector expressing a PET reporter gene, the mutant viral herpes simplex virus type 1-thymidine kinase (HSV1-sr39tk) gene. 1-(2-Fluoro-2-deoxy-beta-D-arabinofuranosyl)-76Br-5-bromouracil (76Br-FBAU) was used as the substrate for the viral tk enzyme. Upon phosphorylation, 76Br-FBAU was retained by cells and imaged by PET. The long half-life of 76Br, 16.2 h, permitted us to perform extended pharmacokinetic and imaging studies.
RESULTS
76Br-FBAU was retained in vascular tissues of the animals with transplanted tk lentiviral vector-transduced CD34+ cells. Elimination of 76Br-FBAU was through renal and hepatic excretion.
CONCLUSION
Noninvasive molecular imaging using PET will help us, in the future, to define the contribution and distribution of cells and their progeny to tissue repair and development.
Topics: Animals; Antigens, CD34; Bromine Radioisotopes; Cell Transplantation; Cyclotrons; Diagnostic Imaging; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Genetic Vectors; Lentivirus; Macaca; Mutation; Positron-Emission Tomography
PubMed: 16818958
DOI: No ID Found -
The Journal of Biological Chemistry Mar 2001The existence of interhalogen compounds was proposed more than a century ago, but no biological roles have been attributed to these highly oxidizing intermediates. In...
The existence of interhalogen compounds was proposed more than a century ago, but no biological roles have been attributed to these highly oxidizing intermediates. In this study, we determined whether the peroxidases of white blood cells can generate the interhalogen gas bromine chloride (BrCl). Myeloperoxidase, the heme enzyme secreted by activated neutrophils and monocytes, uses H2O2 and Cl(-) to produce HOCl, a chlorinating intermediate. In contrast, eosinophil peroxidase preferentially converts Br(-) to HOBr. Remarkably, both myeloperoxidase and eosinophil peroxidase were able to brominate deoxycytidine, a nucleoside, and uracil, a nucleobase, at plasma concentrations of Br(-) (100 microM) and Cl(-) (100 mM). The two enzymes used different reaction pathways, however. When HOCl brominated deoxycytidine, the reaction required Br(-) and was inhibited by taurine. In contrast, bromination by HOBr was independent of Br(-) and unaffected by taurine. Moreover, taurine inhibited 5-bromodeoxycytidine production by the myeloperoxidase-H2O2-Cl(-)- Br(-) system but not by the eosinophil peroxidase-H2O2-Cl(-)-Br(-) system, indicating that bromination by myeloperoxidase involves the initial production of HOCl. Both HOCl-Br(-) and the myeloperoxidase-H2O2-Cl(-)-Br(-) system generated a gas that converted cyclohexene into 1-bromo-2-chlorocyclohexane, implicating BrCl in the reaction. Moreover, human neutrophils used myeloperoxidase, H2O2, and Br(-) to brominate deoxycytidine by a taurine-sensitive pathway, suggesting that transhalogenation reactions may be physiologically relevant. 5-Bromouracil incorporated into nuclear DNA is a well known mutagen. Our observations therefore raise the possibility that transhalogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.
Topics: Bromine; Bromouracil; DNA Damage; Deoxycytidine; Humans; Hydrogen Peroxide; Hypochlorous Acid; Inflammation; Mutagens; Neutrophils; Oxidation-Reduction; Peroxidase; Uracil
PubMed: 11096071
DOI: 10.1074/jbc.M005379200 -
Nucleic Acids Research Jun 1988Decadeoxyribonucleotides containing uracil, 5-bromouracil, 5-cyanouracil and 5-ethyluracil in recognition sequences of restriction endonucleases Bgl II, Sau 3AI, Mbo I... (Comparative Study)
Comparative Study
Synthesis of decadeoxyribonucleotides containing 5-modified uracils and their interactions with restriction endonucleases Bgl II, Sau 3AI and Mbo I (nucleosides and nucleotides 82).
Decadeoxyribonucleotides containing uracil, 5-bromouracil, 5-cyanouracil and 5-ethyluracil in recognition sequences of restriction endonucleases Bgl II, Sau 3AI, Mbo I were synthesized. Decanucleotides containing 5-bromouracil in place of thymine had essentially the same susceptibility to all the restriction endonucleases. Uracil-containing decanucleotides were however very resistant to attack. Decanucleotides containing 5-cyanouracil in the recognition sequence were strongly resistant to hydrolysis by Sau 3AI, but were hydrolysed by Bgl II and Mbo I as well as the parent decanucleotide. Decanucleotides containing 5-ethyluracil were strongly resistant to hydrolysis by Sau 3AI, but were partially resistant to hydrolysis by Bgl II and Mbo I.
Topics: Bacterial Proteins; Bromouracil; Circular Dichroism; DNA Restriction Enzymes; Deoxyribonucleases, Type II Site-Specific; Deoxyribonucleotides; Structure-Activity Relationship; Substrate Specificity; Uracil
PubMed: 2838807
DOI: 10.1093/nar/16.11.4761 -
The Journal of Biological Chemistry Jan 1981Rat liver dihydrothymine dehydrogenase, the rate-limiting enzyme of thymidine and uridine degradation, was purified to homogeneity as judged by polyacrylamide disc gel...
Rat liver dihydrothymine dehydrogenase, the rate-limiting enzyme of thymidine and uridine degradation, was purified to homogeneity as judged by polyacrylamide disc gel electrophoresis, sedimentation velocity, and Ultrogel ACA-34 elution profile. The enzyme has a molecular weight of 220,000 +/- 5,000 as determined by Ultrogel ACA-34 and sedimentation equilibrium The s20,w value of the enzyme was 9.2 S. The isoelectric point was at pH 5.25. The enzyme is composed of two identical subunits of an approximate molecular weight of 110,000 +/- 3,000 as determined by sodium dodecyl sulfate disc gel electrophoresis. The enzyme contains 4 mol of FAD and 3 mol of iron per mol of enzyme. Flavin released from the enzyme by boiling was identified as FAD by absorption spectra and thin layer chromatography, indicating that the enzyme is a flavometal protein. During dialysis, the enzyme was stabilized by 2-mercaptoethanol, but neither NADPH nor thymine was effective. The relative rates of reduction of pyrimidine analogues substituted at position 5 were 5-fluorouracil > 5-bromouracil > 5-diazouracil > 5-iodouracil > 5-nitrouracil, with 5-fluorouracil and 5-diazouracil 70% faster than thymine. Uracil was reduced 25% faster than thymine. The pH optimum for the forward and reverse reactions was 7.4. In the presence of NADPH, the apparent Km was 2.6 microM for thymine and 1.8 microM for uracil. Apparent Km for NADPH was 15 microM with thymine as substrate and 11 microM with uracil. In the reverse reaction, apparent Km values were 43 microM for dihydrothymine and 193 microM for dihydrouracil; apparent Km for NADP+ was 3.8 microM with dihydrothymine as substrate and 2.9 microM with dihydrouracil.
Topics: Amino Acids; Animals; Dihydrouracil Dehydrogenase (NADP); Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Kinetics; Liver; Molecular Weight; Oxidoreductases; Rats; Spectrophotometry; Substrate Specificity; Uracil
PubMed: 7451435
DOI: No ID Found -
Journal of Chromatography Feb 1986The gas chromatographic-mass spectrometric method using selected-ion monitoring (GC-MS-SIM) described here quantitatively determines the amount of DNA thymine...
The gas chromatographic-mass spectrometric method using selected-ion monitoring (GC-MS-SIM) described here quantitatively determines the amount of DNA thymine replacement by 5-bromouracil (BU) after exposure to 5-bromo-2'-deoxyuridine (BUDR) in as few as 10(5) cells. DNA is extracted, enzymatically hydrolyzed, the nucleic acid bases (with added internal standards, 5-iodouracil and 5-chlorouracil) are extracted into ethyl acetate, concentrated and derivatized with bis(trimethylsilyl)trifluoroacetamide. Thymine and BU are then quantitated by GC-MS-SIM. Response is linear to thymine over the range of 100-2000 ng per sample and BU of 1.3-52 ng per sample with a coefficient of variation of less than 10% and an accuracy for seeded samples within 8% of theoretical value. With V79 cells in culture, exposure to increasing BUDR concentrations (0.03-1.0 microM) results in increasing thymine substitution by BU over a range of 1-28%. Other important applications of this technique are mentioned.
Topics: Bromodeoxyuridine; Bromouracil; DNA; Gas Chromatography-Mass Spectrometry; Hydrolysis; Indicators and Reagents; Thymine
PubMed: 3958104
DOI: 10.1016/s0378-4347(00)83686-5 -
Journal of Chromatography Nov 1988A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2'-deoxyuridine (BUdR) and...
A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2'-deoxyuridine (BUdR) and 5-bromouracil (BU) in plasma. BUdR and BU were first extracted with a mixture of ethyl acetate and 2-propanol from plasma presaturated with solid ammonium sulfate. Following evaporation of the organic extract, the remaining residue was reconstituted in saturated ammonium sulfate solution, washed with a mixture of n-pentane-methylene chloride and re-extracted with the original solvent mixture. The organic extract was evaporated, reconstituted in mobile phase and chromatographed on a regular-bore ODS HPLC column using ultraviolet absorbance detection. The BUdR and BU quantification limits were both 0.1 microM, the mean intra-assay coefficients of variation were 5.0 and 5.6%, respectively, and the mean inter-assay coefficients of variation were 5.4 and 10.7%, respectively. This method was used to determine steady-state femoral arterial and hepatic venous plasma concentrations of BUdR and BU in a patient receiving a continuous intravenous infusion of BUdR (20 mg/kg per day).
Topics: Aged; Bromodeoxyuridine; Bromouracil; Chromatography, High Pressure Liquid; Colonic Neoplasms; Humans; Indicators and Reagents; Infusions, Intravenous; Liver Neoplasms; Male; Uracil
PubMed: 3220891
DOI: 10.1016/s0378-4347(00)80648-9 -
Journal of Bacteriology Apr 1974The effect of thymine-5-bromouracil substitution on the regeneration and length of F pili produced by an F(+)Lac(+)/Lac(-)Thy(-) strain of Escherichia coli was studied...
The effect of thymine-5-bromouracil substitution on the regeneration and length of F pili produced by an F(+)Lac(+)/Lac(-)Thy(-) strain of Escherichia coli was studied by electron microscopy. When 5-bromouracil (5BU) incorporation into deoxyribonucleic acid (DNA) was maximal, the modal length of the pilus doubled and the number of pili per cell was approximately 50% that of thymine-grown cells. The ability of 5BU-grown cells to form mating pairs and to be infected by ribonucleic acid (R17) and DNA (M13) male-specific phages was also reduced by approximately 50%. Loss of function was not due to loss of sex factor as 5BU cells retained a sex factor that was susceptible to curing by acridine orange. Elongation of pili on 5BU-grown cells was more sensitive to irradiation at 253.7 nm than on thymine-grown cells, suggesting that DNA is the sensitive target.
Topics: Acridines; Bromouracil; Coliphages; Conjugation, Genetic; Culture Media; DNA, Bacterial; Escherichia coli; Lysogeny; Microscopy, Electron; Radiation Effects; Recombination, Genetic; Thymine; Ultraviolet Rays
PubMed: 4595195
DOI: 10.1128/jb.118.1.175-179.1974