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The 6-oxopurine phosphoribosyltransferases (PRTs) are drug targets for the treatment of parasitic diseases. This is due to the fact that parasites are auxotrophic for the 6-oxopurine bases relying on salvage enzymes for the synthesis of their 6-oxopurine nucleoside monophosphates. In Trypanosoma brucei, the parasite that is the aetiological agent for sleeping sickness, there are three 6-oxopurine PRT isoforms. Two are specific for hypoxanthine and guanine, whilst the third, characterized here, uses all three naturally occurring bases with similar efficiency. Here, we have determined crystal structures for TbrHGXPRT in complex with GMP, XMP and IMP to investigate the structural basis for substrate specificity. The results show that Y201 and E208, not commonly observed within the purine binding pocket of 6-oxopurine PRTs, contribute to the versatility of this enzyme. The structures further show that a nearby water can act as an adaptor to facilitate the binding of XMP and GMP. When GMP binds, a water can accept a proton from the 2-amino group but when XMP binds, the equivalent water can donate its proton to the 2-oxo group. However, when IMP is bound, no water molecule is observed at that location. DATABASE: Coordinates and structure factors were submitted to the Protein Data Bank and have accession codes of 6MXB, 6MXC, 6MXD and 6MXG for the TbrHGXPRT.XMP complex, TbrHGXPRT.GMP complex, TbrHGXPRT.IMP complex, and TbrHGPRT.XMP complex, respectively.

41 p.-7 fig., Objective: To investigate the effect of cholesterol (hypercholesterolemia/7- ketocholesterol) on endoglin expression and regulation with respect to endothelial/vascular dysfunction in vivo and in vitro., Approach and results: In vivo experiments were performed in two-month-old ApoE-/-/LDLR-/- female mice and their wild type C57BL/6J littermates. In in vitro experiments, Human Aortic Endothelial Cells (HAECs) were treated with 7-ketocholesterol (7K). ApoE-/-/LDLR-/- mice developed hypercholesterolemia accompanied by increased circulating levels of P-selectin and endoglin and a disruption of NO metabolism. Functional analysis of aorta demonstrated impaired vascular reactivity and Western blot analysis revealed downregulation of membrane Eng/Smad2/3/eNOS signaling in ApoE-/-/LDLR-/- mice. 7K increased endoglin expression via KLF6, LXR and NF-κB in HAECs. 7K-induced endoglin expression was prevented by the treatment with 2-hydroxypropyl-β-cyclodextrin, PHA-408 or by KLF6 silencing. 7K induced increased adhesion and transmigration of monocytic THP-1 cells, was prevented by endoglin silencing., Conclusions: Hypercholesterolemia altered endoglin expression and signaling, followed by endothelial/vascular dysfunction before formation of atherosclerotic lesions in ApoE-/-/LDLR- /- mice. By contrast, 7-ketocholesterol increased endoglin expression, and induced inflammation in HAECs, which was followed by an increased adhesion and transmigration of monocytes via endothelium, which was prevented by endoglin inhibition. Thus, we propose a relevant role for endoglin in endothelial/vascular dysfunction/inflammation when exposed to cholesterol, This work was supported by project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) cofunded by ERDF, grants of Charles University Grant Agency, GAUK 1158413C, 1216217,Specific University Research SVV/2017/260414 and Czech Health Research Council (AZV CR17-31754A). CB was supported by grants from Consejo Superior de Investigaciones Cientificas(201420E039) and Centro de Investigacion Biomedica en Red de Enfermedades Raras(CIBERER; ISCIII-CB06/07/0038). CIBERER is an initiative of the Instituto de Salud Carlos III(ISCIII) of Spain supported by FEDER funds.

A soluble form of endoglin (sEng) released into the circulation was suggested to be a direct inducer of endothelial dysfunction, inflammation and contributed to the development of hypertension by interfering with TGF-β signaling in cardiovascular pathologies. In the present study, we assessed the hypothesis that high sEng level-induced hypertension via a possible sEng interference with TGF-β signaling pathways may result in inflammatory, structural or fibrotic changes in hearts of Sol-Eng+ mice (mice with high levels of soluble endoglin) fed either chow or high-fat diet. Female Sol-Eng+ mice and their age matched littermates with low plasma levels of sEng were fed either chow or high-fat diet (HFD). Heart samples were subsequently analyzed by histology, qRT-PCR and Western blot analysis. In this study, no differences in myocardial morphology/hypertrophy and possible fibrotic changes between Sol-Eng+ mice and control mice were detected on both chow and HFD. The presence of sEng did not significantly affect the expression of selected members of TGF-β signaling (membrane endoglin, TGFβRII, ALK-5, ALK-1, Id-1, PAI-1 and activated Smad proteins-pSmad 1,5 and pSmad 2,3), inflammation, heart remodeling (PDGFb, Col1A1) and endothelial dysfunction (VCAM-1, ICAM-1) in the hearts of Sol-Eng+ mice compared to control mice on both chow and high-fat diet. High levels of soluble endoglin did not affect microscopic structure (profibrotic and degenerative cardiomyocyte changes), and specific parts of TGF-β signaling, endothelial function and inflammation in the heart of Sol-Eng+ mice fed both chow diet or HFD. However, we cannot rule out a possibility that a long-term chronic exposure (9 months and more) to soluble endoglin alone or combined with other cardiovascular risk factors may contribute to alterations of heart function and structure in Sol-Eng+ mice, which is the topic in our lab in ongoing experiments.

AIMS Statins are known to influence status of adipokines, which play a key role in the pathophysiology of cardiometabolic diseases. As the effect of ezetimibe add-on to statin therapy on the impact of statins on plasma adipokines levels is unclear yet, the aim of this study was to answer this question through meta-analysis of controlled trials. METHODS A systematic review followed by a bibliographic search in PubMed-Medline, SCOPUS, Web of Science and Google Scholar databases was performed. Quantitative data synthesis was performed using a fixed- or random-effects model (based on the level of inter-study heterogeneity) and the generic inverse variance weighting method. Effect sizes were expressed as standardized mean difference (SMD) and 95% confidence interval (CI). RESULTS Meta-analysis of 23 controlled trials did not suggest any significant effect of adding ezetimibe on top of statin therapy on plasma concentrations of adiponectin (SMD: 0.34, 95% CI: -0.28, 0.96, p = 0.288), leptin (SMD: -0.75, 95% CI: -2.35, 0.85, p = 0.360), plasminogen activator inhibitor-1 (SMD: -1.06, 95% CI: -2.81, 0.69, p = 0.236) and interleukin-6 (SMD: 0.30, 95% CI: -0.08, 0.67, p = 0.124). However, significantly greater reductions in plasma concentrations of tumor necrosis factor-α (TNF-α) (SMD: -0.48, 95% CI: -0.87, -0.08, p = 0.018) were achieved with ezetimibe/statin combination therapy. CONCLUSIONS The results suggested that ezetimibe add-on to statin therapy is associated with enhanced TNF-α lowering effect compared with statin monotherapy. Owing to the emerging role of TNF-α in the pathogenesis of metabolic disorders, further investigations are required to unveil the translational relevance of this TNF-α-lowering effect.

The aim of the present study was to describe the currently poorly understood pharmacokinetics (PK) of boldine in control rats (LW, Lewis rats), and Mrp2 transporter-deficient rats (TR(-)). Animals from the LW and TR(-) groups underwent a bolus dose study with 10 mg/kg of boldine applied either orally or intravenously in order to evaluate the major PK parameters. The TR(-) rats demonstrated significantly reduced total clearance with prolonged biological half-life (LW 12+/-4.6 versus TR(-) 20+/-4.4 min), decreased volume of distribution (LW 3.2+/-0.4 l/kg versus TR(-) 2.4+/-0.4 l/kg) and reduced bioavailability (LW 7 % versus TR(-) 4.5 %). Another set of LW and TR(-) rats were used for a clearance study with continuous intravenous administration of boldine. The LW rats showed that biliary and renal clearance formed less than 2 % of the total clearance of boldine. The treatment of samples with beta-glucuronidase showed at least a 38 % contribution of conjugation reactions to the overall clearance of boldine. The TR(-) rats demonstrated reduced biliary clearance of boldine and its conjugates, which was partly compensated by their increased renal clearance. In conclusion, this study presents the PK parameters of boldine and shows the importance of the Mrp2 transporter and conjugation reactions in the elimination of the compound.

Our objective was to investigate the effect of cholesterol [hypercholesterolemia and 7-ketocholesterol (7K)] on endoglin (Eng) expression and regulation with respect to endothelial or vascular dysfunction

14 p.-6 fig., Aims: A soluble form of endoglin (sEng) was proposed to participate in the induction of endothelial dysfunction in small blood vessels. Here, we tested the hypothesis that high levels of sEng combined with a high-fat diet induce endothelial dysfunction in an atherosclerosis-prone aorta., Methods and results: Six-month-old female and male transgenic mice overexpressing human sEng (Sol-Eng+) with low (Sol-Eng+low) or high (Sol-Eng+high) levels of plasma sEng were fed a high-fat rodent diet containing 1.25% cholesterol and 40% fat for 3 months. The plasma cholesterol and mouse sEng levels did not differ in the Sol-Eng+high and Sol-Eng+low mice. The expression of proinflammatory (P-selectin, ICAM-1, pNFκB and COX-2) and oxidative-stress-related markers (HO-1, NOX-1 and NOX-2) in the aortas of Sol-Eng+high female mice was significantly higher than in Sol-Eng+low female mice. Endothelium-dependent vasodilatation induced by acetylcholine was preserved better in the Sol-Eng+ high female mice than in the Sol-Eng+low female mice., Conclusion: These results suggest that high concentrations of sEng in plasma in combination with a high-fat diet induce the simultaneous activation of proinflammatory, pro-oxidative and vasoprotective mechanisms in mice aorta and the balance of these biological processes determines whether the final endothelial phenotype is adaptive or maladaptive., The work was supported by grants from Czech Science Foundation GACR No. GA15-24015S, the Grant Agency of Charles University in Prague No. 1284214/C, No. 1158413C and SVV/2016/260293, the European Regional Development Fund under the Innovative Economy Program of the European Union (grant coordinated by JCET-UJ, No. POIG.01.01.02-00-069/09), the Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to C.B. and SAF2013-45784-R to J.M.L.-N.), the Junta de Castilla y Leon (GR100 to J.M.L.-N.), CIBERER (to C.B.) and Red de Investigación Cooperativa en Enfermedades Renales (REDINREN, RD12/0021/0032 to J.M.L.-N.). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III of Spain supported by European Regional Development Funds (FEDER). The Cardiovascular Phenotyping Unit of the University of Salamanca received the support of FEDER.

Excessive iron accumulation in the liver, which accompanies certain genetic or metabolic diseases, impairs bile acids (BA) synthesis, but the influence of iron on the complex process of BA homeostasis is unknown. Thus, we evaluated the effect of iron overload (IO) on BA turnover in rats. Compared with control rats, IO (8 intraperitoneal doses of 100 mg/kg every other day) significantly decreased bile flow as a consequence of decreased biliary BA secretion. This decrease was associated with reduced expression of Cyp7a1, the rate limiting enzyme in the conversion of cholesterol to BA, and decreased expression of Bsep, the transporter responsible for BA efflux into bile. However, IO did not change net BA content in faeces in response to increased intestinal conversion of BA into hyodeoxycholic acid. In addition, IO increased plasma cholesterol concentrations, which corresponded with reduced Cyp7a1 expression and increased expression of Hmgcr, the rate-limiting enzyme in de novo cholesterol synthesis. In summary, this study describes the mechanisms impairing synthesis, biliary secretion and intestinal processing of BA during IO. Altered elimination pathways for BA and cholesterol may interfere with the pathophysiology of liver damage accompanying liver diseases with excessive iron deposition.

1p., Objective: A soluble form of tissue/membrane endoglin (sEng) circulating in plasma is increased in hypercholesterolemia, atherosclerosis and type II diabetes mellitus patients. Moreover, it has been proposed that sEng might be least partially responsible for the induction of endothelial dysfunction (but not studied in atherosclerosis prone blood vessels so far) and related to the presence of hypercholesterolemia. We provided couple of experiments in order to reveal whether high levels of sEng combined with hypercholesterolemia might affect vascular/endothelial function and inflammation in mice aorta., Methods: Three-month-old female transgenic mice on CBAxC57BL/6J background with high levels of sEng (Sol-Eng+ high HFD) and their littermates with low levels of sEng (Sol-Eng+ low HFD) were fed high fat diet (HFD) for either 3 months or 6 months. Plasma samples were used for biochemical analyses of total cholesterol and ELISA analyses of sEng and inflammatory markers. Functional parameters of aorta were assessed by means of wire myograph 620M. Western Blot analysis endothelial dysfunction/inflammation markers in aorta was performed., Results: 3 months exposure to high sEng levels and HFD resulted in induction of inflammation (increased expression of P-selectin, ICAM-1, pNFκB and COX-2) in aorta of high Sol-Eng+high HFD. Moreover, 6 months exposure to high sEng levels and HFD resulted in impaired KCl induced vasoconstriction, endothelial-dependent relaxation after administration of acetylcholine, endothelial-independent relaxation induced by sodium nitroprusside. In addition, the expressions of endoglin, p-eNOS/eNOS, pSmad2/3/Smad2/3 signaling pathway were significantly lower in Sol-Eng+high HFD group compared to Sol-Eng+low HFD group., Conclusions: Current results show that high levels of soluble endoglin in combination with hypercholesterolemia induce signs of inflammation and endothelial/vascular dysfunction in aorta with possible alteration of membrane endoglin/eNOS signaling. Thus, we might propose that high levels of soluble endoglin presented in patients with hypercholesterolemia, atherosclerosis and type II diabetes mellitus might worsen endothelial dysfunction in combination with hypercholesterolemia, thus soluble endoglin might be considered as a risk factor of cardiovascular diseases., This work was supported by grant from Czech Health Research Council AZV CR number 17-31754A.

32 p.-5 fig.-1 fig. supl. Vitverova, Barbora et al., BACKGROUND AND AIMS:Increased plasma levels of soluble endoglin (sEng) were detected in patients with endothelial dysfunction-related disorders and hypercholesterolemia. In this study, we hypothesized that high levels of sEng accompanied by mild hypercholesterolemia could aggravate endothelial and vessel wall dysfunction and affect endoglin/eNOS signaling in mouse aorta., METHODS:Three-month-old female transgenic mice on CBAxC57BL/6J background, with high levels of sEng (Sol-Eng+high HFD), and their littermates with low levels of sEng (Sol-Eng+low HFD), were fed a high fat diet for six months. Plasma samples were used for biochemical, ELISA and Luminex analyses of total cholesterol, sEng and inflammatory markers. Functional parameters of aorta were assessed with wire myograph 620M. Western blot analyses of membrane endoglin/eNOS signaling and endothelial dysfunction/inflammation markers in aorta were performed., RESULTS:Functional analysis of aorta showed impaired KCl induced vasoconstriction, endothelial-dependent relaxation after the administration of acetylcholine as well as endothelial-independent relaxation induced by sodium nitroprusside in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group. Ach-induced vasodilation after administration of l-NAME was significantly higher in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group. The expression of endoglin, p-eNOS/eNOS, pSmad2/3/Smad2/3 signaling pathway was significantly lower in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group., CONCLUSIONS:The results indicate that long-term hypercholesterolemia combined with high levels of sEng leads to the aggravation of endothelial and vessel wall dysfunction in aorta, with possible alterations of the membrane endoglin/eNOS signaling, suggesting that high levels of soluble endoglin might be considered as a risk factor of cardiovascular diseases., The work was supported by grants from Czech Science foundation (GACR number GA15-24015S), Czech Health Research Council (AZV CR number 17-31754A), the Grant Agency of Charles University in Prague (number 1284214/C, 1158413C and grant SVV/2016/260293), Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to CB Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; to CB), and Instituto de Salud Carlos III (PI16/00460 and Retic RD16/0009/0025, REDINREN-FEDER). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds from the European Commission

Estrogen-induced cholestasis is characterized by impaired hepatic uptake and biliary bile acids secretion because of changes in hepatocyte transporter expression. The induction of heme oxygenase-1 (HMOX1), the inducible isozyme in heme catabolism, is mediated via the Bach1/Nrf2 pathway, and protects livers from toxic, oxidative and inflammatory insults. However, its role in cholestasis remains unknown. Here, we investigated the effects of HMOX1 induction by heme on ethinylestradiol-induced cholestasis and possible underlying mechanisms. Wistar rats were given ethinylestradiol (5 mg/kg s.c.) for 5 days. HMOX1 was induced by heme (15 μmol/kg i.p.) 24 hrs prior to ethinylestradiol. Serum cholestatic markers, hepatocyte and renal membrane transporter expression, and biliary and urinary bile acids excretion were quantified. Ethinylestradiol significantly increased cholestatic markers (P ≤ 0.01), decreased biliary bile acid excretion (39%, P = 0.01), down-regulated hepatocyte transporters (Ntcp/Oatp1b2/Oatp1a4/Mrp2, P ≤ 0.05), and up-regulated Mrp3 (348%, P ≤ 0.05). Heme pre-treatment normalized cholestatic markers, increased biliary bile acid excretion (167%, P ≤ 0.05) and up-regulated hepatocyte transporter expression. Moreover, heme induced Mrp3 expression in control (319%, P ≤ 0.05) and ethinylestradiol-treated rats (512%, P ≤ 0.05). In primary rat hepatocytes, Nrf2 silencing completely abolished heme-induced Mrp3 expression. Additionally, heme significantly increased urinary bile acid clearance via up-regulation (Mrp2/Mrp4) or down-regulation (Mrp3) of renal transporters (P ≤ 0.05). We conclude that HMOX1 induction by heme increases hepatocyte transporter expression, subsequently stimulating bile flow in cholestasis. Also, heme stimulates hepatic Mrp3 expression via a Nrf2-dependent mechanism. Bile acids transported by Mrp3 to the plasma are highly cleared into the urine, resulting in normal plasma bile acid levels. Thus, HMOX1 induction may be a potential therapeutic strategy for the treatment of ethinylestradiol-induced cholestasis.

Iron depletion (ID) has been shown to induce the liver expression of Cyp7a1, the rate-limiting enzyme initiating conversion of cholesterol to bile acids (BA), although the effect on bile acids metabolism and bile production is unknown. Therefore, we investigated changes in bile secretion and BA synthesis during diet-induced iron depletion (ID) in rats. ID increased bile flow along with augmented biliary excretion of bile acids, glutathione, cholesterol and phospholipids. Accordingly, we found transcriptional upregulation of the Cyp7a1, Cyp8b1, and Cyp27a1 BA synthetic enzymes, as well as induction of the Abcg5/8 cholesterol transporters in ID rat livers. In contrast, intravenous infusion of 3H-taurocholate failed to elicit any difference in biliary secretion of this compound in the ID rats. This corresponded with unchanged expression of canalicular rate-limiting transporters for BA as well as glutathione. We also observed that ID substantially changed the spectrum of BA in bile and decreased plasma concentrations of BA and cholesterol. Experiments with differentiated human hepatic HepaRG cells confirmed human CYP7A1 orthologue upregulation resulting from reduced iron concentrations. Results employing a luciferase reporter gene assay suggest that the transcriptional activation of the CYP7A1 promoter under ID conditions works independent of farnesoid X (FXR), pregnane X (PXR) and liver X (LXRα) receptors activation. It can be concluded that this study characterizes the molecular mechanisms of modified bile production as well as cholesterol as along with BA homeostasis during ID. We propose complex upregulation of BA synthesis, and biliary cholesterol secretion as the key factors affected by ID.

