Leoligin is a natural lignan found in Edelweiss (Leontopodium nivale ssp. alpinum). The aim of this study was to examine its influence on cholesterol efflux and to address the underlying mechanism of action. Leoligin increases apo A1- as well as 1% human plasma-mediated cholesterol efflux in THP-1 macrophages without affecting cell viability as determined by resazurin conversion. Western blot analysis revealed that the protein levels of the cholesterol efflux transporters ABCA1 and ABCG1 were upregulated, whereas the SR-B1 protein level remained unchanged upon treatment with leoligin (10 μM, 24 h). Quantitative reverse transcription PCR further uncovered that leoligin also increased ABCA1 and ABCG1 mRNA levels without affecting the half-life of the two mRNAs in the presence of actinomycin D, a transcription inhibitor. Proteome analysis revealed the modulation of protein expression fingerprint in the presence of leoligin. Taken together, these results suggest that leoligin induces cholesterol efflux in THP-1-derived macrophages by upregulating ABCA1 and ABCG1 expression. This novel activity suggests leoligin as a promising candidate for further studies addressing a possible preventive or therapeutic application in the context of atherosclerosis.

• MeSH
• ABC transportéry metabolismus   MeSH
• Asteraceae chemie   MeSH
• ateroskleróza MeSH
• biologický transport MeSH
• daktinomycin farmakologie   MeSH
• lidé MeSH
• lignany chemie   izolace a purifikace   farmakologie   MeSH
• makrofágy metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• molekulární struktura MeSH
• oxaziny metabolismus   MeSH
• polymerázová řetězová reakce MeSH
• sirotčí receptory metabolismus   MeSH
• western blotting MeSH
• xantheny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Recent evidence suggests that energy metabolism contributes to molecular mechanisms controlling stem cell identity. For example, human embryonic stem cells (hESCs) receive their metabolic energy mostly via glycolysis rather than mitochondrial oxidative phosphorylation. This suggests a connection of metabolic homeostasis to stemness. Nicotinamide adenine dinucleotide (NAD) is an important cellular redox carrier and a cofactor for various metabolic pathways, including glycolysis. Therefore, accurate determination of NAD cellular levels and dynamics is of growing importance for understanding the physiology of stem cells. Conventional analytic methods for the determination of metabolite levels rely on linear calibration curves. However, in actual practice many two-enzyme cycling assays, such as the assay systems used in this work, display prominently nonlinear behavior. Here we present a diaphorase/lactate dehydrogenase NAD cycling assay optimized for hESCs, together with a mechanism-based, nonlinear regression models for the determination of NAD(+), NADH, and total NAD. We also present experimental data on metabolic homeostasis of hESC under various physiological conditions. We show that NAD(+)/NADH ratio varies considerably with time in culture after routine change of medium, while the total NAD content undergoes relatively minor changes. In addition, we show that the NAD(+)/NADH ratio, as well as the total NAD levels, vary between stem cells and their differentiated counterparts. Importantly, the NAD(+)/NADH ratio was found to be substantially higher in hESC-derived fibroblasts versus hESCs. Overall, our nonlinear mathematical model is applicable to other enzymatic amplification systems.

• MeSH
• buněčné extrakty MeSH
• elektroforéza kapilární MeSH
• embryonální kmenové buňky metabolismus   MeSH
• kalibrace MeSH
• lidé MeSH
• NAD metabolismus   MeSH
• nelineární dynamika * MeSH
• oxaziny metabolismus   MeSH
• regresní analýza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Little is known about the effects of the cardiovascular drug verapamil (VRP) on metabolic processes in fish. Most calcium channel blockers including VRP are metabolized by cytochrome P450 (CYP450) enzymes. In this study we investigated the in vivo effect of VRP on some CYP450-mediated reactions in juvenile rainbow trout (Oncorhynchus mykiss). Fish were exposed to sublethal concentrations of VRP (0.5, 27 and 270 μg l(-1)) for 0, 21, and 42 day. The following CYP450-mediated reactions were studied in hepatic microsomes: O-dealkylation of ethoxyresorufin, methoxyresorufin, and pentoxyresorufin, hydroxylation of coumarin, tolbutamide, and p-nitrophenol, and O-debenzylation of 7-benzyloxy-4-trifluoromethylcoumarin. The amounts of products of these reactions did not differ among fish exposed to different levels of VRP and control fish. This suggests that the levels of VPR used did not alter catalytic activity of the selected CYP450 enzymes. In conclusion, none of the investigated CYP450-mediated reactions has potential as a biomarker to monitor VRP contamination of the aquatic environment.

• MeSH
• biologické markery metabolismus   MeSH
• blokátory kalciových kanálů toxicita   MeSH
• chemické látky znečišťující vodu toxicita   MeSH
• jaterní mikrozomy účinky léků   enzymologie   metabolismus   MeSH
• játra účinky léků   enzymologie   metabolismus   MeSH
• kumariny metabolismus   MeSH
• nitrofenoly metabolismus   MeSH
• Oncorhynchus mykiss MeSH
• oxaziny metabolismus   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• verapamil toxicita   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Much like other microorganisms, wild yeasts preferentially form surface-associated communities, such as biofilms and colonies, that are well protected against hostile environments and, when growing as pathogens, against the host immune system. However, the molecular mechanisms underlying the spatiotemporal development and environmental resistance of biofilms and colonies remain largely unknown. In this paper, we show that a biofilm yeast colony is a finely tuned, complex multicellular organism in which specialized cells jointly execute multiple protection strategies. These include a Pdr1p-regulated mechanism whereby multidrug resistance transporters Pdr5p and Snq2p expel external compounds solely within the surface cell layers as well as developmentally regulated production by internal cells of a selectively permeable extracellular matrix. The two mechanisms act in concert during colony development, allowing growth of new cell generations in a well-protected internal cavity of the colony. Colony architecture is strengthened by intercellular fiber connections.

