• MeSH
• cytidindifosfátcholin * aplikace a dávkování   terapeutické užití   MeSH
• epizodická paměť MeSH
• fosfatidylcholiny metabolismus   MeSH
• kognice * účinky léků   MeSH
• kognitivní dysfunkce farmakoterapie   prevence a kontrola   MeSH
• kognitivní stárnutí * MeSH
• lidé středního věku MeSH
• lidé MeSH
• randomizované kontrolované studie jako téma MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• výsledek terapie MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fosfatidylcholiny chemie   metabolismus   MeSH
• gating iontového kanálu genetika   MeSH
• genetické vektory chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• interakční proteinové domény a motivy MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• lidé MeSH
• liposomy chemie   metabolismus   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• metoda terčíkového zámku MeSH
• mutace MeSH
• nádorové proteiny chemie   genetika   metabolismus   MeSH
• protein ORAI1 chemie   genetika   metabolismus   MeSH
• protein STIM1 chemie   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• reportérové geny MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• vápník metabolismus   MeSH
• vápníková signalizace * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Most in vivo 31P MR studies are realized on 3T MR systems that provide sufficient signal intensity for prominent phosphorus metabolites. The identification of these metabolites in the in vivo spectra is performed by comparing their chemical shifts with the chemical shifts measured in vitro on high-field NMR spectrometers. To approach in vivo conditions at 3T, a set of phantoms with defined metabolite solutions were measured in a 3T whole-body MR system at 7.0 and 7.5 pH, at 37 °C. A free induction decay (FID) sequence with and without 1H decoupling was used. Chemical shifts were obtained of phosphoenolpyruvate (PEP), phosphatidylcholine (PtdC), phosphocholine (PC), phosphoethanolamine (PE), glycerophosphocholine (GPC), glycerophosphoetanolamine (GPE), uridine diphosphoglucose (UDPG), glucose-6-phosphate (G6P), glucose-1-phosphate (G1P), 2,3-diphosphoglycerate (2,3-DPG), nicotinamide adenine dinucleotide (NADH and NAD+), phosphocreatine (PCr), adenosine triphosphate (ATP), adenosine diphosphate (ADP), and inorganic phosphate (Pi). The measured chemical shifts were used to construct a basis set of 31P MR spectra for the evaluation of 31P in vivo spectra of muscle and the liver using LCModel software (linear combination model). Prior knowledge was successfully employed in the analysis of previously acquired in vivo data.

• MeSH
• adenosindifosfát metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• fosfatidylcholiny metabolismus   MeSH
• fosfatidylethanolaminy metabolismus   MeSH
• fosfáty metabolismus   MeSH
• fosfor metabolismus   MeSH
• játra metabolismus   MeSH
• kosterní svaly metabolismus   MeSH
• lidé MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• pilotní projekty MeSH
• software * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Lipophosphonoxins (LPPOs) are small modular synthetic antibacterial compounds that target the cytoplasmic membrane. First-generation LPPOs (LPPO I) exhibit an antimicrobial activity against Gram-positive bacteria; however they do not exhibit any activity against Gram-negatives. Second-generation LPPOs (LPPO II) also exhibit broadened activity against Gram-negatives. We investigated the reasons behind this different susceptibility of bacteria to the two generations of LPPOs using model membranes and the living model bacteria Bacillus subtilis and Escherichia coli. We show that both generations of LPPOs form oligomeric conductive pores and permeabilize the bacterial membrane of sensitive cells. LPPO activity is not affected by the value of the target membrane potential, and thus they are also active against persister cells. The insensitivity of Gram-negative bacteria to LPPO I is probably caused by the barrier function of the outer membrane with LPS. LPPO I is almost incapable of overcoming the outer membrane in living cells, and the presence of LPS in liposomes substantially reduces their activity. Further, the antimicrobial activity of LPPO is also influenced by the phospholipid composition of the target membrane. A higher proportion of phospholipids with neutral charge such as phosphatidylethanolamine or phosphatidylcholine reduces the LPPO permeabilizing potential.

• MeSH
• antibakteriální látky chemická syntéza   farmakologie   MeSH
• Bacillus subtilis chemie   cytologie   účinky léků   MeSH
• Escherichia coli chemie   cytologie   účinky léků   MeSH
• fosfatidylcholiny analýza   metabolismus   MeSH
• fosfatidylethanolaminy analýza   metabolismus   MeSH
• kationické antimikrobiální peptidy chemická syntéza   farmakologie   MeSH
• lipidové dvojvrstvy MeSH
• membránové potenciály účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• permeabilita buněčné membrány MeSH
• vnější bakteriální membrána chemie   účinky léků   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs' ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.

