Xanthine Dehydrogenase Inhibitor Research Articles

Identification of inhibitors from a functional food-based plant Perillae Folium against hyperuricemia via metabolomics profiling, network pharmacology and all-atom molecular dynamics simulations.

Hyperuricemia (HUA) is a metabolic disorder caused by purine metabolism dysfunction in which the increasing purine levels can be partially attributed to seafood consumption. Perillae Folium (PF), a widely used plant in functional food, has been historically used to mitigate seafood-induced diseases. However, its efficacy against HUA and the underlying mechanism remain unclear. A network pharmacology analysis was performed to identify candidate targets and potential mechanisms involved in PF treating HUA. The candidate targets were determined based on TCMSP, SwissTargetPrediction, Open Targets Platform, GeneCards, Comparative Toxicogenomics Database, and DrugBank. The potential mechanisms were predicted via Gene Ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) analyses. Molecular docking in AutoDock Vina and PyRx were performed to predict the binding affinity and pose between herbal compounds and HUA-related targets. A chemical structure analysis of PF compounds was performed using OSIRIS DataWarrior and ClassyFire. We then conducted virtual pharmacokinetic and toxicity screening to filter potential inhibitors. We further performed verifications of these inhibitors' roles in HUA through molecular dynamics (MD) simulations, text-mining, and untargeted metabolomics analysis. We obtained 8200 predicted binding results between 328 herbal compounds and 25 potential targets, and xanthine dehydrogenase (XDH) exhibited the highest average binding affinity. We screened out five promising ligands (scutellarein, benzyl alpha-D-mannopyranoside, elemol, diisobutyl phthalate, and (3R)-hydroxy-beta-ionone) and performed MD simulations up to 50 ns for XDH complexed to them. The scutellarein-XDH complex exhibited the most satisfactory stability. Furthermore, the text-mining study provided laboratory evidence of scutellarein's function. The metabolomics approach identified 543 compounds and confirmed the presence of scutellarein. Extending MD simulations to 200 ns further indicated the sustained impact of scutellarein on XDH structure. Our study provides a computational and biomedical basis for PF treating HUA and fully elucidates scutellarein's great potential as an XDH inhibitor at the molecular level, holding promise for future drug design and development.

Salt priming induces low-temperature tolerance in sugar beet via xanthine metabolism

To understand the physiological mechanisms involved in xanthine metabolism during salt priming for improving low-temperature tolerance, salt priming (SP), xanthine dehydrogenase inhibitor (XOI), exogenous allantoin (EA), and back-supplemented EA (XOI + EA) treatments were given and the low-temperature tolerance of sugar beet was tested. Under low-temperature stress, salt priming promoted the growth of sugar beet leaves and increased the maximum quantum efficiency of PS II (Fv/Fm). However, during salt priming, either XOI or EA treatment alone increased the content of reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide, in the leaves under low-temperature stress. XOI treatment increased allantoinase activity with its gene (BvallB) expression under low-temperature stress. Compared to the XOI treatment, the EA treatment alone and the XOI + EA treatment increased the activities of antioxidant enzymes. At low temperatures, the sucrose content and the activity of key carbohydrate enzymes (AGPase, Cylnv, and FK) were significantly reduced by XOI compared to the changes under salt priming. XOI also stimulated the expression of protein phosphatase 2C and sucrose non-fermenting1-related protein kinase (BvSNRK2). The results of a correlation network analysis showed that BvallB was positively correlated with malondialdehyde, D-Fructose-6-phosphate, and D-Glucose-6-phosphate, and negatively correlated with BvPOX42, BvSNRK2, dehydroascorbate reductase, and catalase. These results suggested that salt-induced xanthine metabolism modulated ROS metabolism, photosynthetic carbon assimilation, and carbohydrate metabolism, thus enhancing low-temperature tolerance in sugar beet. Additionally, xanthine and allantoin were found to play key roles in plant stress resistance.