Aims Increased plasma soluble endoglin concentrations (sEng) are frequently detected in metabolic disorders accompanied with hypercholesterolemia in serum, but effect of sEng on the cholesterol biochemistry is unknown. Cholesterol and bile acids (BA) are important products of liver metabolism with numerous functions within the organism. Turnover of these substances requires precise regulation due to potential toxicities during their cumulation. In this study, we hypothesized that high sEng levels affect cholesterol homeostasis and BA turnover in mice liver. Main methods Nine-month-old transgenic male mice overexpressing human sEng and wild-type mice underwent plasma, bile, stool, and organ samples analysis by analytical, qRT-PCT and Western blot methods. Key findings sEng mice demonstrated decreased plasma total and LDL cholesterol concentrations due to upregulation of hepatic Sr-b1 and Ldlr receptors, increased liver cholesterol content, and increased Abcg8-mediated cholesterol efflux into bile. sEng also increased conversion of cholesterol into bile acids (BA) via upregulation of Cyp7a1 and increased Mdr1 expression. Plasma concentrations of BA were increased in sEng mice due to their enhanced reabsorption via ileum. Increased hepatic disposition of BA led to their increased biliary excretion coupled with choleretic activity. Significance For the first time, we have shown that high sEng plasma levels affect cholesterol and BA homeostasis on the basis of complex liver and intestinal effects. The significance of these findings for pathophysiology of diseases associated with increased sEng concentrations remains to be elucidated in prospective studies.

34 p.-7 fig., Aims: Endoglin is a transmembrane glycoprotein, that plays an important role in regulating endothelium. Proteolytic cleavage of membrane endoglin releases soluble endoglin (sEng), whose increased plasma levels have been detected in diseases related to the cardiovascular system. It was proposed that sEng might damage vascular endothelium, but detailed information about the potential mechanisms involved is not available. Thus, we hypothesized that sEng contributes to endothelial dysfunction, leading to a pro-inflammatory phenotype by a possible modulation of the TGF-β and/or inflammatory pathways., Main methods: Human umbilical vein endothelial cells (HUVECs) and Human embryonic kidney cell line (HEK293T) were treated with different sEng concentration and time in order to reveal possible effect on biomarkers of inflammation and TGF-β signaling. IL6 and NFκB reporter luciferase assays, quantitative real-time PCR analysis, Western blot analysis and immunofluorescence flow cytometry were used., Key findings: sEng treatment results in activation of NF-κB/IL-6 expression, increased expression of membrane endoglin and reduced expression of Id-1. On the other hand, no significant effects on other markers of endothelial dysfunction and inflammation, including eNOS, peNOSS1177, VCAM-1, COX-1, COX-2 and ICAM-1 were detected., Significance: As a conclusion, sEng treatment resulted in an activation of NF-κB, IL-6, suggesting activation of pro-inflammatory phenotype in endothelial cells. The precise mechanism of this activation and its consequence remains to be elucidated. A combined treatment of sEng with other cardiovascular risk factors will be necessary in order to reveal whether sEng is not only a biomarker of cardiovascular diseases, but also a protagonist of endothelial dysfunction., This work was supported by grants from Czech Science Foundation (GACR 15-24015S,GAUK 1158413C, SVV/2016/260293 and SVV/2017/260414 to Petr Nachtigal), Ministerio de Economía y Competitividad of Spain (SAF2013-43421-R to Carmelo Bernabéu), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIIICB06/07/0038 and ER16PIAC707 to CB). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

The aim of the present study was to investigate the expression, localization, and function of organic cation transporter 3 (Oct3, Slc22a3) and multidrug and toxin extrusion protein 1 (Mate1, Slc47a1) in the rat placenta. Using qRT-PCR and Western blotting techniques, we demonstrated abundant Oct3 and Mate1 mRNA and protein expression achieving significantly higher levels than those in the maternal kidney (positive control). Immunohistochemical visualization revealed preferential localization of Oct3 on the basolateral, i.e., fetus facing side of the placenta, whereas Mate1 positivity was located in the labyrinth area predominantly on the apical, i.e., maternal side of the placenta. To investigate the role of these transporters in the transplacental pharmacokinetics, the in situ method of dually perfused rat term placenta was employed in open- and closed-circuit arrangements; 1-methyl-4-phenylpyridinium (MPP(+)) was used as a model substrate of both Oct3 and Mate1. We provide evidence that Oct3 and Mate1 cause considerable asymmetry between maternal-to-fetal and fetal-to-maternal transport of MPP(+) in favor of fetomaternal direction. Using closed-circuit experimental setup, we further describe the capacity of Oct3 and Mate1 to transport their substrate from fetus to mother even against a concentration gradient. We conclude that Oct3, in a concentration-dependent manner, takes up MPP(+) from the fetal circulation into the placenta, whereas Mate1, on the other side of the barrier, is responsible for MPP(+) efflux from placenta to the maternal circulation. These two transport proteins, thus, form an efficient transplacental eliminatory pathway and play an important role in fetal protection and detoxication.

