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Nucleic Acids Research Apr 2022The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their...
The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU. These appended amino acids are further sculpted by various enzyme classes such as radical SAM isomerases, PLP-dependent decarboxylases, flavin-dependent lyases and acetyltransferases. The combinatorial permutations of thymidine hypermodification genes found in viral metagenomes from geographically widespread sources suggests an untapped reservoir of chemical diversity in DNA hypermodifications.
Topics: Amino Acids; Bacteriophages; DNA; Lyases; Thymidine
PubMed: 34522950
DOI: 10.1093/nar/gkab781 -
Cells Jun 2022Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains...
Tagging proliferating cells with thymidine analogs is an indispensable research tool; however, the issue of the potential in vivo cytotoxicity of these compounds remains unresolved. Here, we address these concerns by examining the effects of BrdU and EdU on adult hippocampal neurogenesis and EdU on the perinatal somatic development of mice. We show that, in a wide range of doses, EdU and BrdU label similar numbers of cells in the dentate gyrus shortly after administration. Furthermore, whereas the administration of EdU does not affect the division and survival of neural progenitor within 48 h after injection, it does affect cell survival, as evaluated 6 weeks later. We also show that a single injection of various doses of EdU on the first postnatal day does not lead to noticeable changes in a panel of morphometric criteria within the first week; however, higher doses of EdU adversely affect the subsequent somatic maturation and brain growth of the mouse pups. Our results indicate the potential caveats in labeling the replicating DNA using thymidine analogs and suggest guidelines for applying this approach.
Topics: Animals; Bromodeoxyuridine; Cell Count; Cell Proliferation; Mice; Neurogenesis; Thymidine
PubMed: 35741018
DOI: 10.3390/cells11121888 -
Future Medicinal Chemistry Apr 2013The compound class of 3-carboranyl thymidine analogues (3CTAs) are boron delivery agents for boron neutron capture therapy (BNCT), a binary treatment modality for... (Review)
Review
The compound class of 3-carboranyl thymidine analogues (3CTAs) are boron delivery agents for boron neutron capture therapy (BNCT), a binary treatment modality for cancer. Presumably, these compounds accumulate selectively in tumor cells via intracellular trapping, which is mediated by hTK1. Favorable in vivo biodistribution profiles of 3CTAs led to promising results in preclinical BNCT of rats with intracerebral brain tumors. This review presents an overview on the design, synthesis, and biological evaluation of first- and second-generation 3CTAs. Boronated nucleosides developed prior to 3CTAs for BNCT and non-boronated N3-substituted thymidine conjugates for other areas of cancer therapy and imaging are also described. In addition, basic features of carborane clusters, which are used as boron moieties in the design and synthesis of 3CTAs, and the biological and structural features of TK1-like enzymes, which are the molecular targets of 3CTAs, are discussed.
Topics: Animals; Boranes; Boron Neutron Capture Therapy; Humans; Molecular Docking Simulation; Neoplasms; Nitrogen; Prodrugs; Thymidine; Thymidine Kinase
PubMed: 23617430
DOI: 10.4155/fmc.13.31 -
Molecules (Basel, Switzerland) Sep 2011Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into... (Review)
Review
Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.
Topics: Animals; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Click Chemistry; DNA; Deoxyuridine; Fluorescent Dyes; Humans; Staining and Labeling; Stem Cell Niche; Thymidine
PubMed: 21921870
DOI: 10.3390/molecules16097980 -
The Journal of Biological Chemistry Nov 2021Detection of thymidine analogues after their incorporation into replicating DNA represents a powerful tool for the study of cellular DNA synthesis, progression through... (Review)
Review
Detection of thymidine analogues after their incorporation into replicating DNA represents a powerful tool for the study of cellular DNA synthesis, progression through the cell cycle, cell proliferation kinetics, chronology of cell division, and cell fate determination. Recent advances in the concurrent detection of multiple such analogues offer new avenues for the investigation of unknown features of these vital cellular processes. Combined with quantitative analysis, temporal discrimination of multiple labels enables elucidation of various aspects of stem cell life cycle in situ, such as division modes, differentiation, maintenance, and elimination. Data obtained from such experiments are critically important for creating descriptive models of tissue histogenesis and renewal in embryonic development and adult life. Despite the wide use of thymidine analogues in stem cell research, there are a number of caveats to consider for obtaining valid and reliable labeling results when marking replicating DNA with nucleotide analogues. Therefore, in this review, we describe critical points regarding dosage, delivery, and detection of nucleotide analogues in the context of single and multiple labeling, outline labeling schemes based on pulse-chase, cumulative and multilabel marking of replicating DNA for revealing stem cell proliferative behaviors, and determining cell cycle parameters, and discuss preconditions and pitfalls in conducting such experiments. The information presented in our review is important for rational design of experiments on tracking dividing stem cells by marking replicating DNA with thymidine analogues.
Topics: Animals; Cell Cycle; Cell Self Renewal; Cell Tracking; DNA Replication; Humans; Stem Cells; Thymidine
PubMed: 34717955
DOI: 10.1016/j.jbc.2021.101345 -
The Journal of Organic Chemistry Oct 2017Thymidine radical cation (1) is produced by ionizing radiation and has been invoked as an intermediate in electron transfer in DNA. Previous studies on its structure and...
Thymidine radical cation (1) is produced by ionizing radiation and has been invoked as an intermediate in electron transfer in DNA. Previous studies on its structure and reactivity have utilized thymidine as a precursor, which limits quantitative product analysis because thymidine is readily reformed from 1. In this investigation, radical cation 1 is independently generated via β-heterolysis of a pyrimidine radical generated photochemically from an aryl sulfide. Thymidine is the major product (33%) from 1 at pH 7.2. Diastereomeric mixtures of thymidine glycol and the corresponding 5-hydroxperoxides resulting from water trapping of 1 are formed. Significantly lower yields of products such as 5-formyl-2'-deoxyuridine that are ascribable to deprotonation from the C5-methyl group of 1 are observed. Independent generation of the N3-methyl analogue of 1 (NMe-1) produces considerably higher yields of products derived from water trapping, and these products are formed in much higher yields than those attributable to the C5-methyl group deprotonation in NMe-1. N3-Methyl-thymidine is, however, the major product and is produced in as high as 70% yield when the radical cation is produced in the presence of excess thiol. The effects of exogenous reagents on product distributions are consistent with the formation of diffusively free radical cations (1, NMe-1). This method should be compatible with producing radical cations at defined positions within DNA.
Topics: Cations; Free Radicals; Molecular Conformation; Thymidine
PubMed: 28994287
DOI: 10.1021/acs.joc.7b02017 -
Chembiochem : a European Journal of... Jun 2021Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The widespread application of...
Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The widespread application of 2-selenopyrimidine nucleosides is currently limited by low yields in established synthetic routes. Herein, we describe the optimization of the synthesis of 2-Se-uridine and 2-Se-thymidine derivatives by thermostable nucleoside phosphorylases in transglycosylation reactions using natural uridine or thymidine as sugar donors. Reactions were performed at 60 or 80 °C and at pH 9 under hypoxic conditions to improve the solubility and stability of the 2-Se-nucleobases in aqueous media. To optimize the conversion, the reaction equilibria in analytical transglycosylation reactions were studied. The equilibrium constants of phosphorolysis of the 2-Se-pyrimidines were between 5 and 10, and therefore differ by an order of magnitude from the equilibrium constants of any other known case. Hence, the thermodynamic properties of the target nucleosides are inherently unfavorable, and this complicates their synthesis significantly. A tenfold excess of sugar donor was needed to achieve 40-48 % conversion to the target nucleoside. Scale-up of the optimized conditions provided four Se-containing nucleosides in 6-40 % isolated yield, which compares favorably to established chemical routes.
Topics: Biocatalysis; Glycosylation; Molecular Structure; Nucleosides; Organoselenium Compounds; Pentosyltransferases; Thermodynamics; Thymidine
PubMed: 33594780
DOI: 10.1002/cbic.202100067 -
Environmental Health Perspectives May 1989Evidence has accumulated showing that active oxygen species participate in at least one stage of tumor promotion. Tumor promoters can induce various types of cells to... (Review)
Review
Evidence has accumulated showing that active oxygen species participate in at least one stage of tumor promotion. Tumor promoters can induce various types of cells to undergo processes that result in formation of active oxygen species. They stimulate polymorphonuclear leukocytes (PMNs) to undergo an oxidative burst that is characterized by rapid formation of .O2- and H2O2. We find that in vitro formation of H2O2 by tumor promoter-activated PMNs correlates with their in vivo first-stage promoting activity. Moreover, two thymidine derivatives are formed in DNA coincubated with tumor promoter-stimulated PMNs: 5-hydroxymethyl-2'-deoxyuridine (HMdU) and thymidine glycol (dTG). The amounts of HMdU and dTG formed correlate with the first-stage tumor-promoting potencies of the agents used for PMN stimulation and with the amount of H2O2 generated. We find that HMdU is also formed in the DNA of HeLa cells coincubated with 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated PMNs, with the amount of HMdU being proportional to that of TPA used. Even in the absence of PMNs, HMdU is increasingly formed in cellular DNA with increased TPA concentration, although at much lower levels than in the presence of PMNs. When rat liver microsomes are incubated with benzo[a]pyrene (BaP), a complete carcinogen, H2O2 is also generated. Production of H2O2 increases linearly with increasing concentrations of BaP. Furthermore, HMdU is formed in DNA exposed to BaP-treated microsomes, and its formation is inhibited by catalase. These results suggest that carcinogen-induced processes generating H2O2 are associated with the first-stage promoting activity of complete carcinogens.
Topics: Animals; Carcinogens; DNA; Humans; Hydrogen Peroxide; Neutrophils; Oxidation-Reduction; Plasma; Thymidine
PubMed: 2667984
DOI: 10.1289/ehp.898145 -
Therapeutic role of nitroglycerin against copper-nitrilotriacetate induced hepatic and renal damage.Human & Experimental Toxicology 2022Earlier we have shown that exposure to copper-nitrilotriacetate (Cu-NTA) manifests toxicity by generating oxidative stress and potent induction of proliferative reaction...
Earlier we have shown that exposure to copper-nitrilotriacetate (Cu-NTA) manifests toxicity by generating oxidative stress and potent induction of proliferative reaction in the liver and kidney. In the study, we look at the impact of nitroglycerin (GTN) administration on Cu-NTA-induced oxidative stress and hyperproliferative response in the liver and kidney. GTN administration intraperitoneally to male Wistar rats after Cu-NTA administration intraperitoneally caused substantial protection against Cu-NTA-induced tissue injury, oxidative stress and hyperproliferative response. Cu-NTA administration at a dose of 4.5 mg/kg body weight produces significant ( < .001) elevation in biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CREA) with a concomitant increase in microsomal lipid peroxidation. Along with these alterations, we discovered a substantial increment in [H]thymidine incorporation into hepatic and renal DNA synthesis ( < .001). Cu-NTA-induced tissue damage and lipid peroxidation in hepatic and renal tissues were inhibited by GTN treatment in a dose-dependent manner ( < .05-0.001). Furthermore, GTN can suppress the hyperproliferative response elicited by Cu-NTA by down-regulating the rate of [H]thymidine incorporation into hepatic and renal DNA ( < .01-0.001). Protective effect of GTN against Cu-NTA was also confirmed by histopathological changes in liver and kidney. This result suggests that GTN may serve as a scavenger for reactive oxygen species (ROS) and reduces toxic metabolites of Cu-NTA, thereby avoiding tissue injury and oxidative stress. Further, administration of NO inhibitor, NG-Nitroarginine methyl ester (L-NAME), exacerbated Cu-NTA induced oxidative tissue damage and cell proliferation. Overall, GTN reduces Cu-NTA-induced tissue damage, oxidative stress, and proliferative response in the rat liver and kidney, according to these findings. On the basis of the above results, present study suggests that GTN may be a potential therapeutic agent for restoration of oxidative damage and proliferation to liver and kidney.
Topics: Rats; Animals; Male; Nitroglycerin; Copper; Rats, Wistar; Kidney; Lipid Peroxidation; Nitrilotriacetic Acid; Oxidative Stress; Liver; Antioxidants; NG-Nitroarginine Methyl Ester; Thymidine; DNA; Ferric Compounds
PubMed: 36305384
DOI: 10.1177/09603271221131312 -
Cells Jan 2023The ability to monitor lymphocyte responses is critical for developing our understanding of the immune response in humans. In the current clinical setting, relying on... (Review)
Review
The ability to monitor lymphocyte responses is critical for developing our understanding of the immune response in humans. In the current clinical setting, relying on the metabolic incorporation of [H] thymidine into cellular DNA via a lymphocyte proliferation test (LPT) is the only method that is routinely performed to determine cell proliferation. However, techniques that measure DNA synthesis with a radioactive material such as [H] thymidine are intrinsically more sensitive to the different stages of the cell cycle, which could lead to over-analyses and the subsequent inaccurate interpretation of the information provided. With cell proliferation assays, the output should preferably provide a direct and accurate measurement of the number of actively dividing cells, regardless of the stimuli properties or length of exposure. In fact, an ideal technique should have the capacity to measure lymphocyte responses on both a quantitative level, i.e., cumulative magnitude of lymphoproliferative response, and a qualitative level, i.e., phenotypical and functional characterization of stimulated immune cells. There are many LPT alternatives currently available to measure various aspects of cell proliferation. Of the nine techniques discussed, we noted that the majority of these LPT alternatives measure lymphocyte proliferation using flow cytometry. Across some of these alternatives, the covalent labelling of cells with a high fluorescence intensity and low variance with minimal cell toxicity while maximizing the number of detectable cell divisions or magnitude of proliferation was achieved. Herein, we review the performance of these different LPT alternatives and address their compatibility with the [H] thymidine LPT so as to identify the "best" alternative to the [H] thymidine LPT.
Topics: Humans; Lymphocytes; Cell Division; Cell Proliferation; DNA; Thymidine
PubMed: 36766728
DOI: 10.3390/cells12030386