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Analysis of polar lipids from eight psychrophilic yeasts (Cryptococcus victoriae, Cystofilobasidium capitatum, Holtermaniella wattica, Mrakiella aquatica, M. cryoconiti, Rhodotorula lignophila, Kondoa malvinella and Trichosporon aggtelekiense) grown at 4-28°C by hydrophilic interaction liquid chromatography/high resolution electrospray ionization tandem mass spectrometry determined 17 classes of lipids and identified dozens of molecular species of phospholipids including their regioisomers. Most of the yeasts were able to grow over the whole temperature range, reaching the highest biomass at 4 or 10°C. On temperature drop to 4°C, all eight strains showed a significant decrease of MUFA and a simultaneous increase of PUFA such as α-linolenic acid, the content of which in the biomass reached up to 20%. We also found alterations in the proportions of individual phospholipids (PI, PE and PC), the PC/PE-ratio decreasing with decreasing temperature. With increasing temperature the content of PoO-PC rose while that of LL-PC decreased, the drop in the content of LL-PC being nearly 100-fold while the content of PoO-PC increased more than twice. A change in temperature brought about changes in molecular species of PC (molecular species PO-PC versus OP-PC) as well as PE, i.e. PO-PE and OP-PE. The phase transition temperature of PO-PC differs from OP-PC by 7°C and the difference between PO-PE and OP-PE is some 10°C; we thus assume that the cell compensates for the adverse temperature effect by changing the fatty acids in the sn-1 and sn-2 positions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The outermost layer of the mammalian skin, the stratum corneum (SC), is a very thin structure and realizes simultaneously the main barrier properties. The penetration barrier for xenobiotica is mostly represented by a complex lipid matrix. There is great interest in the subject of getting information about the arrangement of the lipids, which are mainly ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). SC lipid model membranes containing synthetically derived lipids in a non-physiological ratio were investigated. To compare the study to a former experiment, a methyl-branched ceramide [EOS] species in presence of the ultra-long chain CER[AP], CHOL and behenic acid (23/10/33/33, wt%) was applied. The membrane structure was studied using the very versatile technique of neutron diffraction. We were able to identify a long-periodicity phase (LPP) with a size of 114Å or 118Å with CER[EOS]-br in a ratio of >60wt% of the ceramides. Furthermore, we figured out two additional coexisting short-periodicity phases (SPP) with repeat distances of 48Å and 45Å, respectively. Partial deuterations of CER[EOS]-br and CER[AP] enabled the localization of the molecules within the multiphase system. CER[EOS]-d3 was present in the LPP, but absent in both SPP. CER[AP]-d3 was determined in both short phases but not localized within the LPP. Besides, we revealed influences of humidity and time with respect to the long-periodicity phase., (Copyright © 2016 Elsevier B.V. All rights reserved.)

α-Lactalbumin (aLA) has been shown to form complexes with oleic acid (OA), which may target cancer cells. We recently showed that aLA and several other proteins all form protein-OA complexes called liprotides with a generic structure consisting of a micellar OA core surrounded by a shell of partially denatured protein. Here we report that a heat treatment and an alkaline treatment method both allow us to prepare liprotide complexes composed of aLA and a range of unsaturated fatty acids (FA), provided the FAs contain cis (but not trans) double bonds. All liprotides containing cis-FA form both small and large species, which all consist of partially denatured aLA, though the overall shape of the species differs. Small liprotides have a simple core-shell structure while the larger liprotides are multi-layered, i.e. they have an additional layer of both FA and aLA surrounding the outside of the core-shell structure. All liprotides can transfer their entire FA content to vesicles, releasing aLA as monomers and softening the lipid membrane. The more similar to OA, the more efficiently the different FAs induce hemolysis. We conclude that aLA can take up and transfer a wide variety of FA to membranes, provided they contain a cis-bond. This highlights liprotides as a general class of complexes where both protein and cis-FA component can be varied without departing from a generic (though sometimes multi-layered) core-shell structure., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Two self-sufficient CYP102 family encoding genes (Krac_0936 and Krac_9955) from the bacterium Ktedonobacter racemifer DSM44963, which possesses one of the largest bacterial genomes, have been identified., Methods: Phylogenetic analysis of both the encoded cytochrome P450 enzymes, Krac0936 and Krac9955. Both enzymes were produced and their turnovers with fatty acid substrates assessed in vitro and using a whole-cell oxidation system., Results: Krac0936 hydroxylated