• MeSH
• buňky Hep G2 MeSH
• fokální adhezní kinasa 1 antagonisté a inhibitory   nedostatek   genetika   MeSH
• fosfatidylcholiny metabolismus   MeSH
• genový knockdown MeSH
• hepatocelulární karcinom genetika   metabolismus   patologie   MeSH
• histony antagonisté a inhibitory   nedostatek   genetika   MeSH
• lidé MeSH
• lipidomika MeSH
• lysofosfatidylcholiny metabolismus   MeSH
• metabolismus lipidů * genetika   MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky metabolismus   patologie   MeSH
• nádory jater genetika   metabolismus   patologie   MeSH
• regulace genové exprese u nádorů MeSH
• sekvenování transkriptomu MeSH
• sfingomyeliny metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Membrane phospholipids, including phosphatidylcholine (PC) and phosphatidylethanolamine (PE), consist of distinct fatty acids occupying the sn-1 and sn-2 positions, reflecting the highly regulated nature of lipid biosynthesis. However, little is known about the influence of dietary lipids on the positional nature of fatty acids in tissues, including the enrichment of omega-3 polyunsaturated fatty acid (PUFA) in chicken egg yolk phospholipids. This study was undertaken to characterize the PC and PE species in egg lipids derived from Lohmann hens (n=10/treatment) randomly allocated to either a control (no supplementation), a flaxseed oil (FO) or a marine algal oil (MA) diet. Each of the FO or MA diets supplied three levels of total omega-3 PUFA (0.20, 0.40 and 0.60% of diet) that were provided for 6 weeks. A combination of multiplexed mass spectrometry (MS) experiments are used to determine total, isobaric, and position molecules for PC and PE in egg yolk. The distribution of phospholipids in the yolk was predominantly PC over PE (~72 vs. 23%, respectively) across treatments. The longer chain PUFA existed in the sn-2 position in the PC and PE. Although docosahexaenoic acid (22:6) formed isomers with fatty acids 16:0, 18:0 and 18:1; it was preferentially enriched in the egg in combination with 16:0 with both the FO and MA-fed groups in both lipid pools. All 22:6-containing isomers were enriched by ~2-fold more (P < 0.0001) with MA than FO, however, all isomers exhibited a plateau with the FO-fed group. In addition, the MS analyses of PCs revealed several isobaric species containing eicosapentaenoic acid (EPA, 20:5), however, in the PE, EPA formed only one isomer (i.e. in combination with 16:0). These results may assist to elucidate potential aspects regulating the limited enrichment of omega-3 PUFA, particularly EPA and docosahexaenoic acid (22:6) in chicken eggs.

• MeSH
• biomasa * MeSH
• fosfatidylcholiny metabolismus   MeSH
• fosfatidylethanolaminy metabolismus   MeSH
• krmivo pro zvířata * MeSH
• kur domácí metabolismus   MeSH
• kyseliny mastné omega-3 farmakologie   MeSH
• lipidomika MeSH
• lněný olej farmakologie   MeSH
• vaječný žloutek metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

The literature reports on cationic and anionic phthalocyanines (Pcs) for photodynamic therapy suggest systematically significant differences in activity. In this work, ten different zinc(II) Pcs with carboxylate functions or quaternary nitrogens (hydrophilic anionic, hydrophilic cationic, amphiphilic anionic, and amphiphilic cationic) were investigated, with the aim of revealing reasons for such differences. In vitro assays on HeLa, MCF-7, and HCT-116 cells confirmed higher photoactivity for cationic Pcs (EC50 ∼ 3-50 nM) than for anionic Pcs (EC50 ∼ 0.3-10 μM), the latter being additionally significantly more active in serum-free medium. The environmental pH, binding to serum proteins, interaction with biomembranes, differences in subcellular localization, and relocalization after irradiation were found to be the main factors contributing to the generally lower photoactivity of anionic Pcs than that of the cationic derivatives. This result is not limited only to the presented derivatives and should be considered in the design of novel photosensitizers.

• MeSH
• antitumorózní látky chemická syntéza   metabolismus   farmakologie   účinky záření   MeSH
• fosfatidylcholiny chemie   metabolismus   MeSH
• fotochemoterapie MeSH
• fotosenzibilizující látky chemická syntéza   metabolismus   farmakologie   účinky záření   MeSH
• indoly chemická syntéza   metabolismus   farmakologie   účinky záření   MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• liposomy chemie   metabolismus   MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• sérový albumin hovězí metabolismus   MeSH
• singletový kyslík metabolismus   MeSH
• světlo MeSH
• zinek chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.

