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Salvage of Circulating Pyrimidine Nucleosides in the Rat1

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[CANCER RESEARCH 41, 3010-3017, August 1981] 0008-5472/81 /0041-OOOOS02.00 Salvage of Circulating Pyrimidine Nucleosides in the Rat1 James D. Moyer, James T. Oliver, and Robert E. Handschumacher2 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 [J. D. M., R. E. H.], and Boehringer Ingelheim Research and Development Center, Ridgefield. Connecticut 06877 [J. T. O.] ABSTRACT infusions of radiolabeled tracers to measure the salvage of these nucleosides by various tissues. The contribution of cir A new procedure was developed to measure uridine and culating pyrimidine nucleosides to the nucleotide pools has cytidine in plasma. These nucleosides are present in micro- been examined in relation to the requirements of slowly growing molar concentrations in the plasma of rats, mice, and humans. tissues for the maintenance of pools and also to the needs of Inhibitors of pyrimidine synthesis de novo (pyrazofurin or N- rapidly dividing tissue undertaking net synthesis of RNA and phosphonacetyl-L-aspartate) produce only modest decreases DMA. in the concentration of circulating uridine or cytidine in the rat. Since both uridine and cytidine are rapidly cleared from the MATERIALS AND METHODS circulation of the rat, constant infusions of radiolabeled uridine and cytidine were used to establish a steady-state specific Drugs and Chemicals. Succinic anhydride and pyridine were activity of circulating nucleoside without altering the normal purchased from Pierce Chemical Co. (Rockford, III.) . Nucleo sides, nucleotides, heparin, and 2',3',5'-triacetyl uridine were endogenous concentration. These studies permitted an esti mation of the contribution of circulating pyrimidine nucleoside purchased from Sigma Chemical Co. (St. Louis, Mo.). Acetic anhydride, m-aminophenylboronic acid hemisulfate, and 1- to the nucleotide pools of various rat tissues. Most of the uridine entering the circulation (>70%) is catabolized rather ethyl-3-(3-dimethylaminopropyl) carbodimide hydrochloride than salvaged by formation of nucleotides. Cytidine in the were obtained from Aldrich Chemical Co. (Milwaukee, Wis.). circulation is much more efficiently utilized and is predomi Methanol for HPLC buffers was purchased from Burdick & Jackson Laboratories, Inc., (Muskegon, Mich.). [5-3H]Uridine nantly salvaged. The implication of these results for chemo (27.9 Ci/mmol) and [5-3H]cytidine (27.7 Ci/mmol) were pur therapy based on inhibition of pyrimidine synthesis de novo is discussed. chased from New England Nuclear (Boston, Mass.). HPLC. Chromatography was performed with Altex 110A INTRODUCTION pumps, an Altex 420 microprocessor to generate gradients, and 2 Altex 8-/il flow cell UV detectors monitoring 280 nm and Separate enzymatic pathways exist in mammalian cells to 254 nm with analog output on a dual channel Linear Corp. generate nucleotides by de novo synthesis or by salvage of Model 485 recorder. preformed bases or nucleosides (12,19). Although it has been Anion-Exchange Chromatography of Uridine. This Chro suggested that bone marrow and intestinal mucosa are primar matographie system is a modification of a system described by ily dependent on the salvage pathway to obtain purine nucleo Singhal and Conn (26). The use of the small particle size resin tides (15, 17) and that the liver provides preformed purines has produced greater resolution and sensitivity. A 25- x 0.46- and pyrimidines to other tissues through the circulatory system cm column of HAX-4 anion-exchange resin (Hamilton Co., (15, 16, 23), the extent to which a particular tissue depends Reno, Nev.) was slurry packed, maintained at 50°,and eluted on each pathway has not been firmly established. A more with 0.3 M acetic acid:ammonium acetate (pH 9.7) at 