Nitrogen Metabolism in Non-Nodulated and Nodulated Soybean Plants Related to Ureide Synthesis

Soybean plants can fix atmospheric N2 in the root nodule, a symbiotic organ with rhizobia. The primary forms of N transported from N2 fixation are ureides, allantoate, and allantoin, supplemented with asparagine. The nitrate absorbed in the roots is transported to the shoots in the forms of NO3− and asparagine with a little portion of ureides. The concentrations of N-metabolites were analyzed by capillary electrophoresis after supplying various concentrations of urea, precursors of ureides, and allopurinol, an inhibitor of xanthine dehydrogenase, to investigate the ureide synthesis pathway in the roots. When the non-nodulated soybean plants were treated with 0–5 mM of urea, the concentrations of asparagine and glutamine in the xylem sap and the roots increased remarkably. In addition, allantoate concentration increased with the urea concentrations becoming higher. Allopurinol inhibited the accumulation of allantoate but did not affect the asparagine and glutamine accumulation in roots, stems, leaves, and xylem sap, supporting that allantoate is synthesized by purine degradation in roots the same as in the nodules. When ureide precursors were supplied to the nodulated soybean plants, the concentrations of asparagine and glutamine in the xylem sap and roots increased, suggesting that the ureide precursors were absorbed and assimilated to amides in the roots.

A putative xanthine dehydrogenase is critical for Borrelia burgdorferi survival in ticks and mice.

Borrelia burgdorferi is a pathogenic bacterium and the causative agent of Lyme disease. It is exposed to reactive oxygen species (ROS) in both the vertebrate and tick hosts. While some mechanisms by which B. burgdorferi ameliorates the effects of ROS exposure have been studied, there are likely other unknown mechanisms of ROS neutralization that contribute to virulence. Here, we follow up on a three gene cluster of unknown function, bb_0554, bb_0555, and bb_0556, that our prior unbiased transposon insertional sequencing studies implicated in both ROS survival and survival in Ixodes scapularis. We confirmed these findings through genetic knockout and provide evidence that these genes are co-transcribed as an operon to produce a xanthine dehydrogenase. In agreement with these results, we found that B. burgdorferi exposure to either uric acid (a product of xanthine dehydrogenase) or allopurinol (an inhibitor of xanthine dehydrogenase) could modulate sensitivity to ROS in a bb_0554-bb_0556 dependent manner. Together, this study identifies a previously uncharacterized three gene operon in B. burgdorferi as encoding a putative xanthine dehydrogenase critical for virulence. We propose renaming this locus xdhACB.

MO167AN UNUSUAL COURSE OF A 2,8 DHA CRYSTAL INDUCED NEPHROPATHY

Abstract Background and Aims 2, 8 dihydroxyadenine (DHA) deposition is a less known etiology of crystal-induced nephropathy, caused by a deficiency in a purine salvage enzyme, the adenine phosphoribosyl transferase (APRT). DHA is an insoluble molecule in urine leading to crystal formation, tubular obstruction or stone formation. The disease manifests as a history of urolithiasis, chronic kidney disease and even loss of renal allograft when the disease is undiagnosed in native kidneys. The cornerstone of treatment is the inhibition of xanthine dehydrogenase reducing thus the formation of 2,8-DHA and its renal excretion. Method A 59-year-old obese Lebanese male patient, born to a consanguineously married couple, was admitted to another hospital with desaturation, a history of progressive shortness of breath and a creatinine level of 2,8 mg/dl. He had no hypertension nor diabetes. His family history was positive for a sister with ESRD of unknown etiology. He was discharged on oxygen and continuous airway positive pressure therapy for severe obstructive sleep apnea. His renal function deteriorated leading to a creatinine level of 9.8 mg/dl three months later. There were no signs of systemic disease, no gross hematuria, no fluid overload. His blood pressure was normal. Laboratory work up showed anemia, low grade proteinuria, intermittent microscopic hematuria and negative serological and immunological workup. Kidney ultrasound showed normal size kidneys with no evidence of collecting system dilatation or urolithiasis. Due to this atypical presentation, the patient was admitted for a renal biopsy with a creatinine level of 11 mg/dl upon admission. Results The renal biopsy showed tubulo-interstitial nephritis associated with numerous brown-green crystals by Haematoxylin and eosin of various shapes birefringent under polarized light with the characteristic “maltese cross”. Crystals were found within tubular lumens and cytoplasm, interstitium, and macrophages. These findings were characteristic of 2,8 DHA crystals deposition in the kidney. The patient was started on 120 mg of Febuxostat with a low purine and high fluid diet. A genetic testing showed a pathogenic homozygous variant in the APRT gene which causes an amino acid change from Glycine to Aspartate at position 63. Two weeks later the patient was admitted to the ICU with pneumonia, respiratory failure, a creatinine of 9 mg/dl and severe metabolic encephalopathy. He received 4 sessions of hemodialysis followed by an improvement in his kidney function with a creatinine level down to 3.2 mg/dl a month after his discharge and he remains off dialysis until now. Conclusion Around 400 cases are currently recognized worldwide, emphasizing the under recognition of this autosomal recessive disease. Considering that the homozygoty causing a complete APRT deficiency should range between 1/50 000 and 1/100 000 cases, this would translate in at least 80 000 cases worldwide. The variant found in our patient has previously been described as disease causing for APRT deficiency in four cases. Reviewing the phenotype of these cases we find differences in terms of presentation and evolution, highlighting the variability in the APRT deficiency phenotype and underlining the fact that no correlation between phenotype and genotype was reported to date even for the same type of mutation. This case report shows us that the initiation of an adequate therapy is necessary even at advanced stages of the disease since it can improve our kidney outcome.

