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Analytical Sciences : the International... 2015To evaluate cellular uptake and purine transport, we developed a high-performance liquid chromatography method for intra- and extracellular purine quantification. Our...
To evaluate cellular uptake and purine transport, we developed a high-performance liquid chromatography method for intra- and extracellular purine quantification. Our aim was to develop an effective method for simultaneously quantifying the substrate and metabolites with high sensitivity. C18 columns from different manufacturers were tested for simultaneous quantification of 22 different purine bases, nucleosides, and nucleotides. We used a YMC-Triart C18 column. The analysis conditions, including extraction solutions for the cells and cell culture medium, were optimized to achieve good quantification. Linearity, accuracy, determination limits, and recovery were assessed and showed good performance. The developed HPLC method was successfully applied to the qualitative analysis of 22 different intra- and extracellular purines, demonstrating that it is useful for studying the overall pattern of purine metabolism. This method could also be useful for evaluating metabolic dynamics of purines under a variety of stimulatory conditions of culture cells.
Topics: Chromatography, High Pressure Liquid; Extracellular Space; Hep G2 Cells; Humans; Intracellular Space; Nucleosides; Nucleotides; Purines
PubMed: 26353955
DOI: 10.2116/analsci.31.895 -
Organic Letters Nov 2022A method for the C-H carboxyamidation of purines has been developed that is capable of directly installing primary, secondary, and tertiary amides. Previous Minisci-type...
A method for the C-H carboxyamidation of purines has been developed that is capable of directly installing primary, secondary, and tertiary amides. Previous Minisci-type investigations on purines were limited to alkylations and arylations. Herein, we present the first method for the direct C-H amidation of a wide range of purines: xanthine, guanine, and adenine structures, including guanosine- and adenosine-type nucleosides. The Minisci-type reaction is also metal-free, cheap, operationally simple, scalable, and applicable to late-stage functionalizations of biologically important molecules.
Topics: Purines; Adenine; Guanine; Guanosine; Nucleosides; Purine Nucleosides
PubMed: 36285836
DOI: 10.1021/acs.orglett.2c03206 -
Molecules (Basel, Switzerland) Mar 2019Glycosylation of 6-amino-4-methoxy-1H-pyrazolo[3,4-]pyrimidine and its iodo- and bromo- analogues with the protected ribofuranose and 2'-deoxyribofuranose under...
Glycosylation of 6-amino-4-methoxy-1H-pyrazolo[3,4-]pyrimidine and its iodo- and bromo- analogues with the protected ribofuranose and 2'-deoxyribofuranose under different conditions resulted in the synthesis of ⁸- and ⁸-glycosylated purine nucleosides. Five key intermediate nucleosides, having 6-methoxy, 7-iodo, and 2-bromo groups, were further derivatized to 23 final 8-aza-7-deazapurine nucleoside derivatives. The structures of ⁸- and ⁸-glycosylated products were assigned based on UV and NMR spectra. HMBC analysis of 2D NMR spectra and X-ray crystallographic studies of the representative compounds unambiguously verified the connection of ribose ring to ⁸- or ⁸-position of the purine ring. The anticancer activity of these new compounds was evaluated.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Chemistry Techniques, Synthetic; Humans; Models, Molecular; Molecular Conformation; Molecular Structure; Purine Nucleosides; Purines; Spectrum Analysis; Structure-Activity Relationship
PubMed: 30862058
DOI: 10.3390/molecules24050983 -
International Journal of Molecular... Feb 2020Purines are nitrogen compounds consisting mainly of a nitrogen base of adenine (ABP) or guanine (GBP) and their derivatives: nucleosides (nitrogen bases plus ribose) and... (Review)
Review
Purines are nitrogen compounds consisting mainly of a nitrogen base of adenine (ABP) or guanine (GBP) and their derivatives: nucleosides (nitrogen bases plus ribose) and nucleotides (nitrogen bases plus ribose and phosphate). These compounds are very common in nature, especially in a phosphorylated form. There is increasing evidence that purines are involved in the development of different organs such as the heart, skeletal muscle and brain. When brain development is complete, some purinergic mechanisms may be silenced, but may be reactivated in the adult brain/muscle, suggesting a role for purines in regeneration and self-repair. Thus, it is possible that guanosine-5'-triphosphate (GTP) also acts as regulator during the adult phase. However, regarding GBP, no specific receptor has been cloned for GTP or its metabolites, although specific binding sites with distinct GTP affinity characteristics have been found in both muscle and neural cell lines. Finally, even if the cross regulation mechanisms between the two different purines (ABP and GBP) are still largely unknown, it is now possible to hypothesize the existence of specific signal paths for guanosine-based nucleotides that are capable of modulating the intensity and duration of the intracellular signal, particularly in excitable tissues such as brain and muscle.
Topics: Brain; Embryonic Development; Guanine; Guanine Nucleotides; Guanosine; Guanosine Triphosphate; Humans; Muscles; Nervous System; Nucleosides; Nucleotides; Purines; Receptors, Purinergic
PubMed: 32111063
DOI: 10.3390/ijms21051591 -
Biophysical Journal May 2019The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable...
The skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically relevant mechanical forces lead to small repairable membrane injuries in bone-forming osteoblasts, resulting in release of ATP and stimulation of purinergic (P2) calcium responses in neighboring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, their extracellular diffusion and degradation, and purinergic responses in neighboring cells. After validation using experimental data for intracellular free calcium elevations, ATP, and vesicular release after mechanical stimulation of a single osteoblast, the model was scaled to a tissue-level injury to investigate how purinergic signaling communicates information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration, and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed spatial factors, such as the maximal distance from the injury at which purinergic responses were stimulated. The peak ATP concentration reflected the severity of an individual cell injury, allowing to discriminate between minor and severe injuries that released similar amounts of ATP because of differences in injury repair, and determined temporal aspects of the response, such as signal propagation velocity. ADP-mediated signaling became relevant only in larger tissue-level injuries, conveying information about the distance to the injury site and its geometry. Thus, we identified specific features of extracellular ATP and ADP spatiotemporal signals that depend on tissue mechanoresilience and encode the severity, scope, and proximity of the mechanical stimulus.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Cell Membrane; Mechanical Phenomena; Mice; Mice, Inbred C57BL; Osteoblasts; Purines; Signal Transduction
PubMed: 31053261
DOI: 10.1016/j.bpj.2019.04.012 -
Molecular Plant Pathology Apr 2019The high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall...
The high-affinity cyclic adenosine monophosphate (cAMP) phosphodiesterase MoPdeH is important not only for cAMP signalling and pathogenicity, but also for cell wall integrity (CWI) maintenance in the rice blast fungus Magnaporthe oryzae. To explore the underlying mechanism, we identified MoImd4 as an inosine-5'-monophosphate dehydrogenase (IMPDH) homologue that interacts with MoPdeH. Targeted deletion of MoIMD4 resulted in reduced de novo purine biosynthesis and growth, as well as attenuated pathogenicity, which were suppressed by exogenous xanthosine monophosphate (XMP). Treatment with mycophenolic acid (MPA), which specifically inhibits MoImd4 activity, resulted in reduced growth and virulence attenuation. Intriguingly, further analysis showed that MoImd4 promotes the phosphodiesterase activity of MoPdeH, thereby decreasing intracellular cAMP levels, and MoPdeH also promotes the IMPDH activity of MoImd4. Our studies revealed the presence of a novel crosstalk between cAMP regulation and purine biosynthesis in M. oryzae, and indicated that such a link is also important in the pathogenesis of M. oryzae.
Topics: Cyclic AMP; Fungal Proteins; Gene Expression Regulation, Fungal; Magnaporthe; Purines; Signal Transduction; Virulence
PubMed: 30426699
DOI: 10.1111/mpp.12770 -
Microbiology Spectrum Dec 2021Bacterial biofilms are involved in chronic infections and confer 10 to 1,000 times more resistance to antibiotics compared with planktonic growth, leading to...
The Purine Biosynthesis Pathway Is the Only Commonly Regulated Cellular Pathway during Biofilm Formation in TSB-Based Medium in Staphylococcus aureus and Enterococcus faecalis.
Bacterial biofilms are involved in chronic infections and confer 10 to 1,000 times more resistance to antibiotics compared with planktonic growth, leading to complications and treatment failure. When transitioning from a planktonic lifestyle to biofilms, some Gram-positive bacteria are likely to modulate several cellular pathways, including central carbon metabolism, biosynthesis pathways, and production of secondary metabolites. These metabolic adaptations might play a crucial role in biofilm formation by Gram-positive pathogens such as Staphylococcus aureus and Enterococcus faecalis. Here, we performed a transcriptomic approach to identify cellular pathways that might be similarly regulated during biofilm formation in these bacteria. Different strains and biofilm-inducing media were used to identify a set of regulated genes that are common and independent of the environment or accessory genomes analyzed. Our approach highlighted that the purine biosynthesis pathway was upregulated in biofilms of both species when using a tryptone soy broth-based medium but not so when a brain heart infusion-based medium was used. We did not identify other pathways commonly regulated between both pathogens. Gene deletions and usage of a drug targeting a key enzyme showed the importance of this pathway in biofilm formation of S. aureus. The importance of the purine biosynthesis pathway might reflect an important need for purine during biofilm establishment, and thus could constitute a promising drug target. Biofilms are often involved in nosocomial infections and can cause serious chronic infections if not treated properly. Current anti-biofilm strategies rely on antibiotic usage, but they have a limited impact because of the biofilm intrinsic tolerance to drugs. Metabolism remodeling likely plays a central role during biofilm formation. Using comparative transcriptomics of different strains of Staphylococcus aureus and Enterococcus faecalis, we determined that almost all cellular adaptations are not shared between strains and species. Interestingly, we observed that the purine biosynthesis pathway was upregulated during biofilm formation by both species in a specific medium. The requirement for purine could constitute an interesting new anti-biofilm target with a wide spectrum that could also prevent resistance evolution. These results are also relevant to a better understanding of the physiology of biofilm formation.
Topics: Bacterial Proteins; Biofilms; Biosynthetic Pathways; Culture Media; Enterococcus faecalis; Gram-Positive Bacterial Infections; Humans; Purines; Staphylococcal Infections; Staphylococcus aureus
PubMed: 34935415
DOI: 10.1128/Spectrum.00804-21 -
FEBS Letters Feb 2015A strong purine asymmetry, along with strand-biased gene distribution and the presence of PolC, prevails in Bacillus and some other members of Firmicutes, Fusobacteria...
A strong purine asymmetry, along with strand-biased gene distribution and the presence of PolC, prevails in Bacillus and some other members of Firmicutes, Fusobacteria and Tenericutes. The analysis of protein features in 21 Bacillus species of diverse metabolic, virulence and ecological traits revealed that purine asymmetry in conjunction with lineage/niche specific constraints significantly influences protein evolution in Bacillus. All Bacillus species, except for Se-respiring Bacillus selenitireducens, display distinct strand-specific biases in amino acid usage, which may affect the isoelectric point or surface charge distribution of proteins with prevalence of acidic and basic residues in the leading and lagging strand proteins, respectively.
Topics: Amino Acids; Bacillus; Bacterial Proteins; Purines
PubMed: 25639611
DOI: 10.1016/j.febslet.2015.01.028 -
The FEBS Journal Jan 2009Receptors should be properly analysed in view of the microenvironment in which they are embedded. Therefore, the concept of 'receptosome' was formulated to the complex... (Review)
Review
Receptors should be properly analysed in view of the microenvironment in which they are embedded. Therefore, the concept of 'receptosome' was formulated to the complex interactions taking place between receptors and other proteins at the plasma membrane level, and to explain very heterogeneous or divergent cellular responses to common epigenetic factors and modifications to the extracellular environment. The receptosome thus becomes a molecular network connecting transmitters, hormones or growth factors, to both their specific receptors and unique downstream effector proteins. As an example of receptosome, we introduce here the 'purinome' as molecular complex responsible for the biological effects of extracellular purine and pyrimidine ligands. In addition to a vast heterogeneity of purinergic ligands, the purinome thus consists of ectonucleotide-metabolizing enzymes hydrolysing nucleoside phosphates, purinergic receptors classified as P1 for adenosine/AMP and P2 for nucleosides tri-/diphosphates, nucleoside transporters with both equilibrative and concentrative properties and finally, nucleotide channels and transporters. Notably, these purinergic elements are not independent, but they play tightly concerted actions under physiological conditions. As a whole and not singularly, they trigger, maintain and terminate the purinergic signalling. This signifies that the purinome is not a new, mere definition of juxtaposed purinergic units, but rather the experimental evidence of complex and dynamic molecular cross-talk and cooperation networks. Alteration of this dynamic equilibrium may even participate in many pathological states. As a consequence, to be successful against pathological conditions, the genetic/pharmacological manipulation of purinergic mechanisms must go well beyond single proteins, and be more holistically oriented.
Topics: Animals; Biological Transport; Cell Membrane; Humans; Ligands; Purines; Receptors, Purinergic; Signal Transduction
PubMed: 19076212
DOI: 10.1111/j.1742-4658.2008.06793.x -
Bioorganic & Medicinal Chemistry Letters Jun 2016We report on an extensive structure-activity relationship study of novel PI4K IIIβ inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served...
We report on an extensive structure-activity relationship study of novel PI4K IIIβ inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIβ inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.
Topics: Antiviral Agents; Dose-Response Relationship, Drug; Enterovirus B, Human; HeLa Cells; Hepacivirus; Humans; Microbial Sensitivity Tests; Models, Molecular; Molecular Structure; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Purines; Rhinovirus; Structure-Activity Relationship
PubMed: 27090557
DOI: 10.1016/j.bmcl.2016.04.002