Xdh Gene Research Articles

The presence of xanthine dehydrogenase is crucial for the maturation of the rat kidneys.

The development of the kidney involves essential cellular processes, such as cell proliferation and differentiation, which are led by interactions between multiple signaling pathways. Xanthine dehydrogenase (XDH) catalyzes the reaction producing uric acid in the purine catabolism, which plays a multifaceted role in cellular metabolism. Our previous study revealed that the genetic ablation of the Xdh gene in rats leads to smaller kidneys, kidney damage, decline of renal functions, and failure to thrive. Rats, unlike humans, continue their kidney development postnatally. Therefore, we explored whether XDH plays a critical role in kidney development using SS-/- rats during postnatal development phase. XDH expression was significantly increased from postnatal day 5 to 15 in wild-type but not homozygote rat kidneys. The transcriptomic profile of renal tissue revealed several dysregulated pathways due to the lack of Xdh expression with the remodeling in inflammasome, purinergic signaling, and redox homeostasis. Further analysis suggested that lack of Xdh affects kidney development, likely via dysregulation of epidermal growth factor and its downstream STAT3 signaling. The present study showed that Xdh is essential for kidney maturation. Our data, alongside the previous research, suggests that loss of Xdh function leads to developmental issues, rendering them vulnerable to kidney diseases in adulthood.

Pharmacokinetics of Glutapyrone and its Impact on Expression of the Genes Involved in DNA Repair and Free Radical Production in Intact and Diabetic Rats

Abstract Modification of expression of several genes encoding enzymes involved in radical production and DNA repair by a 1,4-DHP derivative glutapyrone was studied in intact rats and animals with streptozotocin diabetes mellitus. Glutapyrone stimulated iNos mRNA production in both kidneys and blood. The effect was stronger in kidneys of diabetic animals, however in blood the expression was down-regulated. The compound increased the Parp 1 gene expression in kidneys of both intact and diabetic animals; in blood the effect was adverse. Expression of XDh gene was significantly increased by glutapyrone in kidneys of intact and diabetic animals. Administration of the compound to intact animals triggered significant increase of DNA damage in white blood cells assayed by comet assay; in diabetic animals no effect was produced. To explain discrepancies with the formerly described effects of glutapyrone on cultured cells, metabolism of the compound was studied. Glutapyrone is either oxidised or the residue of glutamic acid is removed, glutapyrone turns into AV-153, and the latter is metabolised to smaller compounds. Formation of AV-153, a DNA binder and genotoxic compound in high concentrations, can explain DNA damage in white blood cells and stimulation of DNA repair.

Hemin and iron increase synthesis and trigger export of xanthine oxidoreductase from hepatocytes to the circulation

We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 μM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 μU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control μU/mg). Interestingly, free iron (Fe2+ and Fe3+) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 μM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload.

Xanthine oxidoreductase gene polymorphisms are associated with high risk of sepsis and organ failure

BackgroundSepsis and associated organ failures confer substantial morbidity and mortality. Xanthine oxidoreductase (XOR) is implicated in the development of tissue oxidative damage in a wide variety of respiratory and cardiovascular disorders including sepsis and sepsis-associated acute respiratory distress syndrome (ARDS). We examined whether single nucleotide polymorphisms (SNPs) in the XDH gene (encoding XOR) might influence susceptibility to and outcome in patients with sepsis.MethodsWe genotyped 28 tag SNPs in XDH gene in the CELEG cohort, including 621 European American (EA) and 353 African American (AA) sepsis patients. Serum XOR activity was measured in a subset of CELEG subjects. Additionally, we assessed the functional effects of XDH variants utilizing empirical data from different integrated software tools and datasets.ResultsAmong AA patients, six intronic variants (rs206805, rs513311, rs185925, rs561525, rs2163059, rs13387204), in a region enriched with regulatory elements, were associated with risk of sepsis (P < 0.008–0.049). Two out of six SNPs (rs561525 and rs2163059) were associated with risk of sepsis-associated ARDS in an independent validation cohort (GEN-SEP) of 590 sepsis patients of European descent. Two common SNPs (rs1884725 and rs4952085) in tight linkage disequilibrium (LD) provided strong evidence for association with increased levels of serum creatinine (Padjusted<0.0005 and 0.0006, respectively), suggesting a role in increased risk of renal dysfunction. In contrast, among EA ARDS patients, the missense variant rs17011368 (I703V) was associated with enhanced mortality at 60-days (P < 0.038). We found higher serum XOR activity in 143 sepsis patients (54.5 ± 57.1 mU/mL) compared to 31 controls (20.9 ± 12.4 mU/mL, P = 1.96 × 10− 13). XOR activity was associated with the lead variant rs185925 among AA sepsis patients with ARDS (P < 0.005 and Padjusted<0.01). Multifaceted functions of prioritized XDH variants, as suggested by various functional annotation tools, support their potential causality in sepsis.ConclusionsOur findings suggest that XOR is a novel combined genetic and biochemical marker for risk and outcome in patients with sepsis and ARDS.

