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.

• Klíčová slova
• Exosomes, Lipidomics, Lipids, Liquid chromatography, Mass spectrometry, Plasma, Supercritical fluid chromatography,
• 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
• Názvy látek
• diglyceridy MeSH
• fosfatidylcholiny MeSH
• lysofosfatidylcholiny MeSH
• triglyceridy MeSH

The role of adipokinetic hormone (Drome-AKH) in maintaining the levels of basic nutrients, under starvation conditions, was studied using Drosophila melanogaster mutants with AKH deficiency (Akh1) and AKH abundance (EE-Akh). Our results showed lipids as the main energy reserve in Drosophila, and their physiological level and metabolism were shown to be under the control of AKH. AKH abundance in the body resulted in lower levels of triacylglycerols and diacylglycerols than in the controls, probably due to a more intensive metabolism; interestingly, there was a disproportional representation of fatty acids in triacylglycerols and diacylglycerols in Drosophila. Lower level of glycogen and its partial control by AKH suggest its lesser role as the storage substance. However, maintenance of free carbohydrate level in Drosophila seemed to be critical; when glycogen stores are exhausted, carbohydrates are synthesized from other sources. Protein levels and their alterations, under starvation, did not seem controlled by AKH. AKH-deficient flies were more resistant while AKH-abundant flies were more sensitive to starvation; females were found to be more resistant than males, regardless of the AKH level, probably due to higher body mass and higher amount of nutrients. However, in accordance with the level of all nutrients, that of AKH also gradually decreased with prolonged starvation.

• Klíčová slova
• Drosophila melanogaster mutants, adipokinetic hormone, homeostasis, metabolism, mortality, nutrients, starvation,
• MeSH
• analýza přežití MeSH
• delece genu MeSH
• diglyceridy metabolismus   MeSH
• Drosophila melanogaster genetika   MeSH
• ELISA MeSH
• energetický metabolismus * MeSH
• geneticky modifikovaná zvířata MeSH
• glykogen metabolismus   MeSH
• hladovění metabolismus   MeSH
• hmyzí hormony genetika   metabolismus   MeSH
• křížení genetické MeSH
• kyselina pyrrolidonkarboxylová analogy a deriváty   metabolismus   MeSH
• metabolismus lipidů * MeSH
• metabolismus sacharidů * MeSH
• náhodné rozdělení MeSH
• oligopeptidy genetika   metabolismus   MeSH
• pohlavní dimorfismus MeSH
• proteiny Drosophily genetika   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• triglyceridy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DAKH peptide MeSH Prohlížeč
• diglyceridy MeSH
• glykogen MeSH
• hmyzí hormony MeSH
• kyselina pyrrolidonkarboxylová MeSH
• oligopeptidy MeSH
• proteiny Drosophily MeSH
• triglyceridy MeSH

Using Brassica napus roots we observed statistically significant increase in alternative respiratory pathway in response to exogenous 24-epibrassinolide (EBL) under optimal conditions and salinity. Also we observed activation of phospholipid signaling under the same conditions in response to EBL by measuring levels of lipid second messengers - diacylglycerol (DAG) and phosphatidic acid (PA). We found that brassinosteroids cause closure of stomata in isolated leaf disks while inhibitors of alternative oxidase cancelled these effects. This study demonstrates that BRs activate total respiration rate, alternative respiratory pathway, production of PA and DAG, stimulate stomata closure and growth under optimal conditions and salinity. Also, specific inhibitor of brassinosteroids biosynthesis decreased alternative respiratory pathway and production of lipid messengers in rape plants.

• Klíčová slova
• Alternative oxidase, Brassica napus, Brassinosteroids, Diacylglycerol, Salinity, Stomata cells,
• MeSH
• Brassica napus účinky léků   enzymologie   metabolismus   MeSH
• brassinosteroidy farmakologie   MeSH
• diglyceridy metabolismus   MeSH
• kořeny rostlin účinky léků   enzymologie   metabolismus   MeSH
• kyseliny fosfatidové metabolismus   MeSH
• listy rostlin účinky léků   enzymologie   metabolismus   MeSH
• mitochondriální proteiny metabolismus   MeSH
• oxidoreduktasy metabolismus   MeSH
• průduchy rostlin účinky léků   enzymologie   metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• steroidy heterocyklické farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alternative oxidase MeSH Prohlížeč
• brassinolide MeSH Prohlížeč
• brassinosteroidy MeSH
• diglyceridy MeSH
• kyseliny fosfatidové MeSH
• mitochondriální proteiny MeSH
• oxidoreduktasy MeSH
• rostlinné proteiny MeSH
• steroidy heterocyklické MeSH

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.