Nucleoside transporters are evolutionarily conserved proteins that are essential for normal cellular function. In the present study, we examined the role of equilibrative nucleoside transporter 2 (ent2) inDrosophila. Null mutants ofent2are lethal during late larval/early pupal stages, indicating thatent2is essential for normal development. Hypomorphic mutant alleles ofent2, however, are viable and exhibit reduced associative learning. We additionally used RNA interference to knock downent2expression in specific regions of the CNS and show thatent2is required in the α/β lobes of the mushroom bodies and the antennal lobes. To determine whether the observed behavioral defects are attributable to defects in synaptic transmission, we examined transmitter release at the larval neuromuscular junction (NMJ). Excitatory junction potentials were significantly elevated inent2mutants, whereas paired-pulse plasticity was reduced. We also observed an increase in stimulus dependent calcium influx in the presynaptic terminal. The defects observed in calcium influx and transmitter release probability at the NMJ were rescued by introducing an adenosine receptor mutant allele (AdoR1) into theent2mutant background. The results of the present study provide the first evidence of a role forent2function inDrosophilaand suggest that the observed defects in associative learning and synaptic function may be attributable to changes in adenosine receptor activation.

In the present paper potato plants were cultivated in the presence of ammonium sulphate or elemental sulphur supplementation into the soil to reveal the effects of different sulphur forms on content of nitrogen, phosphorus, potassium, calcium, magnesium and sulphur, and yield of tubers. During the investigation of the influence of different sulphur forms on yield of potato tubers we did not observe significant changes. Average weight of tubers of control plants per one experimental pot was 355 g. Application of sulphur in both forms resulted in moderate potato tubers weight reduction per one experimental pot compared to control group; average value ranged from 320 to 350 g per one experimental pot. Further we treated the plants with two different supplementation of sulphur with cadmium(II) ions (4 mg of cadmium(II) acetate per kilogram of the soil). The significantly lowest cadmium content (p < 0.05) was determined in tissues of plants treated with the highest dosage of elemental sulphur (0.64 mg Cd/kg) compared to control plants (0.82 mg Cd/kg). We also aimed our attention on the cadmium content in proteins, lipids or soluble carbohydrates and ash. Application of sulphate as well as elemental sulphur resulted in significant cadmium content reduction in lipid fraction compared to control plants. In addition to this we quantified content of low molecular mass thiols in potatoes tissues. To determine the thiols content we employed differential pulse voltammetry Brdicka reaction. After twelve days of the treatment enhancing of thiols level was observed in all experimental groups regardless to applied sulphur form and its concentration. Finally we evaluated the effect of sulphur supplementation on Phytophora infestans infection of potato plants.

Much of the knowledge about cryptochrome function in Drosophila stems from analyzing the cryb mutant. Several features of this variant's light responsiveness imply either that CRYb retains circadian-photoreceptive capacities or that additional CRY-independent light-input routes subserve these processes. Potentially to resolve these issues, we generated cry knock-out mutants (cry0's) by gene replacement. They behaved in an anomalously rhythmic manner in constant light (LL). However, cry0 flies frequently exhibited two separate circadian components in LL, not observed in most previous cryb analyses. Temperature-dependent circadian phenotypes exhibited by cry0 flies suggest that CRY is involved in core pacemaking. Further locomotor experiments combined cry0 with an externally blinding mutation (norpAP24), which caused the most severe decrements of circadian photoreception observed so far. cryb cultures were shown previously to exhibit either aperiodic or rhythmic eclosion in separate studies. We found cry0 to eclose in a solidly periodic manner in light:dark cycles or constant darkness. Furthermore, both cry0 and cryb eclosed rhythmically in LL. These findings indicate that the novel cry0 type causes more profound defects than does the cryb mutation, implying that CRYb retains residual activity. Because some norpAP24 cry0 individuals can resynchronize to novel photic regimes, an as-yet undetermined light-input route exists in Drosophila.

Adenosine receptors (AdoR) are members of the G protein-coupled receptor superfamily and mediate extracellular adenosine signaling, but the mechanism of adenosine signaling is still unclear. Here we report the first characterization of an insect AdoR, encoded by the Drosophila gene CG9753. Adenosine stimulation of Chinese hamster ovary cells carrying transiently expressed CG9753 led to a dose-dependent increase of intracellular cAMP and calcium, but untransfected controls showed no such response, showing that CG9753 encodes a functional AdoR. Endogenous CG9753 transcripts were detected in the brain, imaginal discs, ring gland and salivary glands of third-instar Drosophila larvae, and CG9753 overexpression in vivo caused lethality or severe developmental anomalies. These developmental defects were reduced by adenosine depletion, consistent with the proposed function of the CG9753 product as an AdoR. Overexpression of the G protein subunit Galpha(s) or of the catalytic subunit of protein kinase A (PKA) partially mimicked and enhanced the defects caused by ectopic expression of AdoR. Our results suggest that AdoR is an essential part of the adenosine signaling pathway and Drosophila offers a unique opportunity to use genetic analysis to study conserved aspects of the adenosine signaling pathway.

The aim of our study was to investigate whether two potent anti-inflammatory agents, dexamethasone and anakinra, an IL-1 receptor antagonist, may influence acute kidney injury (AKI) and associated drug excretory functions during endotoxemia (LPS) in rats. Ten hours after LPS administration, untreated endotoxemic rats developed typical symptoms of AKI, with reduced GFR, impaired tubular excretion of urea and sodium, and decreased urinary excretion of azithromycin, an anionic substrate for multidrug resistance-transporting proteins. Administration of both immunosuppressants attenuated the inflammatory response, liver damage, AKI, and increased renal clearance of azithromycin mainly by restoration of GFR, without significant influence on its tubular secretion. The lack of such an effect was related to the differential effect of both agents on the renal expression of individual drug transporters. Only dexamethasone increased the urinary clearance of bile acids, in accordance with the reduction of the apical transporter (Asbt) for their tubular reabsorption. In summary, our data demonstrated the potency of both agents used for the prevention of AKI, imposed by endotoxins, and for the restoration of renal drug elimination, mainly by the improvement of GFR. The influence of both drugs on altered tubular functions and the expression of drug transporters was differential, emphasizing the necessity of knowledge of transporting pathways for individual drugs applied during sepsis. The effect of anakinra suggests a significant contribution of IL-1 signaling to the pathogenesis of LPS-induced AKI.

Adenosine deaminases catalyze the deamination of adenosine and deoxyadenosine into their respective inosine nucleosides. Recent sequencing of the genomes of several model organisms and human reveal that Metazoa usually have more than one adenosine deaminase gene. A deficiency in the gene encoding the major enzyme is lethal in mouse and Drosophila and leads to severe combined deficiency (SCID) in human. In these organisms, enzyme deficiency causes increased adenosine/deoxyadenosine concentration in body fluids and some organs. Elevated levels of adenosine and deoxyadenosine are toxic to certain mammalian and insect cells, and it was shown for human and mouse that it is a primary cause of pathophysiological effects. Data suggest that the major role of adenosine deaminases in various taxa is the protection of tissues against increased levels of adenosine and deoxyadenosine. This review also discusses potential roles of adenosine deaminases in Drosophila metamorphosis and the employment of a Drosophila model to study the cell-specific toxicity of elevated nucleoside levels.

The purpose of the present study was to compare the activity of two different clinically available iron chelators on the development of acute liver injury after administration of the bacterial endotoxin (lipopolysaccharide [LPS]) in rats. Lipopolysaccharide was administered either alone or after pretreatment with dexrazoxane (DEX) or deferoxamine (DFO). Control groups received only saline or its combination with either chelator. After 8 h, untreated LPS rats developed liver injury, with signs of inflammation and oxidative stress. Lipopolysaccharide reduced plasma iron concentrations in association with increased production of hepcidin and the reduced liver expression of ferroportin. Administration of chelating agents to LPS animals showed distinct effects. Although both drugs were able to reduce liver iron content, together with corresponding changes in hepcidin and ferroportin expressions, only DFO showed a protective effect against liver injury despite relatively small liver concentrations. In sharp contrast, DEX failed to improve any hallmark of liver injury and even worsened the GSH/GSSG ratio, the indicator of oxidative stress in the tissue. High-performance liquid chromatography-mass spectrometry analysis showed marked liver accumulation of iron-chelating metabolite of DEX (ADR-925), whereas the parent compound was undetectable. Further downregulation of transporters involved in bile formation was observed after DFO in the LPS group as well as in healthy animals. Neither chelator imposed significant liver injury in healthy animals. In conclusion, we demonstrated marked differences in the modulation of endotoxemic liver impairment between two iron chelators, implicating that particular qualities of chelating agents may be of crucial importance.

In our previous study, we described synchronized activity of organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion 1 (MATE1/SLC47A1) transporter in the passage of organic cations across the rat placenta and the role of these transporters in fetal defense; in this study, we hypothesized that changes in placental levels of OCT3 and MATE1 throughout gestation might affect the fetal protection and detoxification. Using quantitative RT-PCR, Western blot analysis, and immunohistochemistry, we were able to detect Oct3/OCT3 and Mate1/MATE1 expression in the rat placenta as early as on Gestation Day (gd) 12 with increasing tendency toward the end of pregnancy. Comparing first versus third trimester human placenta, we observed stable expression of OCT1 and decreasing expression of OCT2 and OCT3 isoforms. Contrary to the current literature, we were able to detect also MATE1/MATE2 isoforms in the human placenta, however, with considerable inter- and intraindividual variability. Using infusion of 1-methyl-4-phenylpyridinium (MPP(+)), a substrate of OCT and MATE transporters, into pregnant dams, we investigated the protective function of the placenta against organic cations at different gds. The highest amount of MPP(+) reached the fetus on gd 12 while from gd 15 onward, maternal-to-fetal transport of MPP(+) decreased significantly. We conclude that increased expression of placental OCT3 and MATE1 along with general maturation of the placental tissues results in significantly lower transport of MPP(+) from mother to fetus. In contrast, decreasing expression of OCT3 and MATE1 in human placenta indicates these transporters may play a role in fetal protection preferentially at earlier stages of gestation.