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

ABSTRACT 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.

Impaired purine homeostasis plays a primary role in trimethoprim-mediated induction of virulence genes in Burkholderia thailandensis.

One of the most commonly prescribed antibiotics against Burkholderia infections is co-trimoxazole, a cocktail of trimethoprim and sulfamethoxazole. Trimethoprim elicits an upregulation of the mal gene cluster, which encodes proteins involved in synthesis of the cytotoxic polyketide malleilactone; trimethoprim does so by increasing expression of the malR gene, which encodes the activator MalR. We report that B. thailandensis grown on trimethoprim exhibited increased virulence against Caenorhabditis elegans. This enhanced virulence correlated with an increase in expression of the mal gene cluster. Notably, inhibition of xanthine dehydrogenase by addition of allopurinol led to similar upregulation of malA and malR, with addition of trimethoprim or allopurinol also resulting in an equivalent intracellular accumulation of xanthine. Xanthine is a ligand for the transcription factor MftR that leads to attenuated DNA binding, and we show using chromatin immunoprecipitation that MftR binds directly to malR. Our gene expression data suggest that malR expression is repressed by both MftR and by a separate transcription factor, which also responds to a metabolite that accumulates on exposure to trimethoprim. Since allopurinol elicits a similar increase in malR/malA expression as trimethoprim, we suggest that impaired purine homeostasis plays a primary role in trimethoprim-mediated induction of malR and in turn malA.

Perfecting a high hypoxanthine phosphoribosyltransferase activity-uricase KO mice to test the effects of purine- and non-purine-type xanthine dehydrogenase (XDH) inhibitors.

Purine metabolism in mice and human differ in terms of uricase (Uox) activity as well as hypoxanthine phosphoribosyltransferase (HPRT) activity. The aim of this study was the establishment of high HPRT activity-Uox knockout (KO) mice as a novel hyperuricaemic model. Then to investigate the effects of purine-type xanthine dehydrogenase (XDH) inhibitor, allopurinol, and non-purine-type XDH inhibitor, topiroxostat, on purine metabolism. A novel hyperuricaemic mouse model was established by mating B6-ChrXCMSM mice with uricase KO mice. The pharmacological effects of allopurinol and topiroxostat were explored by evaluating urate, hypoxanthine, xanthine and creatinine in the plasma and urine of these model mice. Furthermore, we analysed the effect of both drugs on erythrocyte hypoxanthine phosphoribosyltransferase activity. Plasma urate level and urinary urate/creatinine ratio significantly decreased after administration of allopurinol 30 mg·kg-1 or topiroxostat 1 mg·kg-1 for 7 days. The urate-lowering effect was equivalent for allopurinol and topiroxostat. However, the urinary hypoxanthine/creatinine ratio and xanthine/creatinine ratio after treatment with topiroxostat were significantly lower than for allopurinol. In addition, the urinary oxypurine/creatinine ratio was significantly lowered after treatment with topiroxostat, but allopurinol elicited no such effect. Furthermore, allopurinol inhibited mouse erythrocyte hypoxanthine phosphoribosyltransferase, while topiroxostat did not. High hypoxanthine phosphoribosyltransferase activity- uricase KO mice were established as a novel hyperuricaemic animal model. In addition, topiroxostat, a non-purine-type xanthine dehydrogenase inhibitor, elicited a potent plasma urate-lowering effect. However, unlike allopurinol, topiroxostat did not perturb the salvage pathway, resulting in lowered total oxypurine excretion.