Lack of xanthine dehydrogenase leads to a remarkable renal decline in a novel hypouricemic rat model

SummaryUric acid (UA) is the final metabolite in purine catabolism in humans. Previous studies have shown that the dysregulation of UA homeostasis is detrimental to cardiovascular and kidney health. The Xdh gene encodes for the Xanthine Oxidoreductase enzyme group, responsible for producing UA. To explore how hypouricemia can lead to kidney damage, we created a rat model with the genetic ablation of the Xdh gene on the Dahl salt-sensitive rat background (SSXdh−/−). SSXdh−/− rats lacked UA and exhibited impairment in growth and survival. This model showed severe kidney injury with increased interstitial fibrosis, glomerular damage, crystal formation, and an inability to control electrolyte balance. Using a multi-omics approach, we highlighted that lack of Xdh leads to increased oxidative stress, renal cell proliferation, and inflammation. Our data reveal that the absence of Xdh leads to kidney damage and functional decline by the accumulation of purine metabolites in the kidney and increased oxidative stress.

Lack of Xdh Leads to Alterations in Renin‐Angiotensin‐Aldosterone System and Kidney Injury

Uric acid (UA) homeostasis has been elucidated as a key player in chronic kidney disease and cardiovascular diseases, yet definitive mechanisms are unknown. Previous studies in rodents and humans have shown that increased levels of serum UA predict an activated intrarenal Renin‐Angiotensin‐Aldosterone System (RAAS). In hyperuricemic rats, nephropathy was reported with glomerular hypertrophy and interstitial inflammation accompanied by increased renal renin expression. To explore whether hypouricemia leads to RAAS changes and kidney damage, we utilized a novel rat model with genetic ablation of the Xdh gene in the Dahl salt‐sensitive rat background (SSXdh‐/‐). The Xdh gene encodes for the Xanthine Oxidoreductase enzyme group, which produces UA from xanthine and hypoxanthine. We have shown that the SSXdh‐/‐model is hypouricemic and has severe kidney damage. The SSXdh‐/‐ rats demonstrated significant kidney function decline (SSXdh+/+&amp; SSXdh‐/‐respectively, diuresis: 2.7 ± 0.9 &amp; 14.4 ± 5.1 ml; creatinine clearance: 0.51 ± 0.19 &amp; 0.12 ± 0.04 ml/min; and plasma Na+: 136 ± 1.8 &amp; 150 ± 3.7 mmol/l). Quantification of RAAS components was done using liquid chromatography‐tandem mass spectrometry. The comparison of plasma from SSXdh+/+vs SSXdh‐/‐rats revealed significantly lower levels of angiotensin I (650 ± 109, 178 ± 25 pmol/l), Ang II (583 ± 70, 171 ± 16 pmol/l), and renin (indirect measurements using Ang I + Ang II: 1233 ± 172, 349 ± 40 pmol/l). Aldosterone level was increased in SSXdh‐/‐rats (870 ± 115 vs. 4106 ± 1038 pmol/l). The aldosterone/ Ang II ratio was significantly increased in the homozygous rats suggesting enhanced adrenal function (2 ± 0.1, 25 ± 6.3). To further evaluate the contribution of RAAS and its downstream signaling mechanisms, we performed RNA‐Seq analysis on kidney cortex tissue, which showed a number of RAAS‐related genes being differentially expressed between the SSXdh+/+and SSXdh‐/‐ rats. In hypouricemic rats, there was an increase in expression levels of Ace, Ccl2, Fos, Map3k1, Ptger2,and Stat3,and a decrease of Agtr1a, Hsd11b1,and Renb. Therefore, our results show that lack of Xdh/UA leads to kidney injury and functional decline with substantial remodeling in RAAS.