• MeSH
• diglyceridy krev   izolace a purifikace   metabolismus   MeSH
• dospělí MeSH
• exozómy chemie   metabolismus   MeSH
• fosfatidylcholiny krev   izolace a purifikace   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipidomika metody   MeSH
• lysofosfatidylcholiny krev   izolace a purifikace   metabolismus   MeSH
• metabolismus lipidů * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• superkritická fluidní chromatografie metody   MeSH
• triglyceridy krev   izolace a purifikace   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cyanidin and its O-glycosides have many important physiological functions in plants and beneficial effects on human health. Their biological activity is not entirely clear and depends on the structure of the molecule, in particular, on the number and type of sugar substituents. Therefore, in this study the detailed structure-activity relationship (SARs) of the anthocyanins/anthocyanidins in relation to their interactions with lipid bilayer was determined. On the basis of their antioxidant activity and the changes induced by them in size and Zeta potential of lipid vesicles, and mobility and order of lipid acyl chains, the impact of the number and type of sugar substituents on the biological activity of the compounds was evaluated. The obtained results have shown, that 3-O-glycosylation changes the interaction of cyanidin with lipid bilayer entirely. The 3-O-glycosides containing a monosaccharide induces greater changes in physical properties of the lipid membrane than those containing disaccharides. The presence of additional sugar significantly reduces glycoside interaction with model lipid membrane. Furthermore, O-glycosylation alters the ability of cyanidin to scavenge free radicals. This alteration depends on the type of free radicals and the sensitivity of the method used for their determination.

• MeSH
• anthokyaniny metabolismus   MeSH
• fosfatidylcholiny metabolismus   MeSH
• glykosylace MeSH
• peroxid vodíku metabolismus   MeSH
• peroxidace lipidů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH

Background and Aims: The non-specific phospholipase C (NPC) is a new member of the plant phospholipase family that reacts to abiotic environmental stresses, such as phosphate deficiency, high salinity, heat and aluminium toxicity, and is involved in root development, silicon distribution and brassinolide signalling. Six NPC genes (NPC1-NPC6) are found in the Arabidopsis genome. The NPC2 isoform has not been experimentally characterized so far. Methods: The Arabidopsis NPC2 isoform was cloned and heterologously expressed in Escherichia coli. NPC2 enzyme activity was determined using fluorescent phosphatidylcholine as a substrate. Tissue expression and subcellular localization were analysed using GUS- and GFP-tagged NPC2. The expression patterns of NPC2 were analysed via quantitative real-time PCR. Independent homozygous transgenic plant lines overexpressing NPC2 under the control of a 35S promoter were generated, and reactive oxygen species were measured using a luminol-based assay. Key Results: The heterologously expressed protein possessed phospholipase C activity, being able to hydrolyse phosphatidylcholine to diacylglycerol. NPC2 tagged with GFP was predominantly localized to the Golgi apparatus in Arabidopsis roots. The level of NPC2 transcript is rapidly altered during plant immune responses and correlates with the activation of multiple layers of the plant defence system. Transcription of NPC2 decreased substantially after plant infiltration with Pseudomonas syringae, flagellin peptide flg22 and salicylic acid treatments and expression of the effector molecule AvrRpm1. The decrease in NPC2 transcript levels correlated with a decrease in NPC2 enzyme activity. NPC2-overexpressing mutants showed higher reactive oxygen species production triggered by flg22. Conclusions: This first experimental characterization of NPC2 provides new insights into the role of the non-specific phospholipase C protein family. The results suggest that NPC2 is involved in the response of Arabidopsis to P. syringae attack.

• MeSH
• Arabidopsis enzymologie   imunologie   mikrobiologie   MeSH
• fosfatidylcholiny metabolismus   MeSH
• fosfolipasy typu C fyziologie   MeSH
• Golgiho aparát enzymologie   MeSH
• imunita rostlin fyziologie   MeSH
• klonování DNA MeSH
• konfokální mikroskopie MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• nemoci rostlin imunologie   mikrobiologie   MeSH
• proteiny huseníčku fyziologie   MeSH
• protoplasty enzymologie   MeSH
• Pseudomonas syringae * MeSH
• reaktivní formy kyslíku MeSH
• regulace genové exprese u rostlin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH