scholarly journals Profile of Phosphatidylserine Modifications under Nitroxidative Stress Conditions Using a Liquid Chromatography-Mass Spectrometry Based Approach

Molecules ◽  
2018 ◽  
Vol 24 (1) ◽  
pp. 107 ◽  
Author(s):  
Bruna Neves ◽  
Pedro Domingues ◽  
Maria Oliveira ◽  
Maria Domingues ◽  
Tânia Melo
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Head Group ◽  
Fatty Acyl Chain ◽  
Polar Head Group ◽  
Derivatives Of

Nitrated lipids have been detected in vitro and in vivo, usually associated with a protective effect. While nitrated fatty acids have been widely studied, few studies reported the nitration and nitroxidation of the phospholipid classes phosphatidylcholine, and phosphatidylethanolamine. However, no information regarding nitrated and nitroxidized phosphatidylserine can be found in the literature. This work aims to identify and characterize the nitrated and nitroxidized derivatives of 1-palmitoyl-2-oleoyl-sn-3-glycero-phosphoserine (POPS), obtained after incubation with nitronium tetrafluoroborate, by liquid chromatography (LC) coupled to mass spectrometry (MS) and tandem MS (MS/MS). Several nitrated and nitroxidized products were identified, namely, nitro, nitroso, nitronitroso, and dinitro derivatives, as well as some nitroxidized species such as nitrosohydroxy, nitrohydroxy, and nitrohydroperoxy. The fragmentation pathways identified were structure-dependent and included the loss of HNO and HNO2 for nitroso and nitro derivatives, respectively. Combined losses of PS polar head group plus HNO or HNO2 and carboxylate anions of modified fatty acyl chain were also observed. The nitrated POPS also showed antiradical potential, demonstrated by the ability to scavenge the ABTS●+ and DPPH● radicals. Overall, this in vitro model of nitration based on LC-MS/MS provided additional insights into the nitrated and nitroxidized derivatives of PS and their fragmentation fingerprinting. This information is a valuable tool for targeted analysis of these modified PS in complex biological samples, to further explore the new clues on the antioxidant potential of nitrated POPS.

scholarly journals Fatty Acyl Benzamido Antibacterials Based on Inhibition of DnaK-catalyzed Protein Folding

2006 ◽  
Vol 282 (7) ◽  
pp. 4437-4446 ◽  
Author(s):  
Markus Liebscher ◽  
Günther Jahreis ◽  
Christian Lücke ◽  
Susanne Grabley ◽  
Satish Raina ◽  
...  
Keyword(s):  
Protein Folding ◽  
Minimal Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Secondary Amide ◽  
Fatty Acyl Chain ◽  
Isomerase Activity

We have reported that the hsp70 chaperone DnaK from Escherichia coli might assist protein folding by catalyzing the cis/trans isomerization of secondary amide peptide bonds in unfolded or partially folded proteins. In this study a series of fatty acylated benzamido inhibitors of the cis/trans isomerase activity of DnaK was developed and tested for antibacterial effects in E. coli MC4100 cells. Nα-[Tetradecanoyl-(4-aminomethylbenzoyl)]-l-asparagine is the most effective antibacterial with a minimal inhibitory concentration of 100 ± 20 μg/ml. The compounds were shown to compete with fluorophore-labeled σ32-derived peptide for the peptide binding site of DnaK and to increase the fraction of aggregated proteins in heat-shocked bacteria. Despite its inability to serve as a folding helper in vivo a DnaK-inhibitor complex was still able to sequester an unfolded protein in vitro. Structure activity relationships revealed a distinct dependence of DnaK-assisted refolding of luciferase on the fatty acyl chain length, whereas the minimal inhibitory concentration was most sensitive to the structural nature of the benzamido core. We conclude that the isomerase activity of DnaK is a major survival factor in the heat shock response of bacteria and that small molecule inhibitors can lead to functional inactivation of DnaK and thus will display antibacterial activity.

scholarly journals Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement

2016 ◽  
Vol 27 (16) ◽  
pp. 2590-2597 ◽  
Author(s):  
Michael Zick ◽  
William Wickner
Keyword(s):  
Acyl Chain ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
Rab Gtpase ◽  
Lower Phase ◽  
Yeast Vacuole ◽  
Vacuole Fusion ◽  
In Vitro Reconstitution

In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1.

Intestinal microsomes: polyunsaturated fatty acid metabolism and regulation of enterocyte transport properties

1990 ◽  
Vol 68 (5) ◽  
pp. 636-641 ◽  
Author(s):  
M. L. Garg ◽  
M. Keelan ◽  
A. B. R. Thomson ◽  
M. T. Clandinin
Keyword(s):  
Acyl Chain ◽  
Lipid Synthesis ◽  
Fatty Acyl ◽  
Fine Tuning ◽  
Membrane Properties ◽  
Head Group ◽  
Fatty Acyl Chain ◽  
Polar Head Group ◽  
Intestinal Microsomes ◽  
Main Determinants