Investigation of the transport of xanthine dehydrogenase inhibitors by the urate transporter ABCG2

Hyperuricemia induces gout and kidney stones and accelerates the progression of renal and cardiovascular diseases. Adenosine 5′-triphosphate-binding cassette subfamily G member 2 (ABCG2) is a urate transporter, and common dysfunctional variants of ABCG2, non-functional Q126X (rs72552713) and semi-functional Q141K (rs2231142), are risk factors for hyperuricemia and gout. A recent genome wide association study suggested that allopurinol, a serum uric acid-lowering drug that inhibits xanthine dehydrogenase, is a potent substrate of ABCG2. In this study, we aimed to examine the transport of xanthine dehydrogenase inhibitors via ABCG2. Our results show that ABCG2 transports oxypurinol, an active metabolite of allopurinol, whereas allopurinol and febuxostat, a new xanthine dehydrogenase inhibitor, are not substrates of ABCG2. The amount of oxypurinol transported by ABCG2 vesicles significantly increased in the presence of ATP, compared to that observed with mock vesicles. Since the half-life of oxypurinol is longer than that of allopurinol, the xanthine dehydrogenase-inhibiting effect of allopurinol mainly depends on its metabolite, oxypurinol. Our results indicate that the serum level of oxypurinol would increase in patients with ABCG2 dysfunction.

Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes promotes a blood feeding-induced adulticidal activity.

Aedesaegypti has 2 genes encoding xanthine dehydrogenase (XDH). We analyzed XDH1 and XDH2 gene expression by real-time quantitative PCR in tissues from sugar- and blood-fed females. Differential XDH1 and XDH2 gene expression was observed in tissues dissected throughout a time course. We next exposed females to blood meals supplemented with allopurinol, a well-characterized XDH inhibitor. We also tested the effects of injecting double-stranded RNA (dsRNA) against XDH1, XDH2, or both. Disruption of XDH by allopurinol or XDH1 by RNA interference significantly affected mosquito survival, causing a disruption in blood digestion, excretion, oviposition, and reproduction. XDH1-deficient mosquitoes showed a persistence of serine proteases in the midgut at 48 h after blood feeding and a reduction in the uptake of vitellogenin by the ovaries. Surprisingly, analysis of the fat body from dsRNA-XDH1-injected mosquitoes fell into 2 groups: one group was characterized by a reduction of the XDH1 transcript, whereas the other group was characterized by an up-regulation of several transcripts, including XDH1, glutamine synthetase, alanine aminotransferase, catalase, superoxide dismutase, ornithine decarboxylase, glutamate receptor, and ammonia transporter. Our data demonstrate that XDH1 plays an essential role and that XDH1 has the potential to be used as a metabolic target for Ae.aegypti vector control.-Isoe, J., Petchampai, N., Isoe, Y. E., Co, K., Mazzalupo, S., Scaraffia, P. Y. Xanthine dehydrogenase-1 silencing in Aedes aegypti mosquitoes promotes a blood feeding-induced adulticidal activity.

Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy.

Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5-10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo(VI) ion with each one MPT, Mo═O, Mo-O-, and Mo═S ligand, and a weak Mo-O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK ∼ 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK ∼ 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo-OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.