Epigallocatechin gallate (EGCG) has been shown to be protective in various experimental models of liver injury, although opposite effects have also been reported. Since its effect on biliary physiology has not been thoroughly investigated, the present study evaluated effect of EGCG on bile flow and bile acid homeostasis in rats. Compared to controls, EGCG treatment decreased bile flow by 23%. Hepatic paracellular permeability and biliary bile acid excretion were not altered by EGCG administration, but biliary glutathione excretion was reduced by 70%. Accordingly, the main glutathione transporter on the hepatocyte canalicular membrane, multidrug resistance-associated protein 2 (Mrp2), was significantly decreased at the protein level. EGCG administration also doubled plasma bile acid levels compared to controls. While protein levels of the main hepatic bile acid transporters were unchanged, the rate-limiting enzyme in the bile acid synthesis, Cyp7a1, was significantly increased by EGCG. Enhanced bile acid synthesis in these animals was also confirmed by a 2-fold increase in plasma marker 7α-hydroxy-4-cholesten-3-one. In contrast, EGCG markedly downregulated major bile acid transporters (Asbt and Ostα) and regulatory molecules (Shp and Fgf15) in the ileum. When EGCG was coadministered with ethinylestradiol, a potent cholestatic agent, it did not show any additional effect on the induced cholestasis. This study shows ability of EGCG to raise plasma bile acid concentrations, mainly through Cyp7a1 upregulation, and to decrease bile production through reduction in Mrp2-mediated bile acid-independent bile flow. In conclusion, our data demonstrate that under certain conditions EGCG may induce cholestasis.

The beneficial effect of the major green tea catechin, epigallocatechin gallate (EGCG), on cholesterol homeostasis has been studied mainly in relation to the intestinal absorption of cholesterol; however, how EGCG affects cholesterol metabolism in the liver is not entirely known. The present study investigated the effect of EGCG on liver cholesterol metabolism in healthy and ethinylestradiol-treated rats. EGCG treatment reduced plasma total cholesterol in ethinylestradiol-treated animals and very low density lipoprotein cholesterol in both groups receiving EGCG. In healthy rats, despite the decrease in bile flow, EGCG markedly enhanced biliary secretion of cholesterol and phospholipids. These changes were correlated with increased expression of ATP-binding cassette transporter G5 and G8 and scavenger receptor class B type 1, and decreased expression of acyl-CoA:cholesterol acyltransferase. Ethinylestradiol treatment caused marked hepatic cholesterol accumulation with a concomitant liver weight increase and plasma cholesterol reduction. In ethinylestradiol-treated rats, EGCG co-administration attenuated the increase in liver cholesterol and liver weight. Furthermore, EGCG blunted induction of acyl-CoA:cholesterol acyltransferase and raised reduced levels of ATP-binding cassette transporter G5 and G8 and 3-hydroxy-3-methyl-glutaryl-CoA reductase in ethinylestradiol-treated rats. In conclusion, this study has demonstrated for the first time the ability of EGCG to enhance biliary cholesterol secretion and to attenuate ethinylestradiol-induced liver cholesterol accumulation. Changes in the expression of relevant enzymes and transporters suggest evidence of another mechanism that may contribute to the overall effect of EGCG on cholesterol metabolism and imply new physiological consequences of this widely used compound.

The enzyme glucose-6-phosphate dehydrogenase (G6PD) of the bark beetle Ips typographus is derived from a gene that includes eight exons and spans over 7100 nucleotides (nt). By means of two transcription starts, the gene generates two mRNA isoforms that are present in similar amounts in the larvae, pupae and adults. The A isoform includes exon IA of 115 nt, which is followed by intron 1a extending to position 3457 of the gene. The B mRNA isoform begins with exon IB (100 nt) that occupies positions 3291-3390 within the 1a intron. Exons II to VII are included in both mRNA isoforms. The gene contains 31.6% (36.5% in the translated region) of the GC nucleotides. Two transcription starts and the exon/intron organization distinguish bark beetle G6PD from the homologous genes known in other insects. Two enzyme variants were detected in the protein extracts of individual bark beetles but their relationship to the A and B mRNA isoforms is uncertain.

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals., Adenosine deaminase is critically important to survival; congenital deficiency in humans leads to severe immunodeficiency. Here, the authors demonstrate that adenosine deaminase deficiency in flies results in severe developmental defects.

The aim of the current study was to clarify the effect of high sucrose diet (HSD) on bile formation (BF) in rats with hereditary hypertriglyceridemia (HHTg). Potentially positive effects were studied for boldine, a natural choleretic agent. Administration of HSD to HHTg rats led to increased triglyceride deposition in the liver. HSD reduced BF as a consequence of decreased biliary secretion of bile acids (BA) and glutathione. Responsible mechanism was down-regulation of hepatic transporters for BA and glutathione, Bsep and Mrp2, respectively. Moreover, gene expressions of transporters for other constituents of bile, namely Abcg5/8 for cholesterol, Abcb4 for phospholipids, and Oatp1a4 for xenobiotics, were also reduced by HSD. Boldine partially attenuated cholestatic effect of HSD by promotion of biliary secretion of BA through up-regulation of Bsep and Ntcp, and by increase in biliary secretion of glutathione as a consequence of its increased hepatic disposition. This study demonstrates mechanisms of impaired BF during nonalcoholic fatty liver disease induced by HSD. Altered function of responsible transporters suggests also potential for changes in kinetics of drugs, which may complicate pharmacotherapy in subjects with high intake of sucrose, and with fatty liver disease. Sucrose induced alterations in BF may be alleviated by administration of boldine.