Recent evidence has suggested that transport of nutrients from the lumen to the interior of the gastrointestinal epithelium and exit of nutrients from the enterocyte to the circulation is governed by physicochemical properties of brush border and basolateral membranes, respectively. The main determinants of membrane properties are phospholipid, cholesterol, and fatty acyl chain composition (chain length and degree of unsaturation). Lipid synthesis occurs in enterocyte microsomes and the fine tuning of lipid composition is done at other subcellular sites by deacylation–reacylation or by changing the polar head group (e.g., by phosphatidylethanolamine methyltransferase). The present paper will focus on the mechanisms by which enterocyte membranes adapt functional properties in response to external stimuli. It is proposed that under the influence of internal or external stress, the enzymes of lipid metabolism in microsomes are modulated. These changes in lipid synthesis are reflected in other subcellular membranes, changing their physicochemical status and thus transport phenomena. One of the initial events appears to be alteration in desaturase enzyme activity. Our results suggest that desaturase activity and the fatty acyl profiles of the intestinal mucosal phospholipid rapidly respond to physiological conditions such as fasting and dietary fat treatment.Key words: polyunsaturated fatty acids, desaturases, enterocyte, intestinal microsomes, adaptation.

scholarly journals Bile Acids Quantification by Liquid Chromatography–Tandem Mass Spectrometry: Method Validation, Reference Range, and Interference Study

Diagnostics ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 462 ◽  
Author(s):  
Elisa Danese ◽  
Davide Negrini ◽  
Mairi Pucci ◽  
Simone De Nitto ◽  
Davide Ambrogi ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Healthy Subjects ◽  
Reference Range ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Interference Study

Bile acids (BA) play a pivotal role in cholesterol metabolism. Their blood concentration has also been proposed as new prognostic and diagnostic indicator of hepatobiliary, intestinal, and cardiovascular disease. Liquid chromatography tandem mass spectrometry (LC–MS/MS) currently represents the gold standard for analysis of BA profile in biological samples. We report here development and validation of a LC–MS/MS technique for simultaneously quantifying 15 BA species in serum samples. We also established a reference range for adult healthy subjects (n = 130) and performed a preliminary evaluation of in vitro and in vivo interference. The method displayed good linearity, with high regression coefficients (>0.99) over a range of 5 ng/mL (lower limit of quantification, LLOQ) and 5000 ng/mL for all analytes tested. The accuracies were between 85–115%. Both intra- and inter-assay imprecision was <10%. The recoveries ranged between 92–110%. Each of the tested BA species (assessed on three concentrations) were stable for 15 days at room temperature, 4 °C, and −20 °C. The in vitro study did not reveal any interference from triglycerides, bilirubin, or cell-free hemoglobin. The in vivo interference study showed that pools obtained from hyper-cholesterolemic patients and hyper-bilirubinemic patients due to post-hepatic jaundice for benign cholestasis, cholangiocarcinoma and pancreatic head tumors had clearly distinct patterns of BA concentrations compared with a pool obtained from samples of healthy subjects. In conclusion, this study proposes a new suitable candidate method for identification and quantitation of BA in biological samples and provides new insight into a number of variables that should be taken into account when investigating pathophysiological changes of BA in human diseases.

scholarly journals Ectopic expression of ceramide synthase 2 in neurons suppresses neurodegeneration induced by ceramide synthase 1 deficiency

2016 ◽  
Vol 113 (21) ◽  
pp. 5928-5933 ◽  
Author(s):  
Stefka D. Spassieva ◽  
Xiaojie Ji ◽  
Ye Liu ◽  
Kenneth Gable ◽  
Jacek Bielawski ◽  
...  
Keyword(s):  
Acyl Chain ◽  
Strong Association ◽  
Ectopic Expression ◽  
Fatty Acyl ◽  
Chemical Diversity ◽  
Fatty Acyl Chain ◽  
Length Variation ◽  
Ceramide Synthase ◽  
Hydrophobic Moiety

Sphingolipids exhibit extreme functional and chemical diversity that is in part determined by their hydrophobic moiety, ceramide. In mammals, the fatty acyl chain length variation of ceramides is determined by six (dihydro)ceramide synthase (CerS) isoforms. Previously, we and others showed that mutations in the major neuron-specific CerS1, which synthesizes 18-carbon fatty acyl (C18) ceramide, cause elevation of long-chain base (LCB) substrates and decrease in C18 ceramide and derivatives in the brain, leading to neurodegeneration in mice and myoclonus epilepsy with dementia in humans. Whether LCB elevation or C18 ceramide reduction leads to neurodegeneration is unclear. Here, we ectopically expressed CerS2, a nonneuronal CerS producing C22–C24 ceramides, in neurons of Cers1-deficient mice. Surprisingly, the Cers1 mutant pathology was almost completely suppressed. Because CerS2 cannot replenish C18 ceramide, the rescue is likely a result of LCB reduction. Consistent with this hypothesis, we found that only LCBs, the substrates common for all of the CerS isoforms, but not ceramides and complex sphingolipids, were restored to the wild-type levels in the Cers2-rescued Cers1 mutant mouse brains. Furthermore, LCBs induced neurite fragmentation in cultured neurons at concentrations corresponding to the elevated levels in the CerS1-deficient brain. The strong association of LCB levels with neuronal survival both in vivo and in vitro suggests high-level accumulation of LCBs is a possible underlying cause of the CerS1 deficiency-induced neuronal death.

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