1.0 ml/ complete understanding of the balance between alternate path min. Peak height was directly proportional to amount injected ways would help guide the proper chemotherapeutic use of between 0.1 and 5 nmol. The following retention times were PF3 or PALA, inhibitors of the de novo pathway, and p-nitro- observed: cytidine, 2.1 min; adenosine, 4.2 min; thymidine, benzyl-thioinosine (31) or inhibitors of uridine kinase, which 10.8 min; pseudouridine, 17.4 min; deoxyuridine, 17.6 min; may block nucleoside salvage. Quantitating the low levels of uridine, 22 min; PF, 34 min; uracil, 41 min; guanosine, 48 min; circulating pyrimidine nucleosides has been a major difficulty and inosine, 84 min. The Chromatographie efficiency for uridine in studying nucleoside salvage in vivo. Radioimmunoassays was 1200 theoretical plates. Multiple injections of standards (13), microbiological assays (20, 22), and multistep procedures indicated that peak height variation was ±2% (S.D.). involving thin-layer chromatography and derivatization (8, 27, The resin was regenerated after processing 50 biological 29) have been used previously to measure pyrimidine nucleo samples by 30-min treatment with 50% sulfuric acid at 70°. sides. Recently, several HPLC methods have been developed Although human, horse, or mouse plasma samples (pretreated to permit more rapid quantitation of nucleoside levels (10, 14, by borate affinity column separation as described below) gave 25). base line separation in most instances, in 5 of 27 rat plasma In the current study, we have devised a method for quanti extracts, an overlapping peak at 18 min prevented accurate tation of circulating uridine and cytidine and used constant measurement of uridine. Reverse-Phase Chromatography of Nucleosides and 1 Supported by American Cancer Society Grant CH-67T. Bases. A 25- x 0.46-cm Lichrosorb 5-jum particle size column 2 To whom requests for reprints should be addressed. (Altex Co., Berkeley, Calif.) was eluted at 1.0 ml/min with 0.2 3 The abbreviations used are: PF. pyrazofurin; PALA. N-phosphonacetyl-L- M sodium phosphate (pH 6.4) at 25°. The following retention aspartate; HPLC, high-performance liquid chromatography. Received January 19, 1981; accepted April 22, 1981. times were observed: AMP, GMP, UMP, CMP, uric acid, uracil, 3010 CANCER RESEARCH VOL. 41 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1981 American Association for Cancer Research. Circulating Undine and Cytidine in the Rat and cytosine, all <7.5 min; cytidine, 8.5 min; uridine, 14 min; and chromatographed on the HAX-4 column as described deoxycytidine, 14 min; deoxyuridine, 23 min; thymidine, 35 above, and 1-ml fractions were collected and counted in 10 ml min; and adenine, 30 min. Peak height was directly proportional of Formula 963 (New England Nuclear). to amount injected in the range of 0.1 to 1.0 nmol at a sensitivity Disposition of [5-3H]Uridine and [5-3H]Cytidine in the Rat. of 0.005 absorbance units, full scale. Male Sprague-Dawley rats (250 to 350 g) were anesthetized Analysis of RNA Hydrolysate by Anión Exchange. Incor with methoxyflurane, and catheters were placed in the jugular poration of [5-3H]uridine into RNA was assayed by separation vein and the carotid artery. [5-3H]Uridine (27.9 Ci/mmol) was of the 2',3'-mononucleotides resulting from alkaline hydrolysis prepared in sterile 0.9% NaCI solution, and 0.1 mCi was on a 25-cm BAX-4 (Benson Co., Reno, Nev.) column at 50° injected via the jugular vein in a volume of 0.2 ml. At the with a linear gradient from 0.6 to 1.0 M ammonium acetate, pH indicated times, 100 p.\ of blood from the carotid artery were 8.4, for 35 min during a period of 20 min and subsequent mixed with 400 ¡i\of ice-cold 0.5 M HCIO4, and the resulting isocratic elution with 1 M buffer at 2 ml/min. extract was processed and chromatographed sequentially on Cation-Exchange Chromatography of Cytidine. A 25- x the borate affinity resin and on BAX-4 anion-exchange resin as 0.46-cm column packed with BPAN6 resin (Benson Co.) was described above. Radioactivity was determined in 10 ml of eluted at 50°with a flow of 1.5 ml/min with 0.3 M formic acid Formula 963 at 24% efficiency. adjusted to pH 4.0 with ammonium hydroxide. The following Similar experiments were performed with [5-3H]cytidine. The retention times were observed: void, 2.0 min; uracil, 2.4 min; neutralized HCIO4 extract of blood (50 to 200 pi) was chromat uridine, 2.6 min; guanosine, 5.8 min; adenosine, 15 min; cyti- ographed directly on a 25-cm BPAN6 cation-exchange column dine, 24 min; adenine, 55 min; and cytosine, 57 min. For as described above. Fractions of 3 ml (2 min) were collected, quantitation of unlabeled cytidine in extracts, a better resolution and radioactivity was determined by scintillation spectrometry. from the UV-absorbing components was obtained with a 50- x Infusion of [5-3H]Uridine or [5-3H]Cytidine. Catheters con 0.46-cm column eluted at 60°with 0.5 M ammonium formate, taining heparinized (100 units/ml) 0.9% NaCI solution were pH 4.0, at 0.8 ml/min (4000 theoretical plates). Peak height placed in the carotid artery and left jugular vein of a male was proportional to the amount injected over the range of 0.2 Sprague-Dawley rat (250 g) that had been anesthetized with to 2 nmol at a sensitivity of 0.01 absorbance units, full scale. methoxyflurane. The catheters were protected by a spring and Determination of Uridine and Cytidine in Plasma. Plasma attached to swivels to permit sustained infusion in conscious from heparinized blood was mixed with an equal volume of ice- animals. [5-3H]Uridine or [5-3H]cytidine (40pCi/ml) was infused cold 1 M HCIO4 and kept at 4°for 20 min. The precipitated via the jugular vein at 2.3 ml/hr. Carotid blood was obtained at protein was removed, and the supernatant was neutralized to 30-min intervals for determination of radiolabeled nucleoside.
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  • N4-Acetylcytidine and 5-Formylcytidine Are Present in Saccharomyces Cerevisiae Mrnas

    N4-Acetylcytidine and 5-Formylcytidine Are Present in Saccharomyces Cerevisiae Mrnas

    bioRxiv preprint doi: https://doi.org/10.1101/327585; this version posted May 21, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. N4-acetylcytidine and 5-formylcytidine are present in Saccharomyces cerevisiae mRNAs Mehmet Tardu1, Qishan Lin2, and Kristin S. Koutmou1* 1University of Michigan, Department of Chemistry, 930 N University, Ann Arbor, MI 48109, (734) 764-5650, [email protected], 2Mass Spectrometry Consortium for Epitranscriptomics, University at Albany, 1400 Washington Ave, Albany, NY 12222. Abstract Chemical modifications of RNAs have long been appreciated as key modulators of non-coding RNA structure and function. There is an emerging realization that chemical modification of protein-coding mRNAs also plays critical roles in the cell. Nonetheless, of the over 100 known RNA modifications found in biology only a handful have been identified in mRNAs. Here we use an ultra-high performance liquid chromatography- tandem mass spectrometry (UHPLC-MS/MS) method to identify and quantify modifications present in mRNAs of yeast cells. We detect the presence of four modified nucleosides in mRNAs at relatively high abundances: N7-methylguanosine, N6- methyladenosine, N4-acetylcytidine and 5-formylcytidine. Additionally, we investigate how the levels of mRNA modifications change in response to cellular stress. We find that the concentrations of nine mRNA modifications including N6-methyladenosine and N4- acetylcytidine change in response to heat stress, glucose starvation and/or oxidative stress. Our findings suggest that mRNA modification may provide a potential mechanism for cells to rapidly respond to environmental stressors.