straight chain, saturated fatty acids predominantly at the ω-1 and ω-2 positions using NADPH as the cofactor. Krac0936 was less active towards shorter unsaturated fatty acids but longer unsaturated acids were efficiently oxidised. cis,cis-9,12-Octadecadienoic and pentadecanoic acids were the most active substrates tested with Krac0936. Unusually Krac9955 showed very low levels of NAD(P)H oxidation activity though coupling of the reducing equivalents to product formation was high. The product distribution of tridecanoic, tetradecanoic and pentadecanoic acid oxidation by Krac9955 favoured oxidation at the ω-4, ω-5 and ω-6 positions, respectively., Conclusion: Krac0936 and Krac9955 are self-sufficient P450 monooxygenases. Krac0936 has a preference for pentadecanoic acid over other straight chain fatty acids and showed little or no activity with dodecanoic or octadecanoic acids. Krac9955 preferably oxidised shorter fatty acids compared to Krac0936 with tridecanoic having the highest levels of product formation. Unlike Krac0936 and P450Bm3, Krac9995 showed lower activities with unsaturated fatty acids., General Significance: In this study of two of the CYP enzymes from K. racemifer we have shown that this bacterium from the Chloroflexi phylum contains genes which encode new proteins with novel activity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A number of cationic antimicrobial peptides, effectors of innate immunity, are supposed to act at the cytoplasmic membrane leading to permeabilization and eventually membrane disruption. Thereby, interaction of antimicrobial peptides with anionic membrane phospholipids is considered to be a key factor in killing of bacteria. Recently, evidence was provided that killing takes place only when bacterial cell membranes are completely saturated with peptides. This adds to an ongoing debate, which role cell wall components such as peptidoglycan, lipoteichoic acid and lipopolysaccharide may play in the killing event, i.e. if they rather entrap or facilitate antimicrobial peptides access to the cytoplasmic membrane. Therefore, in this review we focused on the impact of Gram-positive cell wall components for the mode of action and activity of antimicrobial peptides as well as in innate immunity. This led us to conclude that interaction of antimicrobial peptides with peptidoglycan may not contribute to a reduction of their antimicrobial activity, whereas interaction with anionic lipoteichoic acids may reduce the local concentration of antimicrobial peptides on the cytoplasmic membrane necessary for sufficient destabilization of the membranes and bacterial killing. Further affinity studies of antimicrobial peptides toward the different cell wall as well as membrane components will be needed to address this problem on a quantitative level. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The rapid development of analytical technology has made lipidomics an exciting new area and this review will focus more on modern approaches to lipidomics than on earlier technology. Although not fully comprehensive for all possible brain lipids, the intent is to at least provide a reference for the analysis of classes of lipids found in brain and nervous tissue. We will discuss problems posed by the brain because of its structural and functional heterogeneity, the development changes it undergoes (myelination, aging, pathology etc.) and its cellular heterogeneity (neurons, glia etc.). Section 2 will discuss the various ways in which brain tissue can be extracted to yield lipids for analysis and section 3 will cover a wide range of techniques used to analyze brain lipids such as chromatography and mass-spectrometry. In Section 4 we will discuss ways of analyzing some of the specific biologically active brain lipids found in very small amounts except in pathological conditions and section 5 looks to the future of experimental lipidomic modification in the brain. This article is part of a Special Issue entitled Brain Lipids., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this work, adrenodoxin (Adx) was expressed on the outer membrane of E. coli by autodisplay and then the iron-sulfur cluster was incorporated into apo-Adx by an anaerobic reconstitution process. For the determination of the redox potentials of the iron-sulfur clusters of the autodisplayed Adx, E. coli cells with autodisplayed Adx were immobilized on a gold electrode modified with a self-assembled monolayer of mercaptoundecanoic acid (MUA). From the repeated cyclic voltammetry (CV) analysis, the E. coli (10mM HEPES buffer, pH7.0) with autodisplayed Adx showed significant changes in shape with an oxidation peak at +0.4V (vs. Ag/AgCl) and a reduction peak at -0.3V (vs. Ag/AgCl) after the reconstitution process for the incorporation of the iron-sulfur cluster. From the repeated CV analysis in the reduction and oxidation potential ranges, the iron-sulfur clusters of the autodisplayed Adx were observed to undergo reversible redox reactions via direct electron transfer to the MUA-modified gold electrode., (Copyright © 2015. Published by Elsevier B.V.)