Kidney Disease in Adenine Phosphoribosyltransferase Deficiency

Adenine phosphoribosyltransferase (APRT) deficiency is a purine metabolism disorder causing kidney stones and chronic kidney disease (CKD). The course of nephrolithiasis and CKD has not been well characterized. The objective of this study was to examine long-term kidney outcomes in patients with APRT deficiency. An observational cohort study. All patients enrolled in the APRT Deficiency Registry of the Rare Kidney Stone Consortium. Kidney stones, acute kidney injury (AKI), stage of CKD, end-stage renal disease, estimated glomerular filtration rate (eGFR), and changes in eGFR. Serum creatinine and eGFR calculated using creatinine-based equations. Of 53 patients, 30 (57%) were females and median age at diagnosis was 37.0 (range, 0.6-67.9) years. Median duration of follow-up was 10.3 (range, 0.0-31.5) years. At diagnosis, kidney stones had developed in 29 (55%) patients and 20 (38%) had CKD stages 3 to 5, including 11 (21%) patients with stage 5. At latest follow-up, 33 (62%) patients had experienced kidney stones; 18 (34%), AKI; and 22 (42%), CKD stages 3 to 5. Of 14 (26%) patients with stage 5 CKD, 12 had initiated renal replacement therapy. Kidney stones recurred in 18 of 33 (55%) patients. The median eGFR slope was -0.38 (range, -21.99 to 1.42) mL/min/1.73m(2) per year in patients receiving treatment with an xanthine dehydrogenase inhibitor and -5.74 (range, -75.8 to -0.10) mL/min/1.73m(2) per year in those not treated prior to the development of stage 5 CKD (P=0.001). Use of observational registry data. Progressive CKD and AKI episodes are major features of APRT deficiency, whereas nephrolithiasis is the most common presentation. Advanced CKD without a history of kidney stones is more prevalent than previously reported. Our data suggest that timely therapy may retard CKD progression.

Unorthodox methods for enhancing solvent production in solventogenic Clostridium species.

While production of biofuels from renewable resources is currently receiving increased attention globally, concerns on availability and sustainability of cheap substrates for their production are growing as well. Lignocellulose-derived sugars (LDS) remain underutilized and merit consideration as a key feedstock. Among other obstacles such as low yield and low solvent titer, mitigation of stresses stemming from lignocellulose-derived microbial inhibitory compounds (LDMICs) that severely impair cell growth and solvent production is a major area of research interest. In addition to attempts at developing LDMIC-tolerant strains via metabolic engineering to enhance utilization of LDS, unconventional approaches that elicit different metabolic perturbations in microorganisms to relieve solvent- and LDMIC-mediated stresses have been explored to increase solvent production from LDS. In this review, the impacts of metabolic perturbations including medium supplementation with glycerol; furfural and 5-hydroxymethyl furfural; allopurinol, an inhibitor of xanthine dehydrogenase; calcium (Ca(2+)) and zinc (Zn(2+)) ions); and artificial electron carriers, methyl viologen and neutral red, on butanol production are discussed. Although these approaches have brought about considerable increases in butanol production, both from LDS and defined glucose-based media, the modes of action for most of these perturbations have yet to be fully characterized. Better understanding of these mechanisms would likely inform development of LDMIC-tolerant, butanol-overproducing strains, as well as possible combinatorial application of these approaches for enhanced butanol production. Hence, delineating the underlying mechanisms of these perturbations deserves further attention.

PharmGKB summary: uric acid-lowering drugs pathway, pharmacodynamics.