• MeSH
• ABC transportéry genetika   metabolismus   MeSH
• biofilmy růst a vývoj   MeSH
• biologické modely MeSH
• delece genu MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• extracelulární matrix fyziologie   MeSH
• galaktokinasa genetika   metabolismus   MeSH
• galaktosa metabolismus   MeSH
• hydroxymethylglutaryl-CoA-reduktasy genetika   metabolismus   MeSH
• měď metabolismus   MeSH
• membránové glykoproteiny genetika   metabolismus   MeSH
• metalothionein genetika   metabolismus   MeSH
• oxaziny metabolismus   MeSH
• permeabilita MeSH
• profiliny genetika   MeSH
• proteiny buněčného cyklu genetika   MeSH
• proteiny spojené s mnohočetnou rezistencí k lékům genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   růst a vývoj   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Reports describing production of reactive oxygen species in neonatal heart are missing. As lysyl oxidase is potentially important source of H(2)O(2), we studied its role during ontogenic development of rat heart. H(2)O(2) was detected in thin sections of developing rat heart by fluorescence microscopy with the use of fluorescence probe 2'-7'-dichlorofluorescin. The experimental design comprised foetuses 21 days after conception, and then the animals sampled on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. Since the day 4 on, H(2)O(2) was produced only extracellularly up to the day 15, between days 30 and 60 intracellular production was detected as well, and in 7-month-old animals only extracellular production was observed. The specific inhibitors of lysyl oxidase almost completely quenched the H(2)O(2)-dependent fluorescence. Starting from day 7, blue autofluorescence specific to oxidized proteins developed in the vessel wall. Intracellular blue autofluorescence specific to autoxidation products developed after day 30. Chloroform extraction diminished the intracellular blue fluorescence, leaving the extracellular fluorescence intact. This confirmed the protein nature of the fluorophores. Lysyl oxidase is significant source of H(2)O(2) in the heart vessel wall during development and H(2)O(2) oxidatively modifies elastin producing protein blue autofluorescence.

• MeSH
• chloroform MeSH
• elastin metabolismus   MeSH
• fluoresceiny metabolismus   MeSH
• fluorescence MeSH
• fluorescenční barviva metabolismus   MeSH
• krysa rodu rattus MeSH
• lysyloxidasa metabolismus   MeSH
• myokard enzymologie   metabolismus   MeSH
• oxaziny metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• potkani Wistar MeSH
• srdce růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVES: The aim of this study was to assess the effect of various flavonoids on the NADPH:cytochrome P450 oxidoreductase (CYPOR) activity in respect of the reduction of different electron acceptors as well as to study an impact of flavonoids on monooxygenation of a model substrate of cytochrome P450 (CYP). DESIGN: The modulation of CYPOR activity was determined spectrophotometrically based on the time course of the reduction of different electron acceptors. The CYP reduction was monitored via its complex formation with CO, having pronounced the absorption maximum at 450 nm. Finally, effect of CYPOR stimulation by 7,8benzoflavone (ANF) on 7pentoxyresorufin Odepentylation was assayed in the microsomal monooxygenation system using the fluorimetric detection of formed resorufin. RESULTS: The stimulation of CYPOR activity via ANF was found to be associated with following electron acceptors: cytochrome c, potassium ferricyanide, cytochrome b5, but not with CYP. Surprisingly, 5,6benzoflavone, a position isomer of ANF, was ineffective in the CYPOR stimulation as well as the other flavonoids tested. In microsomal preparations, ANF did not markedly enhance the reaction rate of monooxygenation of CYP2B4 model substrate. CONCLUSION: Our results document that among all of the tested flavonoids only ANF is able to stimulate CYPOR activity, however, the ANF-mediated stimulation of CYPOR has no impact on the oxidative metabolism catalyzed by CYP system.

• MeSH
• antioxidancia farmakologie   MeSH
• benzoflavony chemie   metabolismus   MeSH
• beta-naftoflavon chemie   metabolismus   MeSH
• časové faktory MeSH
• cytochromy b5 metabolismus   MeSH
• cytochromy c metabolismus   MeSH
• ferrikyanidy metabolismus   MeSH
• flavonoidy chemie   farmakologie   MeSH
• fluorometrie MeSH
• králíci MeSH
• kyselina lipoová farmakologie   MeSH
• mikrozomy účinky léků   enzymologie   metabolismus   MeSH
• NADPH-cytochrom c-reduktasa metabolismus   MeSH
• oxaziny metabolismus   MeSH
• oxid uhelnatý metabolismus   MeSH
• oxidace-redukce účinky léků   MeSH
• spektrofotometrie MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH