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 100 µmol/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.

Modelling of growth kinetics of isolated Pseudomonas sp. and optimisation of parameters for enhancement of xanthine oxidoreductase production by statistical design of experiments.

This report presents the substrate inhibitory effect of xanthine (XN) on microbial growth and optimisation of effective parameters to achieve high enzyme activity of xanthine oxidoreductase (XOR) through statistical design. Three efficient isolated strains (Pseudomonas aeruginosa CEBP1 and CEBP2, Pseudomonas sp. CEB1G) were screened for growth kinetic studies. Substrate inhibitory models (eg. Aiba, Edward) could explain the growth kinetics of CEBP1, CEBP2 and CEB1G very well with various initial [XN] (S0), e.g., 0.1-35 g L-1. Highest XOR activity was obtained at stationary phase when biomass yield was high. Highest catalytic efficiency (kcat/KM) of XOR was obtained by CEBP1 at optimum specific growth rate of 0.082 h-1 and biomass yield of 0.196 g g-1 at S0 = 5 g L-1. The effects of S0, pH and temperature were studied by Box-Behnken experimental design to evaluate the interactive effects of the significant variables influencing XOR production by CEBP1. ANOVA with high correlation coefficient (R2 > 0.99) and lower 'Prob > F'value (< 0.05) validated the second order polynomial model for the enzyme production. The highest XOR activity of 31.2 KU min-1 mg-1 was achieved by CEBP1 under optimised conditions (35 °C; S0=5 g L-1; pH = 7.0) as compared to any report in literature. A sevenfold substrate affinity of the enzyme was observed after purification.

Hexokinase 2 and nuclear factor erythroid 2-related factor 2 transcriptionally coactivate xanthine oxidoreductase expression in stressed glioma cells.

A dynamic network of metabolic adaptations, inflammatory responses, and redox homeostasis is known to drive tumor progression. A considerable overlap among these processes exists, but several of their key regulators remain unknown. To this end, here we investigated the role of the proinflammatory cytokine IL-1ß in connecting these processes in glioma cells. We found that glucose starvation sensitizes glioma cells to IL-1ß-induced apoptosis in a manner that depended on reactive oxygen species (ROS). Although IL-1ß-induced JNK had no effect on cell viability under glucose deprivation, it mediated nuclear translocation of hexokinase 2 (HK2). This event was accompanied by increases in the levels of sirtuin 6 (SIRT6), nuclear factor erythroid 2-related factor 2 (Nrf2), and xanthine oxidoreductase (XOR). SIRT6 not only induced ROS-mediated cell death but also facilitated nuclear Nrf2-HK2 interaction. Recruitment of the Nrf2-HK2 complex to the ARE site on XOR promoter regulated its expression. Importantly, HK2 served as transcriptional coactivator of Nrf2 to regulate XOR expression, indicated by decreased XOR levels in siRNA-mediated Nrf2 and HK2 knockdown experiments. Our results highlight a non-metabolic role of HK2 as transcriptional coactivator of Nrf2 to regulate XOR expression under conditions of proinflammatory and metabolic stresses. Our insights also underscore the importance of nuclear activities of HK2 in the regulation of genes involved in redox homeostasis.

The Stringent Response Induced by Phosphate Limitation Promotes Purine Salvage in Agrobacterium fabrum.

Agrobacterium fabrum induces tumor growth in susceptible plant species. The upregulation of virulence genes that occurs when the bacterium senses plant-derived compounds is enhanced by acidic pH and limiting inorganic phosphate. Nutrient starvation may also trigger the stringent response, and purine salvage is among the pathways expected to be favored under such conditions. We show here that phosphate limitation induces the stringent response, as evidenced by production of (p)ppGpp, and that the xdhCSML operon encoding the purine salvage enzyme xanthine dehydrogenase is upregulated ∼15-fold. The xdhCSML operon is under control of the TetR family transcription factor XdhR; direct binding of ppGpp to XdhR attenuates DNA binding, and the enhanced xdhCSML expression correlates with increased cellular levels of (p)ppGpp. Xanthine dehydrogenase may also divert purines away from salvage pathways to form urate, the ligand for the transcription factor PecS, which in the plant pathogen Dickeya dadantii is a key regulator of virulence gene expression. However, urate levels remain low under conditions that produce increased levels of xdhCSML expression, and neither acidic pH nor limiting phosphate results in induction of genes under control of PecS. Instead, expression of such genes is induced only by externally supplemented urate. Taken together, our data indicate that purine salvage is favored during the stringent response induced by phosphate starvation, suggesting that control of this pathway may constitute a novel approach to modulating virulence. Because bacterial purine catabolism appears to be unaffected, as evidenced by the absence of urate accumulation, we further propose that the PecS regulon is induced by only host-derived urate.

Vascular xanthine oxidoreductase contributes to the antihypertensive effects of sodium nitrite in L-NAME hypertension.

Nitrate and nitrite have emerged as an important novel source of nitric oxide (NO). We have previously demonstrated that sodium nitrite is an antihypertensive compound that exerts antioxidant effects in experimental hypertension. These unpredicted antioxidant effects of nitrite raised the question whether the beneficial effects found were caused by its conversion to NO or simply due to reversal of endothelial dysfunction as a consequence of its antioxidant effects. Here, we evaluated the antihypertensive effects of a daily dose of sodium nitrite for 4 weeks in L-NAME-induced hypertension in rats. We studied the effects of nitrite on markers of NO bioavailability, vascular oxidative stress, and expression of xanthine oxidoreductase. Moreover, we tested if xanthine oxidoreductase inhibition could attenuate the acute hypotensive effects of sodium nitrite in L-NAME hypertensive rats. We found that a single pharmacological dose of sodium nitrite exerts antihypertensive effects in L-NAME-induced hypertension. While the beneficial antihypertensive properties of nitrite were associated with increased levels of NO metabolites, hypertension increased vascular xanthine oxidoreductase expression by approximately 40%, with minor increases in vascular superoxide production. The inhibition of xanthine oxidoreductase by oxypurinol attenuated the acute hypotensive effects of nitrite. Taken together, our results show that nitrite exerts antihypertensive effects in L-NAME hypertensive rats and provide evidence that xanthine oxidoreductase plays an important role in this antihypertensive effect.

Cidea is an essential transcriptional coactivator regulating mammary gland secretion of milk lipids.

Adequate lipid secretion by mammary glands during lactation is essential for the survival of mammalian offspring. However, the mechanism governing this process is poorly understood. Here we show that Cidea is expressed at high levels in lactating mammary glands and its deficiency leads to premature pup death as a result of severely reduced milk lipids. Furthermore, the expression of xanthine oxidoreductase (XOR), an essential factor for milk lipid secretion, is markedly lower in Cidea-deficient mammary glands. Conversely, ectopic Cidea expression induces the expression of XOR and enhances lipid secretion in vivo. Unexpectedly, as Cidea has heretofore been thought of as a cytoplasmic protein, we detected it in the nucleus and found it to physically interact with transcription factor CCAAT/enhancer-binding protein ß (C/EBPß) in mammary epithelial cells. We also observed that Cidea induces XOR expression by promoting the association of C/EBPß onto, and the dissociation of HDAC1 from, the promoter of the Xdh gene encoding XOR. Finally, we found that Fsp27, another CIDE family protein, is detected in the nucleus and interacts with C/EBPß to regulate expression of a subset of C/EBPß downstream genes in adipocytes. Thus, Cidea acts as a previously unknown transcriptional coactivator of C/EBPß in mammary glands to control lipid secretion and pup survival.

Decreased xanthine oxidoreductase (XOR) is associated with a worse prognosis in patients with serous ovarian carcinoma.

OBJECTIVE: Xanthine oxidoreductase (XOR) is a key enzyme in the degradation of DNA, RNA and high-energy phosphates. In the human cancers previously studied, down-regulated XOR identifies patients with unfavorable prognosis. We assessed the clinical relevance of XOR expression in serous ovarian cancer. METHODS: XOR protein was determined in tissue microarrays from 474 patients with serous ovarian cancer and analyzed with respect to clinical parameters and survival. RESULTS: XOR was down regulated in 64% of the tumors as compared to the corresponding normal tissue. Decreased XOR was associated with a poorly differentiated tumor and an abnormal p53 expression, but not with age at diagnosis, FIGO stage, Ki-67 or tumor size. XOR expression was associated with outcome, and the five year ovarian cancer specific survival in patients with strong XOR expression was 59% compared to 44% in those with moderate (hazard ratio, HR; 1.44; P=0.0083) and 26% in patients with lack of XOR (HR, 2.07; P=0.0003). This was also true in patients whose tumors were highly differentiated (HR, 3.67; P=0.008) and in patients with a small (<1cm) residual tumor (HR, 2.62; P=0.017), and in patients whose tumors show a low Ki-67 protein expression (HR, 3.79; P<0.0001). In multivariate survival analysis, absence of XOR emerged as an independent prognostic factor (HR, 1.82; P=0.015). CONCLUSIONS: Decreased XOR is associated with poorer prognosis in patients with serous ovarian cancer especially in those with an otherwise more favorable prognostic profile.

Enhanced xanthine oxidoreductase expression and tissue nitrate reduction in germ free mice.

The nitrate-nitrite-NO pathway is emerging as an alternative to the l-arginine/NO-synthase pathway for the generation of NO in mammals. Bioactivation of the stable nitrate anion involves initial reduction to nitrite by commensal bacteria in the gastrointestinal tract. Nitrite is then further metabolized in blood and tissues to form nitric oxide (NO) and other bioactive nitrogen oxides. In addition to nitrate reduction by bacteria, a functional mammalian nitrate reductase activity was recently explored. It was demonstrated that xanthine oxidoreductase (XOR) and possibly other enzymes can catalyze nitrate reduction under normoxic conditions in vivo. In the present study, we compared nitrate reduction in germ free (GF) and conventional mice. One aim was to see if the complete lack of bacterial nitrate reduction in the GF mice would be associated with an upregulation of mammalian nitrate reductase activity. Sodium nitrate (NaNO(3)) or placebo (NaCl) was injected intraperitoneally and blood and tissues were collected 1.5-2h later for measurements of nitrate and nitrite and in some cases analyses of protein expression. Tissue and plasma levels of nitrate increased to a similar extent in conventional and GF animals after nitrate administration. Plasma nitrite was 3-fold higher in GF mice receiving nitrate compared to placebo while this effect of nitrate was absent in the conventional mice. In GF mice pretreated with the xanthine oxidase inhibitor allopurinol the increase in nitrite was attenuated. The levels of nitrite in the liver and small intestine increased after the nitrate load in GF mice but not in the conventional mice. Anaerobic nitrate reduction to nitrite in intestinal tissue homogenates was also accelerated in GF mice. Studies of tissue protein levels revealed increased expression of XOR in the livers of GF animals. We conclude that XOR expression in tissues is enhanced in germ free mice and this may explain the apparently greater tissue nitrate reductase activity observed in these animals. Future studies will reveal if this represents a compensatory functional response to uphold nitrite homeostasis in the absence of commensal bacteria.

Inhibition of thymic adipogenesis by caloric restriction is coupled with reduction in age-related thymic involution.

Aging of thymus is characterized by reduction in naive T cell output together with progressive replacement of lymphostromal thymic zones with adipocytes. Determining how calorie restriction (CR), a prolongevity metabolic intervention, regulates thymic aging may allow identification of relevant mechanisms to prevent immunosenescence. Using a mouse model of chronic CR, we found that a reduction in age-related thymic adipogenic mechanism is coupled with maintenance of thymic function. The CR increased cellular density in the thymic cortex and medulla and preserved the epithelial signatures. Interestingly, CR prevented the age-related increase in epithelial-mesenchymal transition (EMT) regulators, FoxC2, and fibroblast-specific protein-1 (FSP-1), together with reduction in lipid-laden thymic fibroblasts. Additionally, CR specifically blocked the age-related elevation of thymic proadipogenic master regulator, peroxisome proliferator activated receptor gamma (PPARgamma), and its upstream activator xanthine-oxidoreductase (XOR). Furthermore, we found that specific inhibition of PPARgamma in thymic stromal cells prevented their adipogenic transformation in an XOR-dependent mechanism. Activation of PPARgamma-driven adipogenesis in OP9-DL1 stromal cells compromised their ability to support T cell development. Conversely, CR-induced reduction in EMT and thymic adipogenesis were coupled with elevated thymic output. Compared with 26-mo-old ad libitum fed mice, the T cells derived from age-matched CR animals displayed greater proliferation and higher IL-2 expression. Furthermore, CR prevented the deterioration of the peripheral TCR repertoire diversity in older animals. Collectively, our findings demonstrate that reducing proadipogenic signaling in thymus via CR may promote thymopoiesis during aging.

New evidence for the role of TNF-alpha in liver ischaemic/reperfusion injury.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) plays a key role in causing ischaemia/reperfusion (I/R) injury. I/R also causes activation of xanthine oxidase and dehydrogenase (XDH + XO) system that, via generated free radicals, causes organ damage. We investigated the effect of ischaemia, reperfusion and non-ischaemic prolonged perfusion (NIP) on TNF-alpha and XDH + XO production in an isolated perfused rat liver model. MATERIALS AND METHODS: Rat livers underwent 150 min NIP (control group) or two hours of ischaemia followed by reperfusion (I/R group). TNF-alpha (TNF-alpha mRNA and protein level), XDH + XO production and bile secretion were determined in tissue and effluent at baseline, at 120 min of ischaemia, after 30 min of reperfusion (I/R group) and after 120 and 150 min of prolonged perfusion (control). RESULTS: Unexpectedly, neither ischaemia nor reperfusion had any effect on TNF-alpha production. TNF-alpha in effluent was 11 +/- 4.8 pg mL(-1) at baseline, 7 +/- 3.2 pg mL(-1) at the end of ischaemia, and 13 +/- 5.3 pg mL(-1) after 30 min of reperfusion. NIP, however, caused a significant increase of TNF-alpha synthesis and release. TNF-alpha effluent level after 120 and 150 min of perfusion was 392 +/- 78.7 pg mL(-1) and 408 +/- 64.3 pg mL(-1), respectively. TNF-alpha mRNA in tissue was also significantly elevated compared to baseline levels (1.31 +/- 0.2 P < 0.001 and 1.38 P < 0.002, respectively). Decrease of liver function (expressed by bile secretion) during I/R and NIP was accompanied by significant XDH + XO elevation. CONCLUSION: This is the first evidence that NIP, and not I/R, is the decisive trigger for TNF-alpha production. This study leads to a better understanding of pathogenesis of liver I/R and perfusion damage.

NADPH oxidase inhibition prevents cocaine-induced up-regulation of xanthine oxidoreductase and cardiac dysfunction.

Oxidative stress is involved in the pathogenesis of cocaine-induced cardiomyopathy. In the present study, we aimed to determine the enzymatic sources of reactive oxygen species (ROS) production, namely NADPH oxidase and xanthine oxidoreductase (XOR) in male Wistar rats treated for 7 days with cocaine (2x7.5 mg/kg/day, ip) or cocaine with a NADPH oxidase inhibitor (apocynin, 50 mg/kg/day, po) or a XOR inhibitor (allopurinol, 50 mg/kg/day, po). Cocaine-induced cardiac dysfunction is associated with an increase in NADPH oxidase and XOR activities (59% and 29%, respectively) and a decrease in catalase activity. Apocynin or allopurinol treatment prevents the cocaine-induced cardiac alteration by restoration of cardiac output, stroke volume and fractional shortening. This is associated with a reduction of the myocardial production of superoxide anions and an enhancement of catalase activity. Surprisingly, apocynin treatment prevents XOR up-regulation supporting the hypothesis that NADPH oxidase-derived ROS play a role in modulating ROS production by XOR. These data suggest that NADPH and xanthine oxidase act synergically to form myocardial ROS and clearly demonstrate that their inhibition may be critical in preventing the initiation and progression of cocaine-induced LV dysfunction.

Stress activation of mammary epithelial cell xanthine oxidoreductase is mediated by p38 MAPK and CCAAT/enhancer-binding protein-beta.

Xanthine oxidoreductase (XOR) catalyzes the formation of uric acid from xanthine and hypoxanthine and is recognized as a source of reactive oxygen and nitrogen species. Unexpectedly, XOR was found to play an essential role in milk secretion in the differentiating mammary gland, where it is an integral component of the milk fat globule. XOR gene expression in both mammary glands and differentiating mammary epithelial cells in culture is regulated by the lactogenic hormones prolactin and cortisol. Expression in mammary epithelial cells is also regulated by inflammatory cytokines and induced by cycloheximide. Cycloheximide was found to stimulate XOR gene expression in differentiating HC11 mouse mammary epithelial cells. Activation of XOR gene expression by both cycloheximide and inflammatory cytokines suggested that XOR may be regulated by stress-activated protein kinases, the MAPKs. We demonstrate here that XOR was induced in HC11 cells by low dose cycloheximide and that expression was blocked by inhibitors of p38 MAPK. Accumulation of phospho-p38 was stimulated by low dose cycloheximide. Low dose cycloheximide stress promoted phosphorylation and nuclear accumulation of the CCAAT/enhancer-binding protein-beta (C/EBPbeta) transcription factor, which was blocked by inhibition of p38. Furthermore, C/EBPbeta was found to activate the mouse XOR promoter, and XOR promoter-C/EBPbeta protein complexes were induced by low dose cycloheximide stress. These data demonstrate, for the first time, that mouse mammary epithelial cell XOR is regulated by p38 MAPK. They identify an essential function of the C/EBPbeta transcription factor in mouse XOR expression and suggest a potential role for p38 MAPK activation of C/EBPbeta in mammary epithelial cells.

The plant Mo-hydroxylases aldehyde oxidase and xanthine dehydrogenase have distinct reactive oxygen species signatures and are induced by drought and abscisic acid.

The plant molybdenum-cofactor (Moco) and flavin-containing enzymes, xanthine dehydrogenase (XDH; EC 1.2.1.37) and aldehyde oxidase (AO; EC 1.2.3.1) are thought to play important metabolic roles in purine metabolism and hormone biosynthesis, respectively. Their animal counterparts contribute to reactive oxygen species (ROS) production in numerous pathologies and here we examined these enzymes as potential sources of ROS in plants. Novel in-gel assay techniques and Moco sulfurase mutants, lacking a sulfur ligand in their Moco active center, were employed to demonstrate that the native tomato and Arabidopsis XDHs are capable of producing O, but not H2O2, while the animal counterpart was shown to produce both, O and H2O2. Superoxide production was dependent on Moco sulfuration when using hypoxanthine/xanthine but not NADH as substrates. The activity was inhibited by diphenylene iodonium (DPI), a suicide inhibitor of FAD containing enzymes. Analysis of XDH in an Arabidopsis Atxdh1 T-DNA insertion mutant and RNA interference lines revealed loss of O activity, providing direct molecular evidence that plant XDH generates superoxides. Contrary to XDH, AO activity produced only H2O2 dissimilar to native animal AO, that can produce O as well. Surprisingly, H2O2 accumulation was not sensitive to DPI. Plant ROS production and transcript levels of AO and XDH were rapidly upregulated by application of abscisic acid and in water-stressed leaves and roots. These results, supported by in vivo measurement of ROS accumulation, indicate that plant AO and XDH are possible novel sources for ROS increase during water stress.

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system.

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.

Xanthine oxidoreductase and xanthine oxidase in human cornea.

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.

Xanthine dehydrogenase from Pseudomonas putida 86: specificity, oxidation-reduction potentials of its redox-active centers, and first EPR characterization.

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD+ as the preferred electron acceptor. In the hypoxanthine:NAD+ assay, the specific activity of purified XDH was 26.7 U (mg protein)(-1). Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD+. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an alpha4beta4 structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.

[Establishment of human neuroectodermal tumor cell line (PFSK) with stable expression of xanthine oxidoreductase].

Xanthine Oxidoreductase (XOR) is the key enzyme in purine metabolism and also produces oxygen free radicals. As a continuation of our previous work, in this study, we constructed a retrovirus expression vector (pLNCX2-XOR) containing full length of XOR cDNA. Retrovirus was produced by the virus package cell line PT-67 following the transfection of PT-67 with pLNCX2-XOR and used to infect the human primitive neuroectodermal tumor cell line (PFSK). Infected PFSK cells were selected by G418 to establish cell line with stable expression of XOR. The expression of XOR in the cell line we established was confirmed by RT-PCR, Immunocytochemistry and XOR activity assay.

Lactogenic hormones regulate xanthine oxidoreductase and beta-casein levels in mammary epithelial cells by distinct mechanisms.

Xanthine oxidoreductase (XOR) is a prominent component of the milk lipid globule, whose concentration is selectively increased in mammary epithelial cells during the transition from pregnancy to lactation. To understand how XOR expression is controlled in the mammary gland, we investigated its properties and regulation by lactogenic hormones in cultured HC11 mammary epithelial cells. XOR was purified as the NAD(+)-dependent dehydrogenase by benzamidine-Sepharose chromatography and was shown to be intact and to have biochemical properties similar to those of enzyme from other sources. Treating confluent HC11 cells with prolactin and cortisol produced a progressive, four- to fivefold, increase in XOR activity, while XOR activity in control cells remained constant. Elevated cellular XOR activity was correlated with increased XOR protein and was due to both increased synthesis and decreased degradation of XOR. Prolactin and cortisol increased XOR protein and mRNA in the presence of epidermal growth factor, which blocked the stimulation of beta-casein synthesis by these hormones. Further, hormonal stimulation of XOR was inhibited by genistein (a protein tyrosine kinase inhibitor) and by PD 98059 (a specific inhibitor of the MAP kinase cascade). These findings indicate that lactogenic hormones stimulate XOR and beta-casein expression via distinct pathways and suggest that a MAP kinase pathway mediates their effects on XOR. Our results provide evidence that lactogenic hormones regulate milk protein synthesis by multiple signaling pathways.

Mouse mammary gland xanthine oxidoreductase: purification, characterization, and regulation.

Xanthine oxidoreductase (XOR) has been purified from lactating mouse mammary tissue and its properties and developmental expression have been characterized. XOR was purified 80-fold in two steps using benzamidine-Sepharose affinity chromatography. The purified enzyme had a specific activity of 5.7 U/mg and an activity to flavin ratio of 192. SDS-polyacrylamide gel electrophoresis showed that it was composed of a single (150 kDa) band and N-terminal sequence analysis verified that it was intact mouse XOR. Isoelectric focusing showed that purified XOR was composed of three catalytically active, electrophoretic variants with pI values of 7.55, 7.65, and 7.70. The majority of the XOR activity in both pregnant and lactating mammary glands was shown to exist as NAD+-dependent dehydrogenase (XD form), while the enzyme in freshly obtained mouse milk exits as O2-dependent oxidase (XO form). The activity and protein levels of XOR selectively increased in mammary tissue during pregnancy and lactation. The time course of these increases was biphasic and correlated with the functional maturation of the mammary gland. These results indicate that XOR may have novel, mammary gland-specific functions, in addition to its role in purine metabolism.

Effect of interferon-gamma on purine catabolic and salvage enzyme activities in rats.

To determine whether interferon-gamma affects rat purine catabolic and salvage enzyme activities, rats were injected with interferon-gamma (600000 U/kg, i.p.) and, similarly to a vehicle-injected control group, killed before or after injection at 6, 12, and 24 h. Organ homogenates were prepared and enzymatic reactions with substrates were carried out, after which the products were measured either chromatographically or spectrophotometrically. Western and Northern blotting also were performed. In contrast to the vehicle-injected rats, interferon-gamma-injected rats showed a significant rise in xanthine oxidoreductase activity in the liver, while enzyme activity was unchanged in the spleen, kidney, and lung. Western analysis of hepatic xanthine oxidoreductase showed an increased concentration of this protein 12 and 24 h after interferon-gamma injection. Northern analysis disclosed an enhanced mRNA expression coding for this enzyme, peaking 12 h after injection. Contrastingly, the activities of adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine guanine phosphoribosyltransferase, and adenine phosphoribosyltransferase were not affected by interferon-gamma in any organ tested. While interferon-gamma causes an increased hepatic biosynthesis of xanthine oxidoreductase, the physiologic role of this enzyme induction remains undetermined.