A combination of two chromatographic and two enzymatic methods was used for the analysis of molecular species of lipids from Gram-positive bacteria of the genus Kocuria. Gram-positive bacteria contain a majority of branched fatty acids (FAs), especially iso- and/or anteiso-FAs. Two strains K. rhizophila were cultivated at three different temperatures (20, 28, and 37°C) and the majority phospholipid, i.e., the mixture of molecular species of phosphatidylglycerols (PGs) was separated by means of hydrophilic interaction liquid chromatography (HILIC). After enzymatic hydrolysis of PGs by phospholipase C and derivatization of the free OH group, the sn-1,2-diacyl-3-acetyl triacylglycerols (AcTAGs) were separated by reversed phase HPLC. Molecular species such as i-15:0/i-15:0/2:0, ai-15:0/ai-15:0/2:0, and 15:0/15:0/2:0 (straight chains) were identified by liquid chromatography-positive electrospray ionization mass spectrometry. The tandem mass spectra of both standards and natural compounds containing iso, anteiso and straight chain FAs with the same carbons were identical. Therefore, for identification of the ratio of two regioisomers, i.e. i-15:0/ai-15:0/2:0 vs. ai-15:0/i-15:0/2:0, they were cleavage by pancreatic lipase. The mixture of free fatty acids (FFAs) and 2-monoacylglycerols (2-MAGs) was obtained. After their separation by TLC and esterification and/or transesterification, the fatty acid methyl esters were quantified by GC-MS and thus the ratio of regioisomers was determined. It has been shown that the ratio of PG (containing as majority i-15: 0 / i-15: 0, i-15: 0 / ai-15: 0 and / or ai-15: 0 / i-15: 0 and ai-15: 0 / ai-15: 0 molecular species) significantly affected the membrane flow of bacterial cells cultured at different temperatures.

• MeSH
• Chemistry Techniques, Analytical methods   MeSH
• Chromatography, Liquid * MeSH
• Diglycerides chemistry   isolation & purification   MeSH
• Phospholipids chemistry   MeSH
• Spectrometry, Mass, Electrospray Ionization * MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Fatty Acids chemistry   MeSH
• Micrococcaceae chemistry   MeSH
• Gas Chromatography-Mass Spectrometry MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Journal Article 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
• Diglycerides blood   isolation & purification   metabolism   MeSH
• Adult MeSH
• Exosomes chemistry   metabolism   MeSH
• Phosphatidylcholines blood   isolation & purification   metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Lipidomics methods   MeSH
• Lysophosphatidylcholines blood   isolation & purification   metabolism   MeSH
• Lipid Metabolism * MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Chromatography, Supercritical Fluid methods   MeSH
• Triglycerides blood   isolation & purification   metabolism   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Healthy Volunteers MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• MeSH
• Survival Analysis MeSH
• Gene Deletion MeSH
• Diglycerides metabolism   MeSH
• Drosophila melanogaster genetics   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Energy Metabolism * MeSH
• Animals, Genetically Modified MeSH
• Glycogen metabolism   MeSH
• Starvation metabolism   MeSH
• Insect Hormones genetics   metabolism   MeSH
• Crosses, Genetic MeSH
• Pyrrolidonecarboxylic Acid analogs & derivatives   metabolism   MeSH
• Lipid Metabolism * MeSH
• Carbohydrate Metabolism * MeSH
• Random Allocation MeSH
• Oligopeptides genetics   metabolism   MeSH
• Sex Characteristics MeSH
• Drosophila Proteins genetics   metabolism   MeSH
• Reproducibility of Results MeSH
• Triglycerides metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article 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.

• MeSH
• Brassica napus drug effects   enzymology   metabolism   MeSH
• Brassinosteroids pharmacology   MeSH
• Diglycerides metabolism   MeSH
• Plant Roots drug effects   enzymology   metabolism   MeSH
• Phosphatidic Acids metabolism   MeSH
• Plant Leaves drug effects   enzymology   metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Oxidoreductases metabolism   MeSH
• Plant Stomata drug effects   enzymology   metabolism   MeSH
• Plant Proteins metabolism   MeSH
• Steroids, Heterocyclic pharmacology   MeSH
• Publication type
• Journal Article MeSH

Tiger nut (Cyperus esculentus L.) is a crop traditionally grown in Valencia Region (Spain) and other temperate and tropical regions in the world, where its tubers are commonly consumed as tiger nut milk (horchata). Because of their nutritive potential and original taste, these products are beginning to spread internationally and, as consequence, analytical procedures to assess nutritional profiles, quality control issues are acquiring increasing relevance. The main objective of this study was to use an advance analytical method and chemometrics tools to determine if the ultra-high temperature (UHT) treatment necessary to extend the shelf life of tiger nut milk would affect the profile of nutrients when compared to fresh product. A cold solvent extraction followed by liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) was used. Datasets obtained from UHT and fresh tiger nut milk data were analyzed through an untargeted metabolomics approach to compare chemical patterns, highlighting differences in citric acid esters of mono- diglycerides (CITREM) and monoacylglycerol (MAG) used as emulsifiers of UHT products, and a remarkably higher abundance of biotin, phosphatidic acid (PA) and L-arginine in fresh products. These results showed that untargeted metabolomics through high resolution tandem mass spectrometry allowed fine differences between food products to be found, therefore, the nutrient lost caused by UHT treatment was clearly discerned.

• MeSH
• Amino Acids analysis   MeSH
• Principal Component Analysis MeSH
• Sugars analysis   chemistry   MeSH
• Cyperus chemistry   metabolism   MeSH
• Diglycerides chemistry   MeSH
• Emulsifying Agents chemistry   MeSH
• Food Quality * MeSH
• Fatty Acids analysis   chemistry   MeSH
• Metabolomics * MeSH
• Monoglycerides chemistry   MeSH
• Plant Oils analysis   chemistry   metabolism   MeSH
• Cluster Analysis MeSH
• Tandem Mass Spectrometry MeSH
• Vitamins analysis   MeSH
• Hot Temperature MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Journal Article 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 enzymology   metabolism   radiation effects   MeSH
• Diglycerides chemistry   metabolism   radiation effects   MeSH
• Photochemical Processes radiation effects   MeSH
• Mice MeSH
• Optical Phenomena MeSH
• Protein Kinase C chemistry   metabolism   radiation effects   MeSH
• Signal Transduction radiation effects   MeSH
• Ultraviolet Rays * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article 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.

• MeSH
• Arabidopsis drug effects   enzymology   MeSH
• Benzyl Alcohol pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Diglycerides metabolism   MeSH
• Type C Phospholipases metabolism   MeSH
• Aluminum pharmacology   MeSH
• Ions MeSH
• Plant Roots drug effects   metabolism   MeSH
• Seedlings drug effects   metabolism   MeSH
• Boron Compounds metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• MeSH
• Biosensing Techniques methods   MeSH
• Cell Membrane chemistry   metabolism   MeSH
• Diglycerides metabolism   MeSH
• Fluorescence MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• Phospholipase D metabolism   MeSH
• Photobleaching MeSH
• Phosphatidic Acids analysis   metabolism   MeSH
• Image Processing, Computer-Assisted MeSH
• Qb-SNARE Proteins genetics   metabolism   MeSH
• Qc-SNARE Proteins genetics   metabolism   MeSH
• Pollen Tube genetics   growth & development   metabolism   MeSH
• Recombinant Fusion Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Nicotiana cytology   metabolism   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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
• Chromatography, Thin Layer MeSH
• Diglycerides metabolism   MeSH
• Enzyme Assays methods   MeSH
• Fluorescent Dyes metabolism   MeSH
• Type C Phospholipases metabolism   MeSH
• Reference Standards MeSH
• Plants enzymology   MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Asparagine pharmacology   MeSH
• Autophagy drug effects   MeSH
• Chloroquine pharmacology   MeSH
• Diglycerides metabolism   MeSH
• Hepatocytes metabolism   pathology   drug effects   MeSH
• Liver metabolism   pathology   drug effects   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Fatty Acids, Nonesterified metabolism   MeSH
• Lipolysis MeSH
• Lipoproteins, VLDL metabolism   MeSH
• Lysosomes metabolism   pathology   drug effects   MeSH
• Lysosomal-Associated Membrane Protein 2 metabolism   MeSH
• Oxidation-Reduction MeSH
• Rats, Wistar MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Sirolimus pharmacology   MeSH
• Sterol Esterase metabolism   MeSH
• Triglycerides metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH