[Uric Acid Metabolism, Uric Acid Transporters and Dysuricemia].

Serum urate levels are determined by the balance between uric acid production and uric acid excretion capacity from the kidneys and intestinal tract. Dysuricemia, including hyperuricemia and hypouricemia, develops when the balance shifts towards an increase or a decrease in the uric acid pool. Hyperuricemia is mostly a multifactorial genetic disorder involving several disease susceptibility genes and environmental factors. Hypouricemia, on the other hand, is caused by genetic abnormalities. The main genes involved in dysuricemia are xanthine oxidoreductase, an enzyme that produces uric acid, and the urate transporters urate transporter 1/solute carrier family 22 member 12 (URAT1/SLC22A12), glucose transporter 9/solute carrier family 2 member 9 (GLUT9/SLC2A9) and ATP binding cassette subfamily G member 2 (ABCG2). Deficiency of xanthine oxidoreductase results in xanthinuria, a rare disease with marked hypouricemia. Xanthinuria can be due to a single deficiency of xanthine oxidoreductase or in combination with aldehyde oxidase deficiency as well. The latter is caused by a deficiency in molybdenum cofactor sulfurase, which is responsible for adding sulphur atoms to the molybdenum cofactor required for xanthine oxidoreductase and aldehyde oxidase to exert their action. URAT1/SLC22A12 and GLUT9/SLC2A9 are involved in urate reabsorption and their deficiency leads to renal hypouricemia, a condition that is common in Japanese due to URAT1/SLC22A12 deficiency. On the other hand, ABCG2 is involved in the secretion of urate, and many Japanese have single nucleotide polymorphisms that result in its reduced function, leading to hyperuricemia. In particular, severe dysfunction of ABCG2 leads to hyperuricemia with reduced extrarenal excretion.

Design, synthesis, and biological evaluation of 3-phenyl substituted pyridine derivatives as potential dual inhibitors of XOR and URAT1.

Xanthine oxidoreductase (XOR) and uric acid transporter 1 (URAT1) are two most widely studied targets involved in production and reabsorption of uric acid, respectively. Marketed drugs almost target XOR or URAT1, but sometimes, single agents might not achieve aim of lowering uric acid to ideal value in clinic. Thus, therapeutic strategies of combining XOR inhibitors with uricosuric drugs were proposed and implemented. Based on our initial work of virtual screening, A and B were potential hits for dual-targeted inhibitors on XOR/URAT1. By docking A/B with XOR/URAT1 respectively, compounds I1-7 were designed to get different degree of inhibition effect on XOR and URAT1, and I7 showed the best inhibitory effect on XOR (IC50 = 0.037 ± 0.001 µM) and URAT1 (IC50 = 546.70 ± 32.60 µM). Further docking research on I7 with XOR/URAT1 led to the design of compounds II with the significantly improved inhibitory activity on XOR and URAT1, such as II11 and II15. Especially, for II15, the IC50 of XOR is 0.006 ± 0.000 µM, superior to that of febuxostat (IC50 = 0.008 ± 0.000 µM), IC50 of URAT1 is 12.90 ± 2.30 µM, superior to that of benzbromarone (IC50 = 27.04 ± 2.55 µM). In acute hyperuricemia mouse model, II15 showed significant uric acid lowering effect. The results suggest that II15 had good inhibitory effect on XOR/URAT1, with the possibility for further investigation in in-vivo models of hyperuricemia.

Mechanistic insights into iron-sulfur clusters and flavin oxidation of a novel xanthine oxidoreductase from Sulfobacillus acidophilus TPY.

Xanthine oxidoreductase (XOR) catalyzes the oxidation of purines (hypoxanthine and xanthine) to uric acid. XOR is widely used in various therapeutic and biotechnological applications. In this study, we characterized the biophysical and mechanistic properties of a novel bacterial XOR from Sulfobacillus acidophilus TPY (SaXOR). Our results showed that SaXOR is a heterotrimer consisting of three subunits, namely XoA, XoB, and XoC, which denote the molybdenum cofactor (Moco), 2Fe-2S, and FAD-binding domains, respectively. XoC was found to be stable when co-expressed with XoB, forming an XoBC complex. Furthermore, we prepared a fusion of XoB and XoC via a flexible linker (fusXoBC) and evaluated its function in comparison to that of XoBC. Spectroscopic analysis revealed that XoB harbors two 2Fe-2S clusters, whereas XoC bears a single-bound FAD cofactor. Electron transfer from reduced forms of XoC, XoBC, and fusXoBC to molecular oxygen (O2 ) during oxidative half-reaction yielded no flavin semiquinones, implying ultrafast single-electron transfer from 2Fe-2Sred to FAD. In the presence of XoA, XoBC and fusXoBC exhibited comparable XoA affinity and exploited a shared overall mechanism. Nonetheless, the linkage may accelerate the two-step, single-electron transfer cascade from 2Fe-2Sred to FAD while augmenting protein stability. Collectively, our findings provide novel insights into SaXOR properties and oxidation mechanisms divergent from prior mammalian and bacterial XOR paradigms.

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 dehydrogenase rewires metabolism and the survival of nutrient deprived lung adenocarcinoma cells by facilitating UPR and autophagic degradation.

Xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. The altered expression and activity of XDH are associated with the development and prognosis of multiple types of cancer, while its role in lung adenocarcinoma (LUAD) remains unknown. Herein, we demonstrated that XDH was highly expressed in LUAD and was significantly correlated with poor prognosis. Though inhibition of XDH displayed moderate effect on the viability of LUAD cells cultured in the complete medium, it significantly attenuated the survival of starved cells. Similar results were obtained in XDH-knockout cells. Nucleosides supplementation rescued the survival of starved LUAD cells upon XDH inhibition, while inhibition of purine nucleoside phosphorylase abrogated the process, indicating that nucleoside degradation is required for the XDH-mediated survival of LUAD cells. Accordingly, metabolic flux revealed that ribose derived from nucleoside fueled key carbon metabolic pathways to sustain the survival of starved LUAD cells. Mechanistically, down-regulation of XDH suppressed unfolded protein response (UPR) and autophagic flux in starved LUAD cells. Inhibition of XDH decreased the level of amino acids produced by autophagic degradation, which was accompanied with down-regulation of mTORC1 signaling. Supplementation of amino acids including glutamine or glutamate rescued the survival of starved LUAD cells upon knockout or inhibition of XDH. Finally, XDH inhibitors potentiated the anti-cancer activity of 2-deoxy-D-glucose that induced UPR and/or autophagy in vitro and in vivo. In summary, XDH plays a crucial role in the survival of starved LUAD cells and targeting XDH may improve the efficacy of drugs that induce UPR and autophagy in the therapy of LUAD.

Xanthine oxidoreductase mediates genotoxic drug-induced autophagy and apoptosis resistance by uric acid accumulation and TGF-ß-activated kinase 1 (TAK1) activation.

Autophagy is a highly conserved cellular process that profoundly impacts the efficacy of genotoxic chemotherapeutic drugs. TGF-ß-activated kinase 1 (TAK1) is a serine/threonine kinase that activates several signaling pathways involved in inducing autophagy and suppressing cell death. Xanthine oxidoreductase (XOR) is a rate-limiting enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid and hydrogen peroxide in the purine catabolism pathway. Recent studies showed that uric acid can bind to TAK1 and prolong its activation. We hypothesized that genotoxic drugs may induce autophagy and apoptosis resistance by activating TAK1 through XOR-generated uric acid. Here, we report that gemcitabine and 5-fluorouracil (5-FU), two genotoxic drugs, induced autophagy in HeLa and HT-29 cells by activating TAK1 and its two downstream kinases, AMP-activated kinase (AMPK) and c-Jun terminal kinase (JNK). XOR knockdown and the XOR inhibitor allopurinol blocked gemcitabine-induced TAK1, JNK, AMPK, and Unc51-like kinase 1 (ULK1)S555 phosphorylation and gemcitabine-induced autophagy. Inhibition of the ATM-Chk pathway, which inhibits genotoxic drug-induced uric acid production, blocked gemcitabine-induced autophagy by inhibiting TAK1 activation. Exogenous uric acid in its salt form, monosodium urate (MSU), induced autophagy by activating TAK1 and its downstream kinases JNK and AMPK. Gene knockdown or the inhibitors of these kinases blocked gemcitabine- and MSU-induced autophagy. Inhibition of autophagy by allopurinol, chloroquine, and 5Z-7-oxozeaenol (5Z), a TAK1-specific inhibitor, enhanced gemcitabine-induced apoptosis. Our study uncovers a previously unrecognized role of XOR in regulating genotoxic drug-induced autophagy and apoptosis and has implications for designing novel therapeutic strategies for cancer treatment.