• MeSH
• Caenorhabditis elegans enzymologie   metabolismus   účinky záření   MeSH
• diglyceridy chemie   metabolismus   účinky záření   MeSH
• fotochemické procesy účinky záření   MeSH
• myši MeSH
• optické jevy MeSH
• proteinkinasa C chemie   metabolismus   účinky záření   MeSH
• signální transdukce účinky záření   MeSH
• ultrafialové záření * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2-diacylglycerol MeSH Prohlížeč
• diglyceridy MeSH
• proteinkinasa C MeSH

The first indication of the aluminum (Al) toxicity in plants growing in acidic soils is the cessation of root growth, but the detailed mechanism of Al effect is unknown. Here we examined the impact of Al stress on the activity of non-specific phospholipase C (NPC) in the connection with the processes related to the plasma membrane using fluorescently labeled phosphatidylcholine. We observed a rapid and significant decrease of labeled diacylglycerol (DAG), product of NPC activity, in Arabidopsis seedlings treated with AlCl₃. Interestingly, an application of the membrane fluidizer, benzyl alcohol, restored the level of DAG during Al treatment. Our observations suggest that the activity of NPC is affected by Al-induced changes in plasma membrane physical properties.

• Klíčová slova
• Arabidopsis thaliana, BA, benzyl alcohol, BODIPY, BODIPY, 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene, BY-2, Bright Yellow 2, DAG, diacylglycerol, HP-TLC, high-performance thin-layer chromatography, MS, Murashige-Skoog, NPC, non-specific phospholipase C, PA, phosphatidic acid, PC, phosphatidylcholine, PC-PLC, phosphatidylcholine-specific phospholipase C, PI-PLC, phosphatidylinositol-specific phospholipase C, PIP2, phosphatidylinositol 4, 5-bisphosphate, PLD, phospholipase D, PM, plasma membrane., aluminum toxicity, benzyl alcohol, diacylglycerol, membrane fluidity, non-specific phospholipase C,
• MeSH
• Arabidopsis účinky léků   enzymologie   MeSH
• benzylalkohol farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• diglyceridy metabolismus   MeSH
• fosfolipasy typu C metabolismus   MeSH
• hliník farmakologie   MeSH
• ionty MeSH
• kořeny rostlin účinky léků   metabolismus   MeSH
• semenáček účinky léků   metabolismus   MeSH
• sloučeniny boru metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2-diacylglycerol MeSH Prohlížeč
• 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene MeSH Prohlížeč
• benzylalkohol MeSH
• diglyceridy MeSH
• fosfolipasy typu C MeSH
• hliník MeSH
• ionty MeSH
• sloučeniny boru MeSH

Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells. The biosensor consists of a PA-binding domain of yeast SNARE Spo20p fused to fluorescent proteins. Live-cell imaging of PA dynamics in transiently transformed tobacco (Nicotiana tabacum) pollen tubes was performed using confocal laser scanning microscopy. In growing pollen tubes, PA shows distinct annulus-like fluorescence pattern in the plasma membrane behind the extreme tip. Coexpression studies with markers for other plasmalemma signaling lipids phosphatidylinositol 4,5-bisphosphate and diacylglycerol revealed limited colocalization at the shoulders of the apex. PA distribution and concentrations show distinct responses to various lipid signaling inhibitors. Fluorescence recovery after photobleaching (FRAP) analysis suggests high PA turnover in the plasma membrane. Our data show that a biosensor based on the Spo20p-PA binding domain is suitable for live-cell imaging of PA also in plant cells. In tobacco pollen tubes, distinct subapical PA maximum corroborates its involvement in the regulation of endocytosis and actin dynamics.

• Klíčová slova
• Nicotiana tabacum (tobacco), Spo20p, live-cell microscopy, phosphatidic acid (PA), phospholipase D, pollen tube,
• MeSH
• biosenzitivní techniky metody   MeSH
• buněčná membrána chemie   metabolismus   MeSH
• diglyceridy metabolismus   MeSH
• fluorescence MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfolipasa D metabolismus   MeSH
• fotovybělování MeSH
• kyseliny fosfatidové analýza   metabolismus   MeSH
• počítačové zpracování obrazu MeSH
• proteiny Qb-SNARE genetika   metabolismus   MeSH
• proteiny Qc-SNARE genetika   metabolismus   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• tabák cytologie   metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diglyceridy MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• fosfolipasa D MeSH
• kyseliny fosfatidové MeSH
• proteiny Qb-SNARE MeSH
• proteiny Qc-SNARE MeSH
• rekombinantní fúzní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Spo20 protein, S cerevisiae MeSH Prohlížeč

Plant nonspecific phospholipase C (NPC) is a recently described enzyme which plays a role in membrane rearrangement during phosphate starvation. It is also involved in responses of plants to brassinolide, abscisic acid (ABA), elicitors, and salt. The NPC activity is decreased in cells treated with aluminum. In the case of salt stress, the molecular mechanism of NPC action is based on accumulation of diacylglycerol (DAG) by hydrolysis of phospholipids and conversion of DAG, the product of NPC activity, to phosphatidic acid (PA) that participates in ABA signaling pathways. Here we describe a step-by-step protocol, which can be used to determine in situ or in vitro NPC activity. Determination is based on quantification of fluorescently labeled DAG as a product of cleavage of the fluorescently labeled substrate lipid, phosphatidylcholine. High-performance thin-layer chromatography is used for separation of fluorescent DAG. The spot is visualized with a laser scanner and the relative amounts of fluorescent DAG are quantified using imaging software.

• MeSH
• chromatografie na tenké vrstvě MeSH
• diglyceridy metabolismus   MeSH
• enzymatické testy metody   MeSH
• fluorescenční barviva metabolismus   MeSH
• fosfolipasy typu C metabolismus   MeSH
• referenční standardy MeSH
• rostliny enzymologie   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2-diacylglycerol MeSH Prohlížeč
• diglyceridy MeSH
• fluorescenční barviva MeSH
• fosfolipasy typu C MeSH

We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

• MeSH
• asparagin farmakologie   MeSH
• autofagie * účinky léků   MeSH
• chlorochin farmakologie   MeSH
• diglyceridy metabolismus   MeSH
• hepatocyty účinky léků   metabolismus   patologie   MeSH
• játra účinky léků   metabolismus   patologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• kyseliny mastné neesterifikované metabolismus   MeSH
• lipolýza * MeSH
• lipoproteiny VLDL metabolismus   MeSH
• lyzozomy účinky léků   metabolismus   patologie   MeSH
• membránový protein 2 asociovaný s lyzozomy metabolismus   MeSH
• oxidace-redukce MeSH
• potkani Wistar MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• sirolimus farmakologie   MeSH
• sterolesterasa metabolismus   MeSH
• triglyceridy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• asparagin MeSH
• chlorochin MeSH
• diglyceridy MeSH
• kyseliny mastné neesterifikované MeSH
• LC3 protein, rat MeSH Prohlížeč
• lipoproteiny VLDL MeSH
• membránový protein 2 asociovaný s lyzozomy MeSH
• proteiny asociované s mikrotubuly MeSH
• sirolimus MeSH
• sterolesterasa MeSH
• triglyceridy MeSH

The diacylglycerols (DG) molecular species and their fatty acid (FA) composition were investigated by electrospray mass spectrometry (ESI-MS) and by gas chromatography with flame ionisation detection (GC-FID) in haemolymph of Locusta migratoria after application of adipokinetic hormones Locmi-AKH-I, -II and -III. The analyses showed (1) a heterogeneous distribution of individual DGs in haemolymph after the hormone application. The results revealed that mobilization of the DGs is molecular species-specific with the highest proportion of 34:1 DG (16:0/18:1 - mw 594Da) for all Locmi-AKHs bearing palmitic acid (C16:0) and oleic acid (C18:1) residues, and forming about 20% of the total mobilized DG content. (2) Analysis of fat body triacylglycerols revealed that all Locmi-AKHs mobilize the DGs selectively with the preference of those possessing the C18 and C16 FAs. The fat body FAs with carbon chain longer than 18 did not participate in the mobilization. (3) A distribution of FAs in the DG structures obtained by LC/ESI-MS, and FA analysis by GC-FID after transmethylation indicated a certain degree of Locmi-AKH selectivity toward the mobilized DGs and hence the FAs. The Locmi-AKH-I significantly prefers mobilization of DGs containing unsaturated FAs, while Locmi-AKH-II and -III prefer mobilization of saturated FAs.

• MeSH
• diglyceridy metabolismus   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• hmyzí hormony metabolismus   MeSH
• hmyzí proteiny metabolismus   MeSH
• kyselina pyrrolidonkarboxylová analogy a deriváty   metabolismus   MeSH
• Locusta migratoria metabolismus   MeSH
• oligopeptidy metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adipokinetic hormone MeSH Prohlížeč
• diglyceridy MeSH
• hmyzí hormony MeSH
• hmyzí proteiny MeSH
• kyselina pyrrolidonkarboxylová MeSH
• oligopeptidy MeSH

The mechanism of action by which insulin increases phosphatidic acid (PA) and diacylglycerol (DAG) levels was investigated in cultured hepatoma cells (HEPG2). Insulin stimulated phosphatidylcholine (PC) and phosphatidyl-inositol (PI) degradation through the activation of specific phospholipases C (PLC). The DAG increase appears to be biphasic. The early DAG production seems to be due to PI breakdown, probably through phosphatidyl-inositol-3-kinase (PI3K) involvement, whereas the delayed DAG increase is derived directly from the PC-PLC activity. The absence of phospholipase D (PLD) involvement was confirmed by the lack of PC-derived phosphatidylethanol production. Experiments performed in the presence of R59022, an inhibitor of DAG-kinase, indicated that PA release is the result of the DAG-kinase activity on the DAG produced in the early phase of insulin action.

• MeSH
• cholin metabolismus   MeSH
• chromatografie na tenké vrstvě MeSH
• diglyceridy metabolismus   MeSH
• extracelulární prostor účinky léků   metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosfolipidy fyziologie   MeSH
• hepatocelulární karcinom metabolismus   MeSH
• inzulin farmakologie   fyziologie   MeSH
• kyseliny fosfatidové metabolismus   MeSH
• lidé MeSH
• metabolismus lipidů MeSH
• nádorové buněčné linie MeSH
• nádory jater metabolismus   MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cholin MeSH
• diglyceridy MeSH
• fosfatidylinositol-3-kinasy MeSH
• fosfolipidy MeSH
• inzulin MeSH
• kyseliny fosfatidové MeSH
• tetradekanoylforbolacetát MeSH