-
The FEBS Journal Jan 2020Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch...
Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch repair function of S. pneumoniae has been assigned to the hexA-hexB gene products, an enzyme capable of the direct elimination of noncanonical nucleotides from the cytoplasm has not been described for this bacterium. Our results show that Spr1057, a protein with previously unknown function, is involved in the inactivation of mutagenic pyrimidine nucleotides and was accordingly designated PynA (pyrimidine nucleotidase A). Biochemical assays confirmed the phosphatase activity of the recombinant enzyme and revealed its metal ion dependence for optimal enzyme activity. We demonstrated that PynA forms a homodimer with higher in vitro activity towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate than towards canonical thymidine monophosphate. Furthermore, we showed via in vivo assays that PynA protects cells against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine and prevents the incorporation of the potentially mutagenic 5-bromo-2'-deoxyuridine (5-BrdU) into DNA. Fluctuation analysis performed under S. pneumoniae exposure to 5-BrdU revealed that the pynA null strain accumulates random mutations with high frequency, resulting in a 30-fold increase in the mutation rate. The data support a model in which PynA, a protein conserved in other Gram-positive bacteria, functions as a house-cleaning enzyme by selectively eliminating noncanonical nucleotides and maintaining the purity of dNTP pools, similar to the YjjG protein described for Escherichia coli.
Topics: 5'-Nucleotidase; Bacterial Proteins; Cations; Deoxyuridine; Mutation Rate; Streptococcus pneumoniae; Substrate Specificity; Thymidine Monophosphate
PubMed: 31437335
DOI: 10.1111/febs.15049 -
British Journal of Pharmacology Jul 19971. The aim of this study was to characterize P2 receptors in the arterial vascular bed of human perfused placental cotyledons. Vasoconstrictor responses to bolus...
1. The aim of this study was to characterize P2 receptors in the arterial vascular bed of human perfused placental cotyledons. Vasoconstrictor responses to bolus injections of purine and pyrimidine nucleotides were tested at basal tone, and vasodilator responses in preparations with tone raised by perfusion with prostaglandin F2alpha (PGF2alpha; 10-50 nM). 2. At basal tone, bolus injections of the P2X-selective agonist alpha,beta-methylene ATP (alpha,beta-meATP; 0.5-500 nmol) elicited dose-dependent vasoconstriction. ATP (0.005-5 micromol) also elicited dose-dependent vasoconstriction, but was less potent than alpha,beta-meATP. Vasoconstriction was also elicited by other nucleotides, but only at the highest dose tested (5 micromol): UTP > CTP = ITP (n = 6). GTP and TTP did not cause vasoconstriction. 3. Constrictor responses to bolus injections of alpha,beta-meATP were resistant to desensitization and were not significantly affected when carried out in the presence of 1 microM alpha,beta-meATP added to the perfusate. However, responses to bolus injections of alpha,beta-meATP were partially blocked by perfusion with 10 microM alpha,beta-meATP. In contrast, responses to ATP and UTP were unaffected by 10 microM alpha,beta-meATP. The P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10 and 100 microM) had no significant effect on vasoconstriction mediated by alpha,beta-meATP and ATP. 4. Removal of the endothelium had no significant effect on constrictor responses to alpha,beta-meATP, ATP and UTP. Inhibition of nitric oxide (NO) synthesis with N(G)-nitro-L-arginine methyl ester (L-NAME; 100 microM) had no significant effect on vasoconstriction to ATP and alpha,beta-meATP. 5. In preparations with tone raised with PGF2alpha (10-50 nM) vasodilatation was elicited by nucleotides with the following order of potency: 2MeSATP = ADP >> ATP > UTP > CTP = GTP = ITP = TTP. pD2 values were: 2MeSATP, 10.03+/-0.26 (n=7); ADP, 9.97+/-0.40 (n=5); ATP, 8.89+/-0.18 (n=7); UTP, 7.79+/-0.35 (n=7). Maximal responses to 2MeSATP and ADP were similar and were approximately 40% greater than maximal responses to ATP and UTP. 6. Vasodilator responses to nucleotides were abolished by L-NAME (100 microM) and by removal of the endothelium. 7. In conclusion, contractile responses mediated by alpha,beta-meATP and ATP in human placental smooth muscle are resistant to desensitization and insensitive to PPADS and, thus, show a dissimilar pharmacological profile to the classic smooth muscle P2X1 receptor. There may be two subtypes of smooth muscle P2 receptor based on differential antagonism of alpha,beta-meATP and ATP with alpha,beta-meATP. A smooth muscle P2 receptor mediates vasoconstriction to UTP, and may indicate a further subtype. Endothelium-dependent, NO-dependent, vasodilatation to 2MeSATP and ADP may be mediated by P2Y1 receptors, while endothelial P2Y2 receptors are likely to mediate NO-dependent relaxation to ATP and UTP.
Topics: Adenosine Triphosphate; Adult; Endothelium, Vascular; Enzyme Inhibitors; Female; Humans; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Placenta; Pregnancy; Purine Nucleotides; Pyridoxal Phosphate; Pyrimidine Nucleotides; Receptors, Purinergic P2; Vasoconstrictor Agents
PubMed: 9249247
DOI: 10.1038/sj.bjp.0701262 -
The Journal of Neuroscience : the... Feb 2016Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders....
Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na(v)1.9(-/-) mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Cells, Cultured; Colon; Female; Ganglia, Spinal; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; NAV1.9 Voltage-Gated Sodium Channel; Nociceptors; Purine Nucleotides; Pyrimidine Nucleotides; Receptors, Purinergic P2Y; Species Specificity
PubMed: 26911685
DOI: 10.1523/JNEUROSCI.3369-15.2016 -
Cells Jan 2022The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of...
The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of the same symptoms despite different causes. With the aim of better understanding the pathophysiology and biochemical processes behind dry mouth with different etiologies, we investigated the metabolic profile of 10 HNC patients, 9 pSS patients and 10 healthy controls using high-performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) metabolomics. Principal component analysis (PCA) revealed different metabolic profiles when comparing all subjects included in the study. Both patient groups showed higher ratios of several pyrimidine nucleotides and nucleosides when compared to controls. This finding may indicate that purinergic signaling plays a role in dry mouth conditions. Moreover, significantly increased levels of DL-3-aminoisobutyric acid were found in HNC patients when compared to controls, and a similar tendency was observed in the pSS patients. Furthermore, a dysregulation in amino acid metabolism was observed in both patient groups. In conclusion, metabolomics analysis showed separate metabolic profiles for HNC and pSS patients as compared to controls that could be useful in diagnostics and for elucidating the different pathophysiologies. The demonstrated dysregulation of pyrimidine nucleotides and levels of metabolites derived from amino acids in the patient groups should be studied further.
Topics: Head and Neck Neoplasms; Humans; Metabolomics; Pyrimidine Nucleotides; Saliva; Sjogren's Syndrome; Xerostomia
PubMed: 35159133
DOI: 10.3390/cells11030323 -
Proceedings of the National Academy of... Apr 2018We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers...
We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts in the genome. We find that these photoproducts are removed from the genome primarily by incisions 13-18 nucleotides 5' and 6-7 nucleotides 3' to the UV damage that generate 21- to 27-nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand at early time points followed by predominantly nontranscribed strand repair at later stages. We have also characterized the excision repair level as a function of the transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism.
Topics: DNA Damage; DNA Repair; DNA, Fungal; Genome, Fungal; Pyrimidine Dimers; Saccharomyces cerevisiae; Transcription, Genetic; Ultraviolet Rays
PubMed: 29581276
DOI: 10.1073/pnas.1801687115 -
Pyrimidine nucleotide synthesis in Pseudomonas nitroreducens and the regulatory role of pyrimidines.Microbiological Research Dec 2014Control of pyrimidine biosynthesis in the commercially important, hydrocarbon-utilizing bacterium Pseudomonas nitroreducens ATCC 33634 was investigated. When...
Control of pyrimidine biosynthesis in the commercially important, hydrocarbon-utilizing bacterium Pseudomonas nitroreducens ATCC 33634 was investigated. When glucose-grown wild-type cells were supplemented with uracil or orotic acid, the pyrimidine biosynthetic activities were depressed. Pyrimidine limitation of glucose-grown cells of an orotate phosphoribosyltransferase mutant caused aspartate transcarbamoylase and dihydroorotase activities to increase by about 4-fold while the other enzyme activities about doubled. In succinate-grown phosphoribosyltransferase mutant cells subjected to pyrimidine limitation, transcarbamoylase and dehydrogenase activities rose by about 5-fold while dihydroorotase activity more than tripled. In an OMP decarboxylase mutant, pyrimidine limitation of glucose-grown cells increased transcarbamoylase, dihydroorotase, dehydrogenase and phosphoribosyltransferase activities by 4-, 10-, 6- and 3.8-fold, respectively. Pyrimidine limitation of the succinate-grown decarboxylase mutant cells increased aspartate transcarbamoylase or dihydroorotase by more than 4-fold and the other activities by about 2-fold. Pyrimidine biosynthetic enzyme synthesis appeared to be regulated by pyrimidines with the regulation being influenced by the carbon source present. Aspartate transcarbamoylase activity in Ps. nitroreducens was regulated at the level of enzyme activity since the enzyme was strongly inhibited by UDP, pyrophosphate, ATP and ADP. Overall, the regulation of pyrimidine biosynthesis in Ps. nitroreducens can be used to differentiate it from other taxonomically related species of Pseudomonas.
Topics: Aspartate Carbamoyltransferase; Pseudomonas; Pyrimidine Nucleotides; Pyrimidines
PubMed: 24867376
DOI: 10.1016/j.micres.2014.04.003 -
Scientific Reports Jan 2020The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and...
The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 10 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.
Topics: Click Chemistry; Copper; Cycloaddition Reaction; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Deoxyuracil Nucleotides; HCT116 Cells; HEK293 Cells; Humans; K562 Cells; Rhodamines; Staining and Labeling; Thymine Nucleotides
PubMed: 31953472
DOI: 10.1038/s41598-020-57463-3 -
The Biochemical Journal Mar 1990With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the...
With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 14C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.
Topics: Animals; Chromatography, High Pressure Liquid; Hydrolysis; Kinetics; Liver; Male; Nucleoside Diphosphate Sugars; Pyrimidine Nucleotides; RNA; Rats; Rats, Inbred Strains; Solubility
PubMed: 1691632
DOI: 10.1042/bj2660777 -
Geochemical influences on nonenzymatic oligomerization of prebiotically relevant cyclic nucleotides.RNA (New York, N.Y.) Jun 2020The spontaneous emergence of long RNA molecules on the early Earth, a phenomenon central to the RNA World hypothesis, continues to remain an enigma in the field of...
The spontaneous emergence of long RNA molecules on the early Earth, a phenomenon central to the RNA World hypothesis, continues to remain an enigma in the field of origins of life. Few studies have looked at the nonenzymatic oligomerization of cyclic mononucleotides under neutral to alkaline conditions, albeit in fully dehydrated state. In this study, we systematically investigated the oligomerization of cyclic nucleotides under prebiotically relevant conditions, wherein starting reactants were subjected to repeated dehydration-rehydration (DH-RH) regimes. DH-RH conditions, a recurring geological theme that was prevalent on prebiotic Earth, are driven by naturally occurring processes including diurnal cycles and tidal pool activity. These conditions have been shown to facilitate uphill oligomerization reactions. The polymerization of 2'-3' and 3'-5' cyclic nucleotides of a purine (adenosine) and a pyrimidine (cytidine) was investigated. Additionally, the effect of amphiphiles was also evaluated. Furthermore, to discern the effect of "realistic" conditions on this process, the reactions were also performed using a hot spring water sample from a candidate early Earth environment. Our study showed that the oligomerization of cyclic nucleotides under DH-RH conditions resulted in intact informational oligomers. Amphiphiles increased the stability of both the starting monomers and the resultant oligomers in selected reactions. In the hot spring reactions, both the oligomerization of nucleotides and the back hydrolysis of the resultant oligomers were pronounced. Altogether, this study demonstrates how nonenzymatic oligomerization of cyclic nucleotides, under both laboratory-simulated prebiotic conditions and in a candidate early Earth environment, could have resulted in RNA oligomers of a putative RNA World.
Topics: Cyclic AMP; Cyclic CMP; Hot Springs; Hot Temperature; Phosphatidylcholines; Water
PubMed: 32205323
DOI: 10.1261/rna.074302.119 -
Antiviral Chemistry & Chemotherapy May 2003Beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small... (Comparative Study)
Comparative Study Review
Beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2'-fluorine substitution on the pre-steady state incorporation of 2'-deoxycytidine-5'-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-beta-L-2',3'-dideoxy-3'-thia-5-fluorocytidine (L-FTC, Emtriva), and (+)-beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D-D4FC, Reverset), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2' position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.
Topics: Antiviral Agents; Deoxycytidine Monophosphate; Deoxycytosine Nucleotides; Drug Design; Fluorine; HIV Reverse Transcriptase; Humans; Kinetics; Molecular Structure; Stereoisomerism; Templates, Genetic
PubMed: 14521328
DOI: 10.1177/095632020301400301