Systemic Endothelial Function, Plasma Xanthine Oxidoreductase Activity, and Blood Pressure Variability in Patients with Stable Coronary Artery Disease.

Background and Objectives: Although previous studies showed that an activity of xanthine oxidoreductase (XOR), a rate-limiting enzyme in purine metabolism, beyond the serum uric acid level, was associated with the development of coronary artery disease (CAD), the underlying mechanisms are unclear. Because endothelial dysfunction and a greater blood pressure (BP) variability may play a role, we investigated the relations among the endothelial function, XOR, and BP variability. Materials and Methods: This was a post-hoc study using pooled data of patients with a stable CAD from two prospective investigations, in which the systemic endothelial function was assessed with the reactive hyperemia index (RHI) and the XOR activity was measured. The BP variability was evaluated using BP measurements during the three- and four-day hospitalization. Results: A total of 106 patients with a stable CAD undergoing a percutaneous coronary intervention were included. Of the 106 patients, 46 (43.4%) had a systemic endothelial dysfunction (RHI < 1.67). The multivariable analysis identified a higher body mass index (BMI), female gender, and diabetes as factors associated with an endothelial dysfunction. A higher BMI was also related to an elevated XOR activity, in addition to current smoking. No significant correlation was observed between the RHI and XOR activity. Similarly, the in-hospital BP variability was associated with neither the endothelial function nor XOR. Conclusions: Among patients with a stable CAD, several factors were identified as being associated with a systemic endothelial dysfunction or an elevated XOR activity. However, no direct relations between the endothelial function, XOR, and BP variability were found.

C1QBP regulates apoptosis of renal cell carcinoma via modulating xanthine dehydrogenase (XDH) mediated ROS generation.

Background: Complement component 1 Q subcomponent binding protein (C1QBP) plays a vital role in the progression and metabolism of cancer. Studies have shown that xanthine dehydrogenase (XDH)-derived reactive oxygen species (ROS) accelerates tumor growth, and also induces mutations or produces cytotoxic effects concurrently. However, the role of C1QBP in metabolism, oxidative stress, and apoptosis of renal cell carcinoma (RCC) cells have not yet been explored. Methods: Metabolomics assay was applied to investigate the role of C1QBP in RCC metabolism. C1QBP knockdown and overexpression cells were established via lentiviral infection and subjected to apoptosis and ROS assay in vitro. RNA stability assay was applied to characterize the mechanism of C1QBP regulating XDH transcription. In vivo, orthotopic tumor xenografts assay was performed to investigate the role of C1QBP in RCC progression. Results: Metabolomics investigation revealed that C1QBP dramatically diminished the hypoxanthine content in RCC cells. C1QBP promoted the mRNA and protein expression of hypoxanthine catabolic enzyme XDH. Meanwhile, C1QBP may affect XDH transcription by regulating the mRNA level of XDH transcriptional stimulators IL-6, TNF-α, and IFN-γ. Moreover, the expression of C1QBP and XDH was lower in RCC tumors compared with the tumor-associated normal tissues, and their down-regulation was associated with higher Fuhrman grade. C1QBP significantly increased ROS level, apoptosis, and the expression of apoptotic proteins such as cleaved caspase-3 and bax/bcl2 via regulating XDH. Conclusion: C1QBP promotes the catabolism of hypoxanthine and elevates the apoptosis of RCC cells by modulating XDH-mediated ROS generation.

Beyond Moco Biosynthesis-Moonlighting Roles of MoaE and MOCS2.

Molybdenum cofactor (Moco) biosynthesis requires iron, copper, and ATP. The Moco-containing enzyme sulfite oxidase catalyzes terminal oxidation in oxidative cysteine catabolism, and another Moco-containing enzyme, xanthine dehydrogenase, functions in purine catabolism. Thus, molybdenum enzymes participate in metabolic pathways that are essential for cellular detoxication and energy dynamics. Studies of the Moco biosynthetic enzymes MoaE (in the Ada2a-containing (ATAC) histone acetyltransferase complex) and MOCS2 have revealed that Moco biosynthesis and molybdenum enzymes align to regulate signaling and metabolism via control of transcription and translation. Disruption of these functions is involved in the onset of dementia and neurodegenerative disease. This review provides an overview of the roles of MoaE and MOCS2 in normal cellular processes and neurodegenerative disease, as well as directions for future research.

The double faced role of xanthine oxidoreductase in cancer.

Xanthine oxidoreductase (XOR) is a critical, rate-limiting enzyme that controls the last two steps of purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. It also produces reactive oxygen species (ROS) during the catalytic process. The enzyme is generally recognized as a drug target for the therapy of gout and hyperuricemia. The catalytic products uric acid and ROS act as antioxidants or oxidants, respectively, and are involved in pro/anti-inflammatory actions, which are associated with various disease manifestations, including metabolic syndrome, ischemia reperfusion injury, cardiovascular disorders, and cancer. Recently, extensive efforts have been devoted to understanding the paradoxical roles of XOR in tumor promotion. Here, we summarize the expression of XOR in different types of cancer and decipher the dual roles of XOR in cancer by its enzymatic or nonenzymatic activity to provide an updated understanding of the mechanistic function of XOR in cancer. We also discuss the potential to modulate XOR in cancer therapy.

Xanthine oxidoreductase promotes the progression of colitis-associated colorectal cancer by causing DNA damage and mediating macrophage M1 polarization.

In addition to its pivotal role in purine metabolism, xanthine oxidoreductase (XOR) is one of the key enzymes involved in superoxide radical generation. Oxidative stress has been implicated in the etiology of colorectal cancer, but the contribution of XOR remains unclear. Here we investigated the role of XOR in colitis-associated colorectal cancer (CAC) and the underlying mechanisms. Using clinical samples, we demonstrated that XOR up-regulation was an early event in colonic carcinogenesis. Pharmacological inhibition of XOR effectively delayed the progression of CAC. Moreover, XOR activity positively correlated with tumor necrosis factor-alpha (TNFα) protein levels. Mechanistically, TNFα may activate XOR transcription via activator protein-1 and, thus, promote endogenous hydrogen peroxide generation, resulting in oxidative DNA damage in colon cancer cells. On the other hand, XOR may regulate the TNFα mRNA transcripts by mediating LPS-induced macrophage M1 polarization. Collectively, XOR promotes tumor development by programming the tumor microenvironment and stimulates CAC progression via DNA damage-induced genetic instability.

Omeprazole induces vascular remodeling by mechanisms involving xanthine oxidoreductase and matrix metalloproteinase activation.

Proton pump inhibitors (PPI) are commonly used drugs that may increase the cardiovascular risk by mechanisms not entirely known. We examined whether the PPI omeprazole promotes vascular oxidative stress mediated by xanthine oxidoreductase (XOR) leading to activation of matrix metalloproteinases (MMPs) and vascular remodeling. We studied Wistar rats treated with omeprazole (or vehicle) combined with the XOR inhibitor allopurinol (or vehicle) for four weeks. Systolic blood pressure (SBP) measured by tail-cuff plethysmography was not affected by treatments. Omeprazole treatment increased the aortic cross-sectional area and media/lumen ratio by 25% (P < 0.05). Omeprazole treatment decreased gastric pH and induced vascular remodeling accompanied by impaired endothelium-dependent aortic responses (assessed with isolated aortic ring preparation) to acetylcholine (P < 0.05). Omeprazole increased vascular active MMP-2 expression and activity assessed by gel zymography and in situ zymography, respectively (P < 0.05). Moreover, omeprazole enhanced vascular oxidative stress assessed in situ with the fluorescent dye DHE and with the lucigenin chemiluminescence assay (both P < 0.05). All these biochemical changes caused by omeprazole were associated with increased vascular XOR activity (but not XOR expression assessed by Western blot) and treatment with allopurinol fully prevented them (all P < 0.05). Importantly, treatment with allopurinol prevented the vascular dysfunction and remodeling caused by omeprazole. Our results suggest that the long-term use of omeprazole induces vascular dysfunction and remodeling by promoting XOR-derived reactive oxygen species formation and MMP activation. These findings provide evidence of a new mechanism that may underlie the unfavorable cardiovascular outcomes observed with PPI therapy. Clinical studies are warranted to validate our findings.

Xanthine Oxidoreductase-Mediated Superoxide Production Is Not Involved in the Age-Related Pathologies in Sod1-Deficient Mice.