Disorders involving uric acid including gout, hyperuricemia and resultant kidney failure are prevalent. There are three main groups of pharmaceuticals used for modulating uric acid: 1. Drugs that reduce the generation of uric acid (allopurinol and febuxistat), 2. Those that increase the removal of uric acid (rasburicase, pegloticase), (both depicted in Figure 1) or 3. Those that inhibit the reabsorption of uric acid (depicted in Figure 3). This summary briefly describes the mechanisms of action and the candidate genes and genetic variants associated with response to these drugs. Clinically relevant genetic variants have been identified for allopurinol and rasburicase, and CPIC guidelines have been published that recommend selection of alternative treatments for some individuals. A more extensive description and interactive version can be found for each pathway at www.pharmgkb.org. Figure 1 Uric acid-lowering drugs pathway, Pharmacodynamics Figure 3 Uricosurics pathway, Pharmacodynamics 1) Drugs that reduce the generation of uric acid: allopurinol and febuxostat Pharmacodynamics Allopurinol is a drug indicated for the treatment of gout, prophylaxis of hyperuricemia in patients undergoing chemotherapy, and prevention of kidney stones recurrence [1]. Allopurinol and its metabolite oxypurinol are analogues of hypoxanthine and xanthine, respectively, and work by binding to and inhibiting xanthine dehydrogenase (XDH), preventing the formation of uric acid (Figure 1) [1–5]. Hypoxanthine and xanthine are cleared in the urine or reutilized in the synthesis of nucleotides and nucleic acids [1, 6]. Febuxostat (Uloric®) is a non-purine inhibitor of XDH that can bind to and inhibit both oxidized or reduced forms of XDH (Figure 1) [2, 7, 8]. It is indicated for the treatment of hyperuricemia in patients with gout (but not for asymptomatic hyperuricemia) [9], and is an alternative therapy for patients where allopurinol has been contraindicated due to allergic responses [7, 8, 10]. A downside to allopurinol/febuxostat treatment is that existing high levels of plasma uric acid are not cleared and, as xanthine is less soluble than uric acid, xanthine kidney stones or xanthine nephropathy may result [3, 6].

Pivotal Role of Toll-Like Receptors 2 and 4, Its Adaptor Molecule MyD88, and Inflammasome Complex in Experimental Tubule-Interstitial Nephritis

Tubule-interstitial nephritis (TIN) results in decreased renal function and interstitial inflammation, which ultimately leads to fibrosis. Excessive adenine intake can cause TIN because xanthine dehydrogenase (XDH) can convert this purine into an insoluble compound, which precipitates in the tubuli. Innate immune sensors, such as Toll-like receptors (TLR) and inflammasome complex, play a crucial role in the initiation of inflammation. The aim of this study was to evaluate the roles of TLR-2 and -4, Myd88 and inflammasome complex in an experimental model of TIN. Here, we show that wild-type (WT) mice fed adenine-enriched food exhibited significant renal dysfunction and enhanced cellular infiltration accompanied by collagen deposition. They also presented higher gene and protein expression of pro-inflammatory cytokines. In contrast, TLR-2, -4, MyD88, ASC and Caspase-1 KO mice showed renoprotection associated with expression of inflammatory molecules at levels comparable to controls. Furthermore, treatment of WT animals with allopurinol, an XDH inhibitor, led to reduced levels of uric acid, oxidative stress, collagen deposition and a downregulation of the NF-kB signaling pathway. We concluded that MyD88 signaling and inflammasome participate in the development of TIN. Furthermore, inhibition of XDH seems to be a promising way to therapeutically target the developing inflammatory process.

Ureide metabolism during seedling development in French bean (Phaseolus vulgaris)

French bean (Phaseolus vulgaris) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.

The RNAi-Mediated Silencing of Xanthine Dehydrogenase Impairs Growth and Fertility and Accelerates Leaf Senescence in Transgenic Arabidopsis Plants

Xanthine dehydrogenase (XDH) is a ubiquitous enzyme involved in purine metabolism which catalyzes the oxidation of hypoxanthine and xanthine to uric acid. Although the essential role of XDH is well documented in the nitrogen-fixing nodules of leguminous plants, the physiological importance of this enzyme remains uncertain in non-leguminous species such as Arabidopsis. To evaluate the impact of an XDH deficiency on whole-plant physiology and development in Arabidopsis, RNA interference (RNAi) was used to generate transgenic lines of this species in which AtXDH1 and AtXDH2, the two paralogous genes for XDH in this plant, were silenced simultaneously. The nearly complete reduction in the total XDH protein levels caused by this gene silencing resulted in the dramatic overaccumulation of xanthine and a retarded growth phenotype in which fruit development and seed fertility were also affected. A less severe silencing of XDH did not cause these growth abnormalities. The impaired growth phenotype was mimicked by treating wild-type plants with the XDH inhibitor allopurinol, and was reversed in the RNAi transgenic lines by exogenous supplementation of uric acid. Inactivation of XDH is also associated with precocious senescence in mature leaves displaying accelerated chlorophyll breakdown and by the early induction of senescence-related genes and enzyme markers. In contrast, the XDH protein levels increase with the aging of the wild-type leaves, supporting the physiological relevance of the function of this enzyme in leaf senescence. Our current results thus indicate that XDH functions in various aspects of plant growth and development.