Identification of a new mutation in the human xanthine dehydrogenase responsible for xanthinuria type I.

Hereditary xanthinuria is a rare, autosomal and recessive disorder characterized by severe hypouricemia and increased xanthine excretion, caused by a deficiency of xanthine dehydrogenase/oxidase (XDH/XO, EC: 1.17.1.4/1.17.3.2) in type I, or by a deficiency of XDH/XO and aldehyde oxidase (AOX, EC: 1.2.3.1) in type II. We describe a novel point mutation in the XDH gene in homozygosis found in a patient with very low serum and urine levels of uric acid, together with xanthinuria. He was asymptomatic but renal calculi were discovered during imaging. Additional cases were found in his family and dietary recommendations were made in order to prevent further complications. Hereditary xanthinuria is an underdiagnosed pathology, often found in a routine analysis that shows hypouricemia. It is important for Laboratory Medicine to acknowledge how to guide clinicians in the diagnosis.

Classical Xanthinuria in Nine Israeli Families and Two Isolated Cases from Germany: Molecular, Biochemical and Population Genetics Aspects.

Classical xanthinuria is a rare autosomal recessive metabolic disorder caused by variants in the XDH (type I) or MOCOS (type II) genes. Thirteen Israeli kindred (five Jewish and eight Arab) and two isolated cases from Germany were studied between the years 1997 and 2013. Four and a branch of a fifth of these families were previously described. Here, we reported the demographic, clinical, molecular and biochemical characterizations of the remaining cases. Seven out of 20 affected individuals (35%) presented with xanthinuria-related symptoms of varied severity. Among the 10 distinct variants identified, six were novel: c.449G>T (p.(Cys150Phe)), c.1434G>A (p.(Trp478*)), c.1871C>G (p.(Ser624*)) and c.913del (p.(Leu305fs*1)) in the XDH gene and c.1046C>T (p.(Thr349Ileu)) and c.1771C>T (p.(Pro591Ser)) in the MOCOS gene. Heterologous protein expression studies revealed that the p.Cys150Phe variant within the Fe/S-I cluster-binding site impairs XDH biogenesis, the p.Thr349Ileu variant in the NifS-like domain of MOCOS affects protein stability and cysteine desulfurase activity, while the p.Pro591Ser and a previously described p.Arg776Cys variant in the C-terminal domain affect Molybdenum cofactor binding. Based on the results of haplotype analyses and historical genealogy findings, the potential dispersion of the identified variants is discussed. As far as we are aware, this is the largest cohort of xanthinuria cases described so far, substantially expanding the repertoire of pathogenic variants, characterizing structurally and functionally essential amino acid residues in the XDH and MOCOS proteins and addressing the population genetic aspects of classical xanthinuria.

A Potential Regulatory Role of Xanthine Dehydrogenase (XDH) in the Kidney Development and Damage

There is a clear association of uric acid (UA) dysregulation in cardiovascular and chronic kidney diseases elucidated in the literature. Xanthine dehydrogenase (XDH) encodes for XDH enzyme that catalyzes hypoxanthine oxidation to xanthine and xanthine to UA, the end-product of purine catabolism in humans. UA can act as both an antioxidant scavenging reactive oxygen species (ROS) and conditionally a pro-oxidant. The lack of understanding of UA metabolism and its impact on CKD progression reflects the current controversy regarding the clinical management of the related pathologies. To explore mechanistic insights into UA affecting the kidney, we created a genetic ablation of the Xdh gene in the Dahl salt-sensitive rat background (SSXdh-/-). The absence of Xdh protein expression in SSXdh-/- was confirmed by Western blotting and immunohistochemistry analyses. The knockout rat is underdeveloped (179 ± 6 and 70 ± 7 g: N=9 for both male SSXdh+/+ and SSXdh-/-) and hypouricemic (UA in plasma below the level of detection). RNA-Seq analysis of kidney tissue of the SSXdh-/- and SSXdh+/+ 6-week-old rats revealed 4738 differentially expressed genes. The functional analyses of the obtained data were performed using the ingenuity pathway analysis (IPA) software. The changes in oxidative phosphorylation and the presence of mitochondrial dysfunction were among the top canonical pathways associated with the differentially expressed genes. The cardiac hypertrophy signaling pathway was enhanced the most (z-score 6.5). IPA toxic function analysis suggested the presence of renal damage. Among the upregulated genes were transforming growth factor-β (Tgf-β) and connective tissue growth factor (Ccn2), previously reported to drive the tissue fibrosis. Indeed, SSXdh-/- rats show increased levels of fibrosis in the kidneys. Western blot analysis revealed Ctgf lacking in the SSXdh-/- rats despite the increased mRNA levels. Ctgf is essential for kidney development, and the lack of this protein might explain the underdeveloped kidneys seen in the SSXdh-/- rat. Furthermore, we analyzed the expression of the primary transporters for UA in the kidney: Urat1 and Glut9, encoded by Slc22a12 and Slc2a9 genes, respectively. Western blot analysis revealed that Urat1 is not detectable and Glut9 expression is decreased in SSXdh-/- rats; potential glycosylation of Glut9 in SSXdh-/- rats is also observed. Catalase enzyme encoding Cat gene is also downregulated (log FC=-3.05) in SSXdh-/- rats, indicating the potential ROS production increase. These results provide insights into the regulatory role of the Xdh gene beyond purine catabolism that involves kidney tissue development and kidney damage.

Similar solutions to a common challenge: regulation of genes encoding Ralstonia solanacearum xanthine dehydrogenase.

The stringent response involves accumulation of (p)ppGpp, and it ensures that survival is prioritized. Production of (p)ppGpp requires purine synthesis, and upregulation of an operon that encodes the purine salvage enzyme xanthine dehydrogenase (Xdh) has been observed during stringent response in some bacterial species, where direct binding of ppGpp to a TetR-family transcription factor is responsible for increased xdh gene expression. We show here that the plant pathogen Ralstonia solanacearum has a regulatory system in which the LysR-family transcription factor XanR controls expression of the xan operon; this operon encodes Xdh as well as other enzymes involved in purine salvage, which favor accumulation of xanthine. XanR bound upstream of the xan operon, a binding that was attenuated on addition of either ppGpp or cyclic di-guanosine monophosphate (c-di-GMP). Using a reporter in which enhanced green fluorescent protein (EGFP) is expressed under control of a modified xan promoter, XanR was shown to repress EGFP production. Our data suggest that R. solanacearum features a regulatory mechanism in which expression of genes encoding purine salvage enzymes is controlled by a transcription factor that belongs to a different protein family, yet performs similar regulatory functions.

Construction of recombinant Escherichia coli expressing xylitol-4-dehydrogenase and optimization for enhanced L-xylulose biotransformation from xylitol.

L-Xylulose is a rare ketopentose which inhibits α-glucosidase and is an indicator of hepatitis or liver cirrhosis. This pentose is also a precursor of other rare sugars such as L-xylose, L-ribose or L-lyxose. Recombinant E. coli expressing xylitol-4-dehydrogenase gene of Pantoea ananatis was constructed. A cost-effective culture media were used for L-xylulose production using the recombinant E. coli strain constructed. Response surface methodology was used to optimize these media components for L-xylulose production. A high conversion rate of 96.5% was achieved under an optimized pH and temperature using 20g/L xylitol, which is the highest among the reports. The recombinant E. coli cells expressing the xdh gene were immobilized in calcium alginate to improve recycling of cells. Effective immobilization was achieved with 2% (w/v) sodium alginate and 3% (w/v) calcium chloride. The immobilized E. coli cells retained good stability and enzyme activity for 9 batches with conversion between 53 and 92% which would be beneficial for economical production of L-xylulose.

The Role of Xanthine Dehydrogenase (XDH) and Uric Acid in the Kidney Development and Renal Injury

Uric acid is the end product of purine catabolism in humans and is capable of acting as both an antioxidant scavenging reactive oxygen species and conditionally a pro‐oxidant. Pathophysiological changes in serum uric acid levels (hyperuricemia and hypouricemia) are associated with prevalent diseases, including hypertension and chronic kidney disease as well as rare disorders, such as hereditary xanthinuria. Despite the clear association between uric acid dysregulation and pathology, the mechanisms involved remain unclear. XDH and xanthine oxidase (XO) (together known as Xanthine oxidoreductase (XOR)) is a complex enzyme group that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid. To assess the role of this pathway beyond purine catabolism, we created a genetic ablation of the Xdh gene in the Dahl salt‐sensitive rat background (SSXdh−/−). Sequencing of SSXdh−/− genomic DNA confirmed a 7‐base‐pair‐deletion in exon 4 of the Xdh gene located on chromosome 6. The absence of Xdh protein expression in homozygotes and a reduction in heterozygotes was confirmed by both Western blot and immunohistochemistry analyses. SSXdh−/− rats exhibited early mortality with 7 out of 28 rats not surviving till 6 weeks of age compared to SSXdh+/− and SSXdh+/+ littermates, all of whom survived. Plasma uric acid of 6 weeks old male rats was 0.25±0.03 mg/dl (N=6) in SSXdh+/+ rats and decreased to a level out of the detectable range by the test in the SSXdh−/− group. The 24hr uric acid excretion was also dramatically decreased in SSXdh−/− rats compared to SSXdh+/+ littermates. Masson’s Trichrome staining revealed severe renal damage with an increase in fibrosis (1 and 14% (N=3 and 2 for male SSXdh+/+ and SSXdh−/−, respectively), and dilated tubules in the SSXdh−/− rats. Total body weight was decreased in SSXdh−/− rats, indicating a failure to thrive compared to control rats (190 ± 4 and 58 ± 8 g; N=6 and 4 for male SSXdh+/+ and SSXdh−/−). Additionally, In SSXdh−/− rats at 6 weeks, Western blot analysis showed a lack of connective tissue growth factor (CTGF), which is known to be critical in the nephrogenesis. No apparent sex‐specific differences in development or kidney injury were identified. Our results suggest that the genetic ablation of Xdh caused uric acid depletion and severe renal damage. Therefore, XOR may be essential for the development of the kidney and survival of the rats by regulating purine catabolism.Support or Funding InformationSupported by the Department of Veteran Affairs Grant I01 BX004024 and the National Institutes of Health Grants R35 HL‐135749.

An ancestral variant causing type I xanthinuria in Turkmen and Arab families is predicted to prevail in the Afro-Asian stone-forming belt.

Classical xanthinuria is a rare autosomal recessive metabolic disorder characterized by lack of xanthine dehydrogenase activity that often manifests as xanthine urolithiasis and risk of drug toxicity. Variants in the XDH or HMCS gene underlie classical xanthinuria type I and type II, respectively. Here we present two Israeli Arab families affected by type I xanthinuria in whom a c.2164A>T (Lys722Ter) variant in the XDH gene, previously reported in a Turkish family of Turkmen origin, was identified. Analysis of polymorphic markers surrounding the variant site revealed common haplotypes spanning 0.6 Mbp shared by all three, and 1.7 Mbp shared by two of the studied families. By applying Bayesian methods to a simple model of crossover events through generations in the chromosomes carrying the variant, the most recent common ancestor of these families was found to be 179 (95% credible limit 70) generations old. The estimated antiquity of the variant, the historical genealogy of the affected families and the history and present day dispersion of their people strongly suggest prevalence of this variant in the Afro‐Asian stone‐forming belt. As far as we are aware, this is a first report of an ancient variant causing xanthinuria with potential wide geographical dispersion.

Association between Serum Urate and Risk of Hypertension in Menopausal Women with XDH Gene.

Elevated serum urate (sUA) concentrations have been associated with an increased risk of hypertension. We aimed to examine the association of sUA concentration on the risk of hypertension in pre- and post-menopausal women and investigated the association between the polymorphism of the xanthine dehydrogenase gene and the risk of hypertension. Among 7294 women, 1415 premenopausal and 5879 postmenopausal women were recruited. Anthropometric parameters as risk factors of hypertension were identify by logistic regression models. In addition, we investigated an association between xanthine dehydrogenase gene and sUA and their combined associations on the risk of hypertension. Body mass index (BMI) and waist circumference (WC) were significantly increased in accordance to the increase of sUA levels (p < 0.001). Multivariate logistic regression analysis showed postmenopausal women with a high sUA and high BMI were 3.18 times more likely to have hypertension than in those with normal and lower sUA (Odds ratio: 3.18, 95% confidence interval: 2.54–3.96). Postmenopausal women with a high WC were 1.62 times more likely to have hypertension than in those with normal and lower sUA. Subjects with the AG genotype of rs206860 was found to be at lower risk of hypertension (odd ratio: 0.287, 95% confidence interval: 0.091–0.905, p = 0.033). This cross-sectional study indicated a high sUA is associated with a higher risk of hypertension in postmenopausal women. Further well-designed prospective studies in other populations are warranted to validate our results.

Halenia coreana (Gentianaceae), a new species from Korea: Evidence from morphological and molecular data

A new species of Halenia (Gentianaceae) from Korea, H. coreana S.M.Han, H.Won &amp; C.E.Lim, is recognized based on morphological and molecular data, and its description and illustration are provided. It is distinct from H. corniculata in having long, narrower and incurved spurs and attenuated leaf apex. Molecular sequence data of nuclear ribosomal ITS region, nuclear XDH gene, and chloroplast rbcL gene also strongly support its species status.

Identification of oxidative stress–related Xdh gene as a di(2‐ethylhexyl)phthalate (DEHP) target and the use of melatonin to alleviate the DEHP‐induced impairments in newborn mouse ovaries

This study, using an in vitro ovary culture model, investigates the mechanisms through which di(2-ethylhexyl)phthalate (DEHP) impairs germ cell cyst breakdown and primordial follicle assembly. The results indicate the latter effects exerted by 10 or 100µmol/L DEHP in cultured newborn ovaries were associated with increased levels of reactive oxygen species (ROS) and apoptosis. Based on a transcriptome analysis, we found the expression of the oxidative stress-related gene Xdh (xanthine dehydrogenase) was significantly upregulated in DEHP-cultured ovaries. Two treatments, namely Xdh RNAi or the addition of melatonin to the ovary culture, inhibited the increase in Xdh expression and ROS levels caused by DEHP and, at the same time, reduced apoptosis and the impairment of primordial follicle assembly in the treated ovaries. Together, the results identify Xdh gene as one of the major targets of DEHP in newborn ovaries and that the consequent increased level of ROS is possibly responsible for the increment of apoptosis and primordial follicle assembly impairment. At the same time, they highlight that melatonin alleviates the effects of DEHP as with other endocrine-disrupting compounds on the ovary.

De novo transcriptome and phytochemical analyses reveal differentially expressed genes and characteristic secondary metabolites in the original oolong tea (Camellia sinensis) cultivar \u2018Tieguanyin\u2019 compared with cultivar \u2018Benshan\u2019

BackgroundThe two original plants of the oolong tea cultivar (‘Tieguanyin’) are “Wei shuo” ‘Tieguanyin’—TGY (Wei) and “Wang shuo” ‘Tieguanyin’—TGY (Wang). Another cultivar, ‘Benshan’ (BS), is similar to TGY in its aroma, taste, and genetic make-up, but it lacks the “Yin Rhyme” flavor. We aimed to identify differences in biochemical characteristics and gene expression among these tea plants.ResultsThe results of spectrophotometric, high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) analyses revealed that TGY (Wei) and TGY (Wang) had deeper purple-colored leaves and higher contents of anthocyanin, catechins, caffeine, and limonene compared with BS. Analyses of transcriptome data revealed 12,420 differentially expressed genes (DEGs) among the cultivars. According to a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the flavonoid, caffeine, and limonene metabolic pathways were highly enriched. The transcript levels of the genes involved in these three metabolic pathways were not significantly different between TGY (Wei) and TGY (Wang), except for two unigenes encoding IMPDH and SAMS, which are involved in caffeine metabolism. The comparison of TGY vs. BS revealed eight up-regulated genes (PAL, C4H, CHS, F3’H, F3H, DFR, ANS, and ANR) and two down-regulated genes (FLS and CCR) in flavonoid metabolism, four up-regulated genes (AMPD, IMPDH, SAMS, and 5′-Nase) and one down-regulated XDH gene in caffeine metabolism; and two down-regulated genes (ALDH and HIBADH) in limonene degradation. In addition, the expression levels of the transcription factor (TF) PAP1 were significantly higher in TGY than in BS. Therefore, high accumulation of flavonoids, caffeine, and limonene metabolites and the expression patterns of their related genes in TGY might be beneficial for the formation of the “Yin Rhyme” flavor.ConclusionsTranscriptomic, HPLC, and GC-MS analyses of TGY (Wei), TGY (Wang), and BS indicated that the expression levels of genes related to secondary metabolism and high contents of catechins, anthocyanin, caffeine, and limonene may contribute to the formation of the “Yin Rhyme” flavor in TGY. These findings provide new insights into the relationship between the accumulation of secondary metabolites and sensory quality, and the molecular mechanisms underlying the formation of the unique flavor “Yin Rhyme” in TGY.

Modulation of Xanthine metabolism ameliorates inflammation and accelerates diabetic wound healing

Abstract Wound healing in diabetic patients is impaired because of dysregulated innate immune response that results in a chronic inflammatory state. It has been shown by our group and others that metabolites can contribute to a state of chronic inflammation in diabetic tissues. In a murine model of Type-2 diabetes (diet-induced obese mice; DIO), we assessed role of uric acid in driving macrophage-mediated inflammation and delayed wound healing. Using liquid chromatography-mass spectrometry, we show that macrophages from DIO mice have reduced levels of xanthine, a precursor in the uric acid pathway. Xanthine is converted to uric acid via Xanthine oxidase/dehydrogenase (XOR) encoded by the Xdh gene. DIO macrophages express higher levels of Xdh and increased levels of uric acid, suggesting increased XOR activity. Further, exogenous addition of uric acid led to increased production of IL-1β by macrophages. Importantly, pharmacologic inhibition of XOR with Allopurinol resulted in reduced levels of IL-1β expression by macrophages. In order to examine this in vivo, DIO mice were treated with Allopurinol leading to significantly improved wound healing. In summary, our data suggest that the xanthine metabolic pathway can be targeted therapeutically to accelerate wound healing in diabetic patients.

Erratum to: A Vitis vinifera xanthine dehydrogenase gene, VvXDH, enhances salinity tolerance in transgenic Arabidopsis

Xanthine dehydrogenase (EC1.1.1.204; XDH) plays an important role in purine catabolism that catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and of xanthine to uric acid. Long attributed to its role in recycling and remobilization of nitrogen, recently, XDH is implicated in plant stress responses and acclimation, such research efforts, however, have thus far been restricted to Arabidopsis XDH-knockdown/knockout studies. This study, using an ectopic overexpression approach, is expected to provide novel findings. In this study, a XDH gene from Vitis vinifera, named VvXDH, was synthesized and overexpressed in Arabidopsis, the transgenic Arabidopsis showed enhanced salt tolerance. The VvXDH gene was investigated and the results demonstrated the explicit role of VvXDH in conferring salt stress by increasing allantoin accumulation and activating ABA signaling pathway, enhancing ROS scavenging in transgenic Arabidopsis. In addition, the water loss and chlorophyll content loss were reduced in transgenic plants; the transgenic plants showed higher proline level and lower MDA content than that of wild-type Arabidopsis, respectively. In conclusion, the VvXDH gene has the potential to be applied in increasing allantoin accumulation and enhancing the tolerance to abiotic stresses in Arabidopsis and other plants.

Enhanced xylitol production: Expression of xylitol dehydrogenase from Gluconobacter oxydans and mixed culture of resting cell

Xylitol has numerous applications in food and pharmaceutical industry, and it can be biosynthesized by microorganisms. In the present study, xdh gene, encoding xylitol dehydrogenase (XDH), was cloned from the genome of Gluconobacter oxydans CGMCC 1.49 and overexpressed in Escherichia coli BL21. Sequence analysis revealed that XDH has a TGXXGXXG NAD(H)-binding motif and a YXXXK active site motif, and belongs to the short-chain dehydrogenase/reductase family. And then, the enzymatic properties and kinetic parameter of purified recombinant XDH were investigated. Subsequently, transformations of xylitol from d-xylulose and d-arabitol, respectively, were studied through mixed culture of resting cells of G.oxydans wild-type strain and recombinant strain BL21-xdh. We obtained 28.80g/L xylitol by mixed culture from 30g/L d-xylulose in 28h. The production was increased by more than three times as compared with that of wild-type strain. Furthermore, 25.10g/L xylitol was produced by the mixed culture from 30g/L d-arabitol in 30h with a yield of 0.837g/g, and the max volumetric productivity of 0.990g/Lh was obtained at 22h. These contrast to the fact that wild-type strain G.oxydans only produced 8.10g/L xylitol in 30h with a yield of 0.270g/g. To our knowledge, these values are the highest among the reported yields and productivity efficiencies of xylitol from d-arabitol with engineering strains.