Determination of total potentially available nucleosides in bovine milk

Bovine colostrum and milk samples were collected from two herds over the course of the first month post-partum, pooled for each herd by stage of lactation and total potentially available nucleosides were determined. Sample analysis consisted of parallel enzymatic treatments, phenylboronate clean-up, and liquid chromatography to quantify contributions of nucleosides, monomeric nucleotides, nucleotide adducts, and polymeric nucleotides to the available nucleosides pool. Bovine colostrum contained high levels of nucleosides and monomeric nucleotides, which rapidly decreased as lactation progressed into transitional milk. Mature milk was relatively consistent in nucleoside and monomeric nucleotide concentrations from approximately the tenth day post-partum. Differences in concentrations between summer-milk and winter-milk herds were largely attributable to variability in uridine and monomeric nucleotide concentrations

Interaction of low-energy electrons with the purine bases, nucleosides, and nucleotides of DNA

The authors report results from computational studies of the interaction of low-energy electrons with the purine bases of DNA, adenine and guanine, as well as with the associated nucleosides, deoxyadenosine and deoxyguanosine, and the nucleotide deoxyadenosine monophosphate. Their calculations focus on the characterization of the pi* shape resonances associated with the bases and also provide general information on the scattering of slow electrons by these targets. Results are obtained for adenine and guanine both with and without inclusion of polarization effects, and the resonance energy shifts observed due to polarization are used to predict pi* resonance energies in associated nucleosides and nucleotides, for which static-exchange calculations were carried out. They observe slight shifts between the resonance energies in the isolated bases and those in the nucleosides

Phosphorylation of purine and pyrimidine nucleosides by isolated rat liver mitochondria

Formation of 5'-AMP, 5'-GMP, 5'-CMP and 5'UMP was confirmed in isolated rat liver mitochondria incubated with alpha-ketoglutarate, inorganic phosphate, purine nucleoside and pyrimidine nucleoside. Increased incorporation of 32Pi into ATP, GTP and UTP was observed by adding purine- and pyrimidine nucleosides. The phosphorylation of nucleosides was inhibited severely by arsenite and affected slightly by the addition of nuclear or post-mitochondrial fraction.</p

Unexpected Accumulation of ncm\u3csup\u3e5\u3c/sup\u3eU and ncm\u3csup\u3e5\u3c/sup\u3es\u3csup\u3e2\u3c/sup\u3eU in a \u3cem\u3etrm9\u3c/em\u3e Mutant Suggests an Additional Step in the Synthesis of mcm\u3csup\u3e5\u3c/sup\u3eU and mcm\u3csup\u3e5\u3c/sup\u3es\u3csup\u3e2\u3c/sup\u3eU

Background Transfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um), 5-methoxycarbonylmethyl-uridine (mcm5U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The formation of mcm5 and ncm5 side chains involves a complex pathway, where the last step in formation of mcm5 is a methyl esterification of cm5 dependent on the Trm9 and Trm112 proteins. Methodology and Principal Findings Both Trm9 and Trm112 are required for the last step in formation of mcm5 side chains at wobble uridines. By co-expressing a histidine-tagged Trm9p together with a native Trm112p in E. coli, these two proteins purified as a complex. The presence of Trm112p dramatically improves the methyltransferase activity of Trm9p in vitro. Single tRNA species that normally contain mcm5U or mcm5s2U nucleosides were isolated from trm9Δ or trm112Δ mutants and the presence of modified nucleosides was analyzed by HPLC. In both mutants, mcm5U and mcm5s2U nucleosides are absent in tRNAs and the major intermediates accumulating were ncm5U and ncm5s2U, not the expected cm5U and cm5s2U. Conclusions Trm9p and Trm112p function together at the final step in formation of mcm5U in tRNA by using the intermediate cm5U as a substrate. In tRNA isolated from trm9Δ and trm112Δ strains, ncm5U and ncm5s2U nucleosides accumulate, questioning the order of nucleoside intermediate formation of the mcm5 side chain. We propose two alternative explanations for this observation. One is that the intermediate cm5U is generated from ncm5U by a yet unknown mechanism and the other is that cm5U is formed before ncm5U and mcm5U

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients

Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.DFG, 390540038, EXC 2008: UniSysCatDFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

Synthesis of 1,4-Disubstituted Mono and Bis-triazolocarbo-acyclonucleoside Analogues of 9-(4-Hydroxybutyl)guanine by Cu(I)-Catalyzed Click Azide-Alkyne Cycloaddition

A series of novel mono-1,2,3-triazole and bis-1,2,3-triazole acyclonucleoside analogues of 9-(4-hydroxybutyl)guanine was prepared via copper(I)-catalyzed 1,3-dipolar cycloaddition of N-9 propargylpurine, N-1-propargylpyrimidines/as-triazine with the azido-pseudo-sugar 4-azidobutylacetate under solvent-free microwave conditions, followed by treatment with K2CO3/MeOH, or NH3/MeOH. All compounds studied in this work were screened for their antiviral activities [against human rhinovirus (HRV) and hepatitis C virus (HCV)] and antibacterial activities against a series of Gram positive and negative bacteria

Human immunodeficiency virus rebound after suppression to < 400 copies/mL during initial highly active antiretroviral therapy regimens, according to prior nucleoside experience and duration of suppression

This study evaluated 1433 human immunodeficiency virus (HIV)-infected patients starting highly active antiretroviral therapy (HAART), 409 (28%) of whom had prior nucleoside experience and achieved an HIV load of <400 copies/mL by 24 weeks of therapy. Three hundred seven patients experienced virus rebound during a total of 2773.3 person-years of follow-up. There was a higher rate of virus rebound among the patients with pre-HAART nucleoside experience (relative hazard [RH], 2.86; 95% confidence interval, 2.22-3.84; P < .0001) and a decreasing rate of virus rebound with increasing duration of virus suppression (i.e., time since achieving a virus load of <400 HIV RNA copies/mL) among both the nucleoside-experienced and naive patients (P < .0001), but the difference between the groups persisted into the third year of follow-up (P = .0007). Even patients who had experienced <2 months of nucleoside therapy before beginning HAART had an increased risk of virus rebound (RH, 1.95; P = .009). It appears that only a small period of pre-HAART nucleoside therapy is sufficient to confer a disadvantage, in terms of risk of virus rebound, that persists for several years