Oxidative stress contributes to methotrexate-induced small intestinal toxicity in rats

Background: Gastrointestinal toxicity is one of the most serious side effects in the methotrexate (MTX) treatment. However, the mechanism of the toxicity has not been completely clarified, which may be the reason why symptomatic therapy is carried out. On the other hand, the oxidative stress is known to play an important role in various diseases and drug-induced side effects. In this study the focus was on the oxidative stress in order to clarify the mechanism of MTX-induced small intestinal damage, especially neutrophil infiltration. Methods: MTX (20 mg/kg body wt) was administered to rats intravenously. Mucosal homogenates were prepared from the small intestine and used for assay of biochemical parameters, by which induction of oxidative stress and neutrophil infiltration were evaluated. N-acetylcysteine (NAC; 80 mg/kg body wt), an antioxidant or sodium tungstate (tungsten; 0.7 g/kg body wt), an inhibitor of xanthine dehydrogenase (XD)/xanthine oxidase (XO) known as an important source of reactive oxygen species (ROS) was given to rats with MTX to investigate the contribution of ROS to neutrophil infiltration. Results: The MTX treatment of rats induced the oxidative stress in the small intestine. The ROS production was seen preceding an increase of myeloperoxidase activity, which suggested neutrophil infiltration. Both treatments of NAC and tungsten prevented the MTX-induced ROS production and neutrophil infiltration. Conclusions: These results suggest that oxidative stress plays an important role in the MTX-induced small intestinal damage, especially neutrophil infiltration. Thus, the modulation of oxidative stress would be useful in reducing intestinal damage in MTX treatment.

Crystal Structures of the Active and Alloxanthine-Inhibited Forms of Xanthine Dehydrogenase from Rhodobacter capsulatus

Xanthine dehydrogenase (XDH), a complex molybdo/iron-sulfur/flavoprotein, catalyzes the oxidation of hypoxanthine to xanthine followed by oxidation of xanthine to uric acid with concomitant reduction of NAD +. The 2.7 Å resolution structure of Rhodobacter capsulatus XDH reveals that the bacterial and bovine XDH have highly similar folds despite differences in subunit composition. The NAD + binding pocket of the bacterial XDH resembles that of the dehydrogenase form of the bovine enzyme rather than that of the oxidase form, which reduces O 2 instead of NAD +. The drug allopurinol is used to treat XDH-catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of XDH. The 3.0 Å resolution structure of the XDH-alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new XDH inhibitors.

Effect of allopurinol treatment on biotrophic growth of some powdery mildew fungi in their specific hosts

When supplied via roots to tobacco host plants, aqueous solutions of allopurinol, a specific inhibitor of xanthine oxidase and dehydrogenase, markedly reduced or completely prevented the visible development of tobacco powdery mildew fungus Erysiphe cichoracearum(E. orontii) at concentrations as low as 25–50μM administered for periods as short as 2 days. The duration of this allopurinol-dependent protection from the pathogen was of at least 20 days after the last administration. If supplied as foliar sprays at concentrations up to 1600μM, allopurinol was not absorbed by the leaf tissues and did not affect the fungal infection. Histological observations revealed that on tobacco plants treated with allopurinol via roots, a sharp reduction inE. cichoracearum mycelial development started only 48h after inoculation (after formation of the first haustoria) and was accompanied by a strong delay and reduction of sporulation. The proportion of haustoria partially or entirely embedded in thick translucent sheaths (possibly callosic in nature) increased significantly in allopurinol irrigated plants reaching about 41% of the total haustoria (compared to 9% in controls) 7 days post-inoculation. No effects of allopurinol supply was observed on Blumeria graminis and Erysiphe trifolii development on wheat and clover plants, respectively. The present results are consistent with the view that allopurinol affects pathogen growth and sporulation by inhibition of xanthine dehydrogenase in the plant, the pathogen or both organisms.