Purines enrich root-associated Pseudomonas and improve wild soybean growth under salt stress.

The root-associated microbiota plays an important role in the response to environmental stress. However, the underlying mechanisms controlling the interaction between salt-stressed plants and microbiota are poorly understood. Here, by focusing on a salt-tolerant plant wild soybean (Glycine soja), we demonstrate that highly conserved microbes dominated by Pseudomonas are enriched in the root and rhizosphere microbiota of salt-stressed plant. Two corresponding Pseudomonas isolates are confirmed to enhance the salt tolerance of wild soybean. Shotgun metagenomic and metatranscriptomic sequencing reveal that motility-associated genes, mainly chemotaxis and flagellar assembly, are significantly enriched and expressed in salt-treated samples. We further find that roots of salt stressed plants secreted purines, especially xanthine, which induce motility of the Pseudomonas isolates. Moreover, exogenous application for xanthine to non-stressed plants results in Pseudomonas enrichment, reproducing the microbiota shift in salt-stressed root. Finally, Pseudomonas mutant analysis shows that the motility related gene cheW is required for chemotaxis toward xanthine and for enhancing plant salt tolerance. Our study proposes that wild soybean recruits beneficial Pseudomonas species by exudating key metabolites (i.e., purine) against salt stress.

Hypothermia increases adenosine monophosphate and xanthosine monophosphate levels in the mouse hippocampus, preventing their reduction by global cerebral ischemia.

Global cerebral ischemia (GCI) caused by clinical conditions such as cardiac arrest leads to delayed neuronal death in the hippocampus, resulting in physical and mental disability. However, the mechanism of delayed neuronal death following GCI remains unclear. To elucidate the mechanism, we performed a metabolome analysis using a mouse model in which hypothermia (HT) during GCI, which was induced by the transient occlusion of the bilateral common carotid arteries, markedly suppressed the development of delayed neuronal death in the hippocampus after reperfusion. Fifteen metabolites whose levels were significantly changed by GCI and 12 metabolites whose levels were significantly changed by HT were identified. Furthermore, the metabolites common for both changes were narrowed down to two, adenosine monophosphate (AMP) and xanthosine monophosphate (XMP). The levels of both AMP and XMP were found to be decreased by GCI, but increased by HT, thereby preventing their decrease. In contrast, the levels of adenosine, inosine, hypoxanthine, xanthine, and guanosine, the downstream metabolites of AMP and XMP, were increased by GCI, but were not affected by HT. Our results may provide a clue to understanding the mechanism by which HT during GCI suppresses the development of delayed neuronal death in the hippocampus.

K2Cr2O7-induced DNA damage in HT1080 cells: Electrochemical signal response mechanism.

The existing DNA damage detection technology cannot meet the current detection requirements. It is critical to build new methods and discover novel biomarkers. In this study, alkaline comet and 8-OHDG ELISA assays were used to identify DNA damage in HT-1080 cells exposed to K2Cr2O7, and electrochemical behaviors of HT-1080 cells with DNA damage was studied. With an increase in K2Cr2O7 exposure time, two electrochemical signals from HT-1080 cells at 0.69 and 1.01 V steadily grew before decreasing after reaching their highest values. The electrochemical signal's initial response time and peak time decreased as the concentration of K2Cr2O7 increased. The duration of the high dose group was 0.5 and 1 h, while the low dose group was 1.5 and 6 h. Western blotting analysis revealed that DNA damage increased the expression of proteins involved in catabolism and de novo purine synthesis, particularly de novo purine synthesis. Expressions of PRPP amidotransferase, IMPDH, and ADA were all higher than those of ADSS, XOD, and GDA, which resulted in larger concentrations of hypoxanthine, guanine, and xanthine, and in turn improved electrochemical signaling. These findings suggest that intracellular purine identified by linear scan voltammetry is predicted to evolve as a marker of early DNA damage.

Investigation of the Inhibition Mechanism of Xanthine Oxidoreductase by Oxipurinol: A Computational Study.