Reactive oxygen species (ROS) metabolism is regulated by the oxygen-mediated enzyme reaction and antioxidant mechanism within cells under physiological conditions. Xanthine oxidoreductase (XOR) exhibits two inter-convertible forms (xanthine oxidase (XO) and xanthine dehydrogenase (XDH)), depending on the substrates. XO uses oxygen as a substrate and generates superoxide (O2•-) in the catalytic pathway of hypoxanthine. We previously showed that superoxide dismutase 1 (SOD1) loss induced various aging-like pathologies via oxidative damage due to the accumulation of O2•- in mice. However, the pathological contribution of XO-derived O2•- production to aging-like tissue damage induced by SOD1 loss remains unclear. To investigate the pathological significance of O2•- derived from XOR in Sod1-/- mice, we generated Sod1-null and XO-type- or XDH-type-knock-in (KI) double-mutant mice. Neither XO-type- nor XDH-type KI mutants altered aging-like phenotypes, such as anemia, fatty liver, muscle atrophy, and bone loss, in Sod1-/- mice. Furthermore, allopurinol, an XO inhibitor, or apocynin, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, failed to improve aging-like tissue degeneration and ROS accumulation in Sod1-/- mice. These results showed that XOR-mediated O2•- production is relatively uninvolved in the age-related pathologies in Sod1-/- mice.

Small-Molecule Inhibitors of Reactive Oxygen Species Production.

Reactive oxygen species (ROS) are involved in physiological cellular processes including differentiation, proliferation, and apoptosis by acting as signaling molecules or regulators of transcription factors. The maintenance of appropriate cellular ROS levels is termed redox homeostasis, a balance between their production and neutralization. High concentrations of ROS may contribute to severe pathological events including cancer, neurodegenerative, and cardiovascular diseases. In recent years, approaches to target the sources of ROS production directly in order to develop tool compounds or potential therapeutics have been explored. Herein, we briefly outline the major sources of cellular ROS production and comprehensively review the targeting of these by small-molecule inhibitors. We critically assess the value of ROS inhibitors with different mechanisms-of-action, including their potency, mode-of-action, known off-target effects, and clinical or preclinical status, while suggesting future avenues of research in the field.

Renal Reabsorptive Transport of Uric Acid Precursor Xanthine by URAT1 and GLUT9.

Xanthine and hypoxanthine are intermediate metabolites of uric acid and a source of reactive oxidative species (ROS) by xanthine oxidoreductase (XOR), suggesting that facilitating their elimination is beneficial. Since they are reabsorbed in renal proximal tubules, we investigated their reabsorption mechanism by focusing on the renal uric acid transporters URAT1 and GLUT9, and examined the effect of clinically used URAT1 inhibitor on their renal clearance when their plasma concentration is increased by XOR inhibitor. Uptake study for [3H]xanthine and [3H]hypoxanthine was performed using URAT1- and GLUT9-expressing Xenopus oocytes. Transcellular transport study for [3H]xanthine was carried out using Madin-Darby canine kidney (MDCK)II cells co-expressing URAT1 and GLUT9. In in vivo pharmacokinetic study, renal clearance of xanthine was estimated based on plasma concentration and urinary recovery. Uptake by URAT1- and GLUT9-expressing oocytes demonstrated that xanthine is a substrate of URAT1 and GLUT9, while hypoxanthine is not. Transcellular transport of xanthine in MDCKII cells co-expressing URAT1 and GLUT9 was significantly higher than those in mock cells and cells expressing URAT1 or GLUT9 alone. Furthermore, dotinurad, a URAT1 inhibitor, increased renal clearance of xanthine in rats treated with topiroxostat to inhibit XOR. It was suggested that xanthine is reabsorbed in the same manner as uric acid through URAT1 and GLUT9, while hypoxanthine is not. Accordingly, it is expected that treatment with XOR and URAT1 inhibitors will effectively decrease purine pools in the body and prevent cell injury due to ROS generated during XOR-mediated reactions.

New insights into purine metabolism in metabolic diseases: role of xanthine oxidoreductase activity.

Xanthine oxidoreductase (XOR) consists of two different forms, xanthine dehydrogenase and xanthine oxidase (XO), and is a rate-limiting enzyme of uric acid production from hypoxanthine and xanthine. Uric acid is the end product of purine metabolism in humans and has a powerful antioxidant effect. The lack of ascorbic acid, known as vitamin C, in hominoids has been thought to cause a compensatory increase in uric acid as an antioxidant by unfunctional gene mutation of uricase to a pseudogene. Because XO is involved in an increase in reactive oxygen species (ROS) by generating superoxide and hydrogen peroxide, inadequate activation of XOR promotes oxidative stress-related tissue injury. Plasma XOR activity is associated with obesity, smoking, liver dysfunction, hyperuricemia, dyslipidemia, insulin resistance, and adipokines, indicating a novel biomarker of metabolic disorders. However, XOR activity in adipose tissue is low in humans unlike in rodents, and hypoxanthine is secreted from human adipose tissue. The concentration of hypoxanthine, but not xanthine, is independently associated with obesity in a general population, indicating differential regulation of hypoxanthine and xanthine. Treatment with an XOR inhibitor can decrease uric acid for preventing gout, reduce production of XO-related ROS, and promote reutilization of hypoxanthine and ATP production through the salvage pathway. It has recently been suggested that discontinuation of an XOR inhibitor causes adverse cardiovascular outcomes as XOR inhibitor withdrawal syndrome, possibly due to cardiac disturbance of conduction and contraction by reduced ATP production. New insights into purine metabolism, including the role of XOR activity in the past 5 yr, are mainly discussed in this review.

Potential roles of nitrate and nitrite in nitric oxide metabolism in the eye.

Nitric oxide (NO) signaling has been studied in the eye, including in the pathophysiology of some eye diseases. While NO production by nitric oxide synthase (NOS) enzymes in the eye has been characterized, the more recently described pathways of NO generation by nitrate (NO3-) and nitrite (NO2-) ions reduction has received much less attention. To elucidate the potential roles of these pathways, we analyzed nitrate and nitrite levels in components of the eye and lacrimal glands, primarily in porcine samples. Nitrate and nitrite levels were higher in cornea than in other eye parts, while lens contained the least amounts. Lacrimal glands exhibited much higher levels of both ions compared to other organs, such as liver and skeletal muscle, and even to salivary glands which are known to concentrate these ions. Western blotting showed expression of sialin, a known nitrate transporter, in the lacrimal glands and other eye components, and also xanthine oxidoreductase, a nitrate and nitrite reductase, in cornea and sclera. Cornea and sclera homogenates possessed a measurable amount of nitrate reduction activity. These results suggest that nitrate ions are concentrated in the lacrimal glands by sialin and can be secreted into eye components via tears and then reduced to nitrite and NO, thereby being an important source of NO in the eye.

Inflammatory response of uric acid produced by Porphyromonas gingivalis gingipains.

Uric acid is a potential metabolite that serves as a danger-associated molecular pattern (DAMP) and induces inflammatory responses in sterile environments. Porphyromonas gingivalis is a keystone periodontopathogen, and its gingipain proteases play a critical role in the pathogenesis of periodontitis. In this study, we demonstrate that P. gingivalis gingipains play a role in THP-1 macrophage uric acid production by increasing the expression and activity of xanthine oxidoreductase (XOR). Uric acid sodium salt induces caspase-1 activation, cell death, and the expression of proinflammatory cytokines, including IL-1α, IL-6, and IL-8, in the human keratinocyte HOK-16B cell line. Our results suggest that gingipain-induced uric acid can mediate inflammation in periodontal tissue cells.

TDP-43 facilitates milk lipid secretion by post-transcriptional regulation of Btn1a1 and Xdh.

Milk lipid secretion is a critical process for the delivery of nutrition and energy from parent to offspring. However, the underlying molecular mechanism is less clear. Here we report that TDP-43, a RNA-binding protein, underwent positive selection in the mammalian lineage. Furthermore, TDP-43 gene (Tardbp) loss induces accumulation of large lipid droplets and severe lipid secretion deficiency in mammary epithelial cells to outside alveolar lumens, eventually resulting in lactation failure and pup starvation within three weeks postpartum. In human milk samples from lactating women, the expression levels of TDP-43 is positively correlated with higher milk output. Mechanistically, TDP-43 exerts post-transcriptional regulation of Btn1a1 and Xdh mRNA stability, which are required for the secretion of lipid droplets from epithelial cells to the lumen. Taken together, our results highlights the critical role of TDP-43 in milk lipid secretion, providing a potential strategy for the screening and intervention of clinical lactation insufficiency.