Quantitative RP high-performance liquid chromatography of thymine and thymidine (original) (raw)
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Applied and Environmental Microbiology, 1988
The relationship between bacterial growth and incorporation of [methyl-3lHlthymidine in oligotrophic lake water cultures was investigated. Prescreening, dilution, and addition of organic and inorganic nutrients were treatments used to prevent bacterivory and stimulate bacterial growth. Growth in unmanipulated samples was estimated through separate measurements of grazing losses. Both bacterial number and biovolume growth responses were measured, and incorporation of [3H]thymidine in both total macromolecules and nucleic acids was assayed. The treatments had significant effects on conversion factors used to relate thymidine incorporation to bacterial growth. Cell number-based factors ranged from 1.1 x 1018 to 38 x 1018 cells mol of total thymidine incorporation-l and varied with treatment up to 10-fold for the same initial bacterial assemblage. In contrast, cell biovolume-based conversion factors were similar for two treatment groups across a 16-fold range of [3H]thymidine incorporation rates: 5.54 x 10'7 ,Im3 mol of total thymidine incorporation-' and 15.2 x 1017 ,um3 mol of nucleic acid incorporation-'. Much of the variation in cell number-based conversion factors was related to changes in apparent mean cell volume of produced bacteria. Phosphorus addition stimulated [3H]thymidine incorporation more than it increased bacterial growth, which resulted in low conversion factors.
Applied and environmental microbiology, 1988
The relationship between bacterial growth and incorporation of [methyl-H]thymidine in oligotrophic lake water cultures was investigated. Prescreening, dilution, and addition of organic and inorganic nutrients were treatments used to prevent bacterivory and stimulate bacterial growth. Growth in unmanipulated samples was estimated through separate measurements of grazing losses. Both bacterial number and biovolume growth responses were measured, and incorporation of [H]thymidine in both total macromolecules and nucleic acids was assayed. The treatments had significant effects on conversion factors used to relate thymidine incorporation to bacterial growth. Cell number-based factors ranged from 1.1 x 10 to 38 x 10 cells mol of total thymidine incorporation and varied with treatment up to 10-fold for the same initial bacterial assemblage. In contrast, cell biovolume-based conversion factors were similar for two treatment groups across a 16-fold range of [H]thymidine incorporation rates:...
Journal of Chromatography A, 1980
Cyclobutadipyrimidines (pyrimidine dimers, Pyr < > Pyr) (the abbreviation system used is that proposed in ref. 1) represent the major class of DNA lesions induced by the action of fm and near uhraviolet (I_JV) light on biological systemsz4. The four stereoisomeric thymine dimers (Thy < > Thy) produced by UV irradiation of Thy have been separated by thin-layer chromatography (ILQs and by gas-liquid chromatography (GLC) after N-methylation 6. Surprisingly, in view of the large number of separations of mixtures of nucleic acid components7-'o and modified bases or nuckosides"-'5 by high-performance liquid chromatography (HPLC) using chemically bonded reversed-phase packings, to our knowledge the only reported separation of pyrimidine dimers (cis_svn Thy < > Thy and Ura < > Thy) by HPLCF was by ion exchange. This paper reports the deveIopment of an HPLC method for rapid and complete separation of the four Thy c> Thy dimers on a conventional octadecyl reversed-phase column and on the new radial-PAK A cartridge with the Waters radial compression separaticn system (RC 55). Separations of the optically active stereoisomers as well as the meso pairs of cyclobutadithymidine (thymidine dimers, Thd < > Thd) by reversed-phase HPLC and two-dimensional TLC are also described. MAERIAIS AND METHODS High-performance Iiquid citronzfzrography A Model HIOOA dual-piston pump, a Model U6K universal injector (Waters Assoc.
Applied and Environmental Microbiology, 1990
The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited signfficant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which .95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-3H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [3H]thymidine experiments and compared the degree of nonspecific labeling by [3Hladenine with that derived from [3H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.
ACTA Pharmaceutica Sciencia, 2018
INTRODUCTION Nucleosides are key compounds involved in major biological processes, such as nucleic acids and proteins synthesis, cell signaling, enzyme regulation, and metabolism. Nucleoside and their derivatives have emerged as molecules with ABSTRACT In search of new leads toward potent antibacterial agents; therefore, a series of thymidine analogues were synthesized by direct acylation method and furnished the 5´-O-acyl thymidine derivatives in good yield. A number of acyl derivatives were prepared in order to obtain a series of newer components for antibacterial screening experiments. The synthesized compounds were characterized by their FTIR, 1 HNMR spectral data and elemental analysis. These thymidine derivatives were evaluated for in vitro antibacterial screening studies against a number of human pathogenic microorganisms by disc diffusion method. The study revealed that most of the tested chemicals exhibited moderate to good antibacterial activities. It was also observed that the test chemical 2-bromobenzoyl derivative 11 very significantly inhibited the growth of all Gram-positive and Gram-negative bacterial strains used. For comparative studies, antibacterial activity of standard antibiotics, Azithromycin was also carried out against these microorganisms. Hence, these thymidine derivatives can be used to discover antibacterial agents that may serve as leads in the development of new pharmaceuticals research activities.
A synthetic hapten for induction of thymine-dimer-specific antibodies
European Journal of Biochemistry, 1984
High specificity and sensitivity of thymine cyclobutane dimer (thy[]thy) detection were obtained by a radioimmunoassay. Attempts to raise thy[]thy-monospecific antibodies with antigens produced according to conventional methods were unsuccessful. Thy[]thy-specific antibodies could only be raised by using a new strategy to bind thy[]thy to protein: thymine was activated by trimethylsilylation and alkylated at N1 yielding N1thyminebutanoic acid which was dimerised by ultraviolet treatment. The resulting derivative of thymine cyclobutane dimer was coupled to bovine serum albumin by the active-ester method. The new strategy appears to be generally applicable for binding haptens, such as DNA bases, photoproducts etc. to proteins via a derivative containing a carboxyl group. Immunisation of rabbits with the thy[]thy-bovine-serum-albumin conjugate prepared by the new method resulted in a highly specific antiserum which allows detection of thy[]thy down to 0.06 p mol