Xanthine oxidoreductase (XOR) is an enzyme found in various organisms. It converts hypoxanthine to xanthine and urate, which are crucial steps in purine elimination in humans. Elevated uric acid levels can lead to conditions like gout and hyperuricemia. Therefore, there is significant interest in developing drugs that target XOR for treating these conditions and other diseases. Oxipurinol, an analogue of xanthine, is a well-known inhibitor of XOR. Crystallographic studies have revealed that oxipurinol directly binds to the molybdenum cofactor (MoCo) in XOR. However, the precise details of the inhibition mechanism are still unclear, which would be valuable for designing more effective drugs with similar inhibitory functions. In this study, molecular dynamics and quantum mechanics/molecular mechanics calculations are employed to investigate the inhibition mechanism of XOR by oxipurinol. The study examines the structural and dynamic effects of oxipurinol on the pre-catalytic structure of the metabolite-bound system. Our results provide insights on the reaction mechanism catalyzed by the MoCo center in the active site, which aligns well with experimental findings. Furthermore, the results provide insights into the residues surrounding the active site and propose an alternative mechanism for developing alternative covalent inhibitors.

Xanthine-induced deficits in hippocampal behavior and abnormal expression of hemoglobin genes.

The prevalence of mental disorders such as depression and anxiety is high and often comorbid with other diseases. Chronic stress is a common risk factor for these disorders, but the mechanisms behind their development are not yet fully understood. Metabolomics has revealed a close association between purine and pyrimidine metabolism and depression and anxiety, with increased levels of serum xanthine observed in both humans and mice. Xanthine is known as purine metabolism, and this compound shows several biological activities, but the impact of xanthine on our brain function is still unclear. The hippocampus, which plays a crucial role in memory and learning, is also implicated in the pathophysiology of depression and anxiety. Here, we investigated the effects of xanthine intraperitoneal administration on spatial memory and anxiety-like behavior in mice. The findings indicated that xanthine administration induced a deficit of hippocampus-dependent spatial memory and a tendency to anxiety-like behavior in mice. RNA-seq analysis showed that xanthine administration upregulated hemoglobin (Hb) genes involved in oxygen transport in the hippocampus. The upregulated Hb genes occurred in the neuronal cells, and in vitro experiments revealed that both Hba-a1 derived from mice and HBA2 derived from humans were upregulated by xanthine treatment. These observations suggest that the xanthine-induced Hb in the hippocampus could be related to spatial memory deficit and anxiety. This study sheds light on the direct effects of xanthine on the brain and its potential role in the development of depression and anxiety symptoms caused by chronic stress.

Xanthine-derived reactive oxygen species exacerbates adipose tissue disorders in male db/db mice induced by real-ambient PM2.5 exposure.

Epidemiological and experimental data have associated exposure to fine particulate matter (PM2.5) with various metabolic dysfunctions and diseases, including overweight and type 2 diabetes. Adipose tissue is an energy pool for storing lipids, a necessary regulator of glucose homeostasis, and an active endocrine organ, playing an essential role in developing various related diseases such as diabetes and obesity. However, the molecular mechanisms underlying PM2.5-impaired functions in adipose tissue have rarely been explored. In this work, metabolomics based on liquid chromatography-mass spectrometry was performed to study the adverse impacts of PM2.5 exposure on brown adipose tissue (BAT) and white adipose tissue (WAT) in the diabetic mouse model. We found the effects of PM2.5 exposure by comparing the different metabolites in both adipose tissues of male db/db mice using real-ambient PM2.5 exposure. The results showed that PM2.5 exposure changed the purine metabolism in mice, especially the dramatic increase of xanthine content in both WAT and BAT. These changes led to significant oxidative stress. Then the results from real-time quantitative polymerase chain reaction showed that PM2.5 exposure could cause the production of inflammatory factors in both adipose tissues. Moreover, the increased reactive oxygen species (ROS) promoted triglyceride accumulation in WAT and inhibited its decomposition, causing increased WAT content in db/db mice. In addition, PM2.5 exposure significantly suppressed thermogenesis and affected energy metabolism in the BAT of male db/db mice, which may deteriorate insulin sensitivity and blood glucose regulation. This research demonstrated the impact of PM2.5 on the adipose tissue of male db/db mice, which may be necessary for public health.

Elemental profile identifies metallurgical pollution in epiphytic lichen Xanthoria parietina and (hypo)xanthine correlates with metals.

The accumulation of 55 elements in lichens under the heap of a former nickel smelter (village Dolná Streda, Slovakia) and at eight sites at different distances from the heap plus six sites throughout Slovakia was studied to determine the elemental profile. The major metals in the heap sludge and in the lichens below the heap (Ni, Cr, Fe, Mn, and Co) were surprisingly low in lichens from both the near and far vicinity of the heap (4-25 km), indicating limited airborne spread. However, two different sites with metallurgical activity (another site near the ferroalloy producer in Orava) typically contained the highest amount of individual elements, including rare earth elements, Th, U, Ag, Pd, Bi and Be, and their separation from other sites was confirmed by PCA and HCA analyses. In addition, the amounts of Cd, Ba and Re were highest at sites without a clear source of pollution and further monitoring is needed. It was also an unexpected finding that the enrichment factor calculated using UCC values was increased (often considerably >10) for 12 elements at all 15 sites, indicating eventual anthropogenic contamination with P, Zn, B, As, Sb, Cd, Ag, Bi, Pd, Pt, Te and Re (and other EF values were locally increased). Metabolic analyses showed a negative correlation between some metals and metabolites (ascorbic acid, thiols, phenols and allantoin), but slightly positive (amino acids) or highly positive correlation with purine derivatives hypoxanthine and xanthine. The data suggest that lichens adapt their metabolism to excessive metal loading and that epiphytic lichens are suitable for identifying metal contamination even at apparently clean sites.

The role of xanthine dioxygenase in the biosynthetic pathway of 2-aza-8-oxohypoxanthine of Lepista sordida.

2-Azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), discovered as causal substances of fairy rings are known to be endogenous in the fairy ring-forming Lepista sordida. In this study, we showed that xanthine dioxygenase, an a-ketoglutarate-dependent dioxygenase, might catalyze the conversion of AHX to AOH in the fungus. Furthermore, this enzyme is the first reported molybdopterin-independent protein of hypoxanthine metabolism.

Intermediate products of purine metabolism in an experimental model of pancreatic necrosis.

BACKGROUND AND AIM: Determine the level of purines in the blood plasma of experimental animals at three stages of induced pancreatic necrosis. Find out the potential of purines as predictors of the severity of pancreatitis. METHODS: The experiment was carried out on white outbred rabbits. The pancreatic necrosis was modeled by introducing self-bile into the pancreatic parenchyma. The pancreas of rabbits, after isolation, was subjected to microscopic description. Blood was also taken from rabbits to determine the plasma levels of adenine, guanine, hypoxanthine, xanthine, and uric acid. RESULTS: 12 hours after the administration of self-bile, the level of xanthine significantly increases and the concentration of uric acid in the blood plasma increases by 3 times. 24 hours after the introduction of self-bile, there is a slight decrease in the level of adenine, xanthine and uric acid, and the indicators of purine metabolism remain elevated. 48 hours after the introduction of self-bile, the levels of guanine, hypoxanthine and xanthine are reduced. CONCLUSIONS: The concentration indices of absolute and relative intermediate products of purine metabolism were increased at the initial stage of pancreatic necrosis. The activity of enzymes and metabolites of purine metabolism involved in the formation of reactive oxygen species and free radicals increased. The hypothesis that intermediate products of purine metabolism can be predictors of pancreatic necrosis was confirmed.

Xanthine oxidoreductase activity is correlated with hepatic steatosis.

The enzyme xanthine oxidoreductase (XOR) catalyzes the synthesis of uric acid (UA) from hypoxanthine and xanthine, which are products of purine metabolism starting from ribose-5-phosphate. Several studies suggested a relationship between hyperuricemia and hepatic steatosis; however, few previous studies have directly examined the relationship between XOR activity and hepatic steatosis. A total of 223 subjects with one or more cardiovascular risk factors were enrolled. The liver-to-spleen (L/S) ratio on computed tomography and the hepatic steatosis index (HSI) were used to assess hepatic steatosis. We used a newly developed highly sensitive assay based on [13C2, 15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry to measure plasma XOR activity. Subjects with the L/S ratio of < 1.1 and the HSI of < 36 had increased XOR activity and serum UA levels. Independent of insulin resistance and serum UA levels, multivariate logistic regression analysis revealed that plasma XOR activity was associated with the risk of hepatic steatosis as assessed by the L/S ratio and HSI. According to the findings of this study, plasma XOR activity is associated with hepatic steatosis independent of insulin resistance and serum UA levels.

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2'-deoxyguanosine.

The aptamer portions of previously reported riboswitch classes that sense guanine, adenine, or 2'-deoxyguanosine are formed by a highly similar three-stem junction with distinct nucleotide sequences in the regions joining the stems. The nucleotides in these joining regions form the major features of the selective ligand-binding pocket for each aptamer. Previously, we reported the existence of additional, rare variants of the predominant guanine-sensing riboswitch class that carry nucleotide differences in the ligand-binding pocket, suggesting that these RNAs have further diversified their structures and functions. Herein, we report the discovery and analysis of three naturally occurring variants of guanine riboswitches that are narrowly distributed across Firmicutes. These RNAs were identified using comparative sequence analysis methods, which also revealed that some of the gene associations for these variants are atypical for guanine riboswitches or their previously known natural variants. Binding assays demonstrate that the newfound variant riboswitch representatives recognize xanthine, guanine, or 2'-deoxyguanosine, with the guanine class exhibiting greater discrimination against related purines than the more common guanine riboswitch class reported previously. These three additional variant classes, together with the four previously discovered riboswitch classes that employ the same three-stem junction architecture, reveal how a simple structural framework can be diversified to expand the range of purine-based ligands sensed by RNA.

Biogenic Guanine Crystals Are Solid Solutions of Guanine and Other Purine Metabolites.

Highly reflective crystals of the nucleotide base guanine are widely distributed in animal coloration and visual systems. Organisms precisely control the morphology and organization of the crystals to optimize different optical effects, but little is known about how this is achieved. Here we examine a fundamental question that has remained unanswered after over 100 years of research on guanine: what are the crystals made of? Using solution-state and solid-state chemical techniques coupled with structural analysis by powder XRD and solid-state NMR, we compare the purine compositions and the structures of seven biogenic guanine crystals with different crystal morphologies, testing the hypothesis that intracrystalline dopants influence the crystal shape. We find that biogenic "guanine" crystals are not pure crystals but molecular alloys (aka solid solutions and mixed crystals) of guanine, hypoxanthine, and sometimes xanthine. Guanine host crystals occlude homogeneous mixtures of other purines, sometimes in remarkably large amounts (up to 20% of hypoxanthine), without significantly altering the crystal structure of the guanine host. We find no correlation between the biogenic crystal morphology and dopant content and conclude that dopants do not dictate the crystal morphology of the guanine host. The ability of guanine crystals to host other molecules enables animals to build physiologically "cheaper" crystals from mixtures of metabolically available purines, without impeding optical functionality. The exceptional levels of doping in biogenic guanine offer inspiration for the design of mixed molecular crystals that incorporate multiple functionalities in a single material.

Initiation of cytosolic plant purine nucleotide catabolism involves a monospecific xanthosine monophosphate phosphatase.

In plants, guanosine monophosphate (GMP) is synthesized from adenosine monophosphate via inosine monophosphate and xanthosine monophosphate (XMP) in the cytosol. It has been shown recently that the catabolic route for adenylate-derived nucleotides bifurcates at XMP from this biosynthetic route. Dephosphorylation of XMP and GMP by as yet unknown phosphatases can initiate cytosolic purine nucleotide catabolism. Here we show that Arabidopsis thaliana possesses a highly XMP-specific phosphatase (XMPP) which is conserved in vascular plants. We demonstrate that XMPP catalyzes the irreversible entry reaction of adenylate-derived nucleotides into purine nucleotide catabolism in vivo, whereas the guanylates enter catabolism via an unidentified GMP phosphatase and guanosine deaminase which are important to maintain purine nucleotide homeostasis. We also present a crystal structure and mutational analysis of XMPP providing a rationale for its exceptionally high substrate specificity, which is likely required for the efficient catalysis of the very small XMP pool in vivo.

Hepatotoxicity or hepatoprotection of emodin? Two sides of the same coin by 1H-NMR metabolomics profiling.

Emodin is the major anthraquinone component of many important traditional Chinese herbs, such as Rheum palmatum L. and Polygonum multiflorum Thunb. They have been popular health products but recently aroused concerns about their hepatotoxicity, which are believed to be arising from the contained anthraquinones, such as emodin. However, emodin exerts potent hepatoprotective ability, such as anti-fibrotic, anti-oxidative, and anti-inflammatory effects. In this study, 1H NMR based metabolomics approach, complemented with histopathological observation, biochemical measurements, western blotting analysis and real-time quantitative PCR (RT-qPCR), was applied to interpret the paradox of emodin (30 mg/kg, 10 mg/kg BW) using both healthy mice (male, ICR) and chronic CCl4-injured mice (0.1 mL/kg, 0.35% CCl4, 3 times a week for a month). Emodin exerted a weight loss property associated with its lipid-lowing effects, which helped alleviate CCl4-induced steatosis. Emodin effectively ameliorated CCl4-induced oxidative stress and energy metabolism dysfunction in mice liver via regulating glucose, lipid and amino acid metabolism, and inhibited excessive inflammatory response. In healthy mice, emodin only exhibited hepatoxicity on high-dosage by disturbing hepatic anti-oxidant homeostasis, especially GSH and xanthine metabolism. This integrated metabolomics approach identified the bidirectional potential of emodin, which are important for its rational use.

Identification of New Specificity Determinants in Bacterial Purine Nucleobase Transporters based on an Ancestral Sequence Reconstruction Approach.

The relation of sequence with specificity in membrane transporters is challenging to explore. Most relevant studies until now rely on comparisons of present-day homologs. In this work, we study a set of closely related transporters by employing an evolutionary, ancestral-reconstruction approach and reveal unexpected new specificity determinants. We analyze a monophyletic group represented by the xanthine-specific XanQ of Escherichia coli in the Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2 (NAT/NCS2) family. We reconstructed AncXanQ, the putative common ancestor of this clade, expressed it in E. coli K-12, and found that, in contrast to XanQ, it encodes a high-affinity permease for both xanthine and guanine, which also recognizes adenine, hypoxanthine, and a range of analogs. AncXanQ conserves all binding-site residues of XanQ and differs substantially in only five intramembrane residues outside the binding site. We subjected both homologs to rationally designed mutagenesis and present evidence that these five residues are linked with the specificity change. In particular, we reveal Ser377 of XanQ (Gly in AncXanQ) as a major determinant. Replacement of this Ser with Gly enlarges the specificity of XanQ towards an AncXanQ-phenotype. The ortholog from Neisseria meningitidis retaining Gly at this position is also a xanthine/guanine transporter with extended substrate profile like AncXanQ. Molecular Dynamics shows that the S377G replacement tilts transmembrane helix 12 resulting in rearrangement of Phe376 relative to Phe94 in the XanQ binding pocket. This effect may rationalize the enlarged specificity. On the other hand, the specificity effect of S377G can be masked by G27S or other mutations through epistatic interactions.

Plumbagin can potently enhance the activity of xanthine oxidase: in vitro, in vivo and in silico studies.

BACKGROUND: Abnormally elevated xanthine oxidase (XO) activity has been verified to cause various pathological processes, such as gout, oxidative stress injury and metabolic syndrome. Thus, XO activators may exhibit above potential toxicological properties. Plumbagin (PLB) is an important active compound in traditional Chinese medicine (TCM), while its obvious toxic effects have been reported, including diarrhea, skin rashes and hepatic toxicity. However, the potential toxicity associated with enhancement of XO activity has not been fully illuminated so far. METHODS: The present study investigated the effect of PLB on XO activity by culturing mouse liver S9 (MLS9), human liver S9 (HLS9), XO monoenzyme system with PLB and xanthine. Then, the molecular docking and biolayer interferometry analysis were adopted to study the binding properties between PLB and XO. Finally, the in vivo acceleration effect also investigated by injected intraperitoneally PLB to KM mice for 3 days. RESULTS: PLB could obviously accelerate xanthine oxidation in the above three incubation systems. Both the Vmax values and intrinsic clearance values (CLint, Vmax/Km) of XO in the three incubation systems increased along with elevated PLB concentration. In addition, the molecular docking study and label-free biolayer interferometry assay displayed that PLB was well bound to XO. In addition, the in vivo results showed that PLB (2 and 10 mg/kg) significantly increased serum uric acid levels and enhanced serum XO activity in mice. CONCLUSION: In summary, this study outlines a potential source of toxicity for PLB due to the powerful enhancement of XO activity, which may provide the crucial reminding for the PLB-containing preparation development and clinical application.

Xanthine-derived metabolites enhance chlorophyll degradation in cotyledons and seedling growth during nitrogen deficient condition in Brassica rapa.

Nitrogen (N) deficiency is a main environmental factor that induces early senescence. Cotyledons provide an important N source during germination and early seedling development. In this study, we observed that N deficient condition enhanced gene expression involved in purine catabolism in cotyledons of Chinese cabbage (Brassica rapa ssp. Pekinensis). Seedlings grown with added allopurinol, an inhibitor of xanthine dehydrogenase, in the growth medium showed reduced chlorophyll degradation in cotyledons and lower fresh weight, compared with seedlings grown on normal medium. On the basis of these results, we speculated that xanthine-derived metabolites might affect both seedling growth and early senescence in cotyledons. To confirm this, seedlings were grown with exogenous xanthine to analyze the role of xanthine-derived metabolites under N deficient condition. Seedlings with xanthine as the sole N-source grew faster, and more cotyledon chlorophyll was broken down, compared with seedlings grown without xanthine. The expression levels of senescence- and purine metabolism-related genes in cotyledons were higher than those in seedlings grown without xanthine. These results indicate the possibility that xanthine plays a role as an activator in both purine catabolism and chlorophyll degradation in cotyledons under N deficient condition.

Molecular Pathways and Pigments Underlying the Colors of the Pearl Oyster Pinctada margaritifera var. cumingii (Linnaeus 1758).

The shell color of the Mollusca has attracted naturalists and collectors for hundreds of years, while the molecular pathways regulating pigment production and the pigments themselves remain poorly described. In this study, our aim was to identify the main pigments and their molecular pathways in the pearl oyster Pinctada margaritifera-the species displaying the broadest range of colors. Three inner shell colors were investigated-red, yellow, and green. To maximize phenotypic homogeneity, a controlled population approach combined with common garden conditioning was used. Comparative analysis of transcriptomes (RNA-seq) of P. margaritifera with different shell colors revealed the central role of the heme pathway, which is involved in the production of red (uroporphyrin and derivates), yellow (bilirubin), and green (biliverdin and cobalamin forms) pigments. In addition, the Raper-Mason, and purine metabolism pathways were shown to produce yellow pigments (pheomelanin and xanthine) and the black pigment eumelanin. The presence of these pigments in pigmented shell was validated by Raman spectroscopy. This method also highlighted that all the identified pathways and pigments are expressed ubiquitously and that the dominant color of the shell is due to the preferential expression of one pathway compared with another. These pathways could likely be extrapolated to many other organisms presenting broad chromatic variation.

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1 Receptor in Living Cells.

The adenosine A1 receptor (A1AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A1AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A1AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A1AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A1AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A1AR molecular pharmacology and signaling in living cells.