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder resulting from defective ABCD1 transport protein. ABCD1 mediates peroxisomal uptake of free very-long-chain fatty acids (VLCFA) as well as their CoA-esters. Consequently, VLCFA accumulate in patients' plasma and tissues, which is considered as pathogenic X-ALD triggering factor. Clinical symptoms are mostly manifested in neural tissues and adrenal gland. Here, we investigate astrocytes from wild-type control and a genetic X-ALD mouse model (Abcd1-knockout), exposed to supraphysiological VLCFA (C22:0, C24:0 and C26:0) concentrations. They exhibit multiple impairments of energy metabolism. Furthermore, brain mitochondria from Abcd1(-/-) mice and wild-type control respond similarly to VLCFA with increased ROS generation, impaired oxidative ATP synthesis and diminished Ca(2+) uptake capacity, suggesting that a defective ABCD1 exerts no adaptive pressure on mitochondria. In contrast, astrocytes from Abcd1(-/-) mice respond more sensitively to VLCFA than wild-type control astrocytes. Moreover, long-term application of VLCFA induces high ROS generation, and strong in situ depolarization of mitochondria, and, in Abcd1(-/-) astrocytes, severely diminishes the capability to revert oxidized pyridine nucleotides to NAD(P)H. In addition, observed differences in responses of mitochondria and astrocytes to the hydrocarbon chain length of VLCFA suggest that detrimental VLCFA activities in astrocytes involve defective cellular functions other than mitochondria. In summary, we clearly demonstrate that VLCFA increase the vulnerability of Abcd1(-/-) astrocytes., (Copyright © 2015 Elsevier B.V. All rights reserved.)

N-Acylserotonins (NASTs), present in the mammalian gastro-intestinal tract and central nervous tissues, exhibit significant biological and pharmacological activities. In the present study, a homologous series of NASTs have been synthesized and characterized. Differential scanning calorimetric studies show that in the dry and hydrated states the transition temperatures, enthalpies, and entropies of NASTs exhibit odd-even alternation. Both odd and even chain length NASTs independently display linear dependence of the transition enthalpies and entropies on the chain length under dry as well as hydrated conditions, suggesting that the molecular packing and intermolecular interactions in each series (odd or even) are likely to be similar for NASTs with different acyl chain lengths in the dry state as well as in the hydrated state. Powder X-ray diffraction studies indicated that the incremental increase in the d-spacing per CH₂group is 1.023 Å, suggesting that the lipid acyl chains are most likely packed in an interdigitated fashion. Results of computational studies are consistent with this and suggest that the acyl chains of the NASTs are tilted with respect to the bilayer normal. Incorporation of N-myristoylserotonin (NMST) into dimyristoylphosphatidylcholine (DMPC) membranes did not significantly affect the phase transition properties at low mole fractions (1-5 mol%), although distinct decrease in the chain-melting transition temperature and increase in the pretransition temperature were observed at higher contents (7.5-30 mol%), suggesting that NMST increases the stability of the tilted gel phase (L(β)') but destabilizes the ripple phase (P(β)'). These observations provide a thermodynamic basis for understanding the functional role of NASTs in their parent tissues., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The stratum corneum (SC) plays a fundamental role in the barrier function of the skin. The SC consists of corneocytes embedded in a lipid matrix. The main lipid classes in the lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to examine the effect of the chain length of FFAs on the thermotropic phase behavior and mixing properties of SC lipids. Fourier transform infrared spectroscopy and Raman imaging spectroscopy were used to study the mixing properties using either protonated or deuterated FFAs. We selected SC model lipid mixtures containing only a single CER, CHOL and either a single FFA or a mixture of FFAs mimicking the FFA SC composition. The single CER consists of a sphingoid base with 18 carbon atoms and an acyl chain with a chain length of 24 carbon atoms. When using lignoceric acid (24 carbon atoms) or a mixture of FFAs, the CER and FFAs participated in mixed crystals, but hydration of the mixtures induced a slight phase separation between CER and FFA. The mixed crystalline structures did not phase separate during storage even up to a time period of 3months. When using palmitic acid (16 carbon atoms), a slight phase separation was observed between FFA and CER. This phase separation was clearly enhanced during hydration and storage. In conclusion, the thermotropic phase behavior and the mixing properties of the SC lipid mixtures were shown to strongly depend on the chain length and chain length distribution of FFAs, while hydration enhanced the phase separation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is an antitumor drug that regulates membrane lipid composition and structure. An important effect of this drug is the restoration of sphingomyelin (SM) levels in cancer cell membranes, where the SM concentration is lower than in non-tumor cells. It is well known that free fatty acid concentration in cell membranes is lower than 5%, and that fatty acid excess is rapidly incorporated into phospholipids. In a recent work, we have considered the effect of free 2OHOA in model membranes in liquid ordered (Lo) and liquid disordered (Ld) phases, by using all-atom molecular dynamics. This study concerns membranes that are modified upon incorporation of 2OHOA into different phospholipids. 2OHOA-containing phospholipids have a permanent effect on lipid membranes, making a Ld membrane surface more compact and less hydrated, whereas the opposite effect is observed in Lo domains. Moreover, the hydroxyl group of fatty acid chains increases the propensity of Ld model membranes to form hexagonal or other non-lamellar structures. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This review deals with the effects of synthetic and natural fatty acids on the biophysical properties of membranes, and on their implication on cell function. Natural fatty acids are constituents of more complex lipids, like triacylglycerides or phospholipids, which are used by cells to store and obtain energy, as well as for structural purposes. Accordingly, natural and synthetic fatty acids may modify the structure of the lipid membrane, altering its microdomain organization and other physical properties, and provoking changes in cell signaling. Therefore, by modulating fatty acids it is possible to regulate the structure of the membrane, influencing the cell processes that are reliant on this structure and potentially reverting pathological cell dysfunctions that may provoke cancer, diabetes, hypertension, Alzheimer's and Parkinson's disease. The so-called Membrane Lipid Therapy offers a strategy to regulate the membrane composition through drug administration, potentially reverting pathological processes by re-adapting cell membrane structure. Certain fatty acids and their synthetic derivatives are described here that may potentially be used in such therapies, where the cell membrane itself can be considered as a target to combat disease. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This paper describes two synthetic lipid models designed to replace human stratum corneum (SC) in studies of the impact of volatile organic chemicals on the molecular organization of the skin barrier lipids. The models built upon previously developed self-assembled lipid membranes which have composition and 3D organization similar to those of the lipid matrix in SC. In one model the target chemicals were incorporated in the lipids before their self-assembly, and in the other one they were applied on top of a preformed lipid membrane. The chemicals could be incorporated within the model membranes in quantities close to those reached within human SC upon heavy surface loading. The dose-dependent effects of the chemicals on the lateral molecular organization in the models were qualitatively identical to those observed by infrared spectroscopy in human SC. The models facilitated the interpretation of X-ray diffraction profiles used to determine the nature of the interactions between the chemicals and the lipid lamellae and the position of the exogenous molecules within the unit cell of the lipid phases. These model systems are suitable for in vitro studies in the areas of skin biophysics, dermatology, transdermal drug delivery, and risk assessment., (© 2013.)

Background: The molecular details of fatty acid (FA) interactions with albumin are fundamental to understanding transport in the plasma and cellular utilization of these key nutrients and building blocks of membranes., Scope of Review: This review focuses on the development and application of NMR methods to study FA binding to albumin [bovine (BSA) and human (HSA)]. The key strategy was to use (13)C enrichment of a specific carbon in the FA as a non-perturbing probe to permit visualization of the small ligand complexed to the very large protein. NMR contributions to illuminating molecular interactions and FA dynamics are summarized from three decades of studies., Major Conclusions: Our early studies detected multiple binding sites that we hypothesized were distinguished because of the unique tertiary structure of the protein in close proximity to the FA labeled carbon in each site. Later crystallographic structures revealed the presence of polar and charged amino acid side chains near the carboxyl carbon of the FA and unique tertiary structures lining all of the FA binding pockets. In collaboration with the crystallography group, several FA sites in the crystalline state were matched with NMR resonances in the solution state. With the newest application of NMR, 2D NMR spectroscopy detected nine binding sites, and three were located in the crystal structure through displacement of drugs with identified sites., General Significance: NMR spectroscopy utilizing the FA as a probe allows characterization of site-specific interactions, molecular motions within binding sites, the order of filling and removal of FA from sites. This article is part of a Special Issue entitled Serum Albumin., (© 2013 Elsevier B.V. All rights reserved.)

Background: Binding affinity for human serum albumin (HSA) is one of the most important factors affecting the distribution and free blood concentration of many ligands. The effect of fatty acids (FAs) on HSA-ligand binding has long been studied. Since the elucidation of the 3-dimensional structure of HSA, molecular simulation approaches have been applied to studies of the structure-function relationship of HSA-FA binding., Scope of Review: We review current insights into the effects of FA binding on HSA, focusing on the biophysical insights obtained using molecular simulation approaches such as docking, molecular dynamics (MD), and binding free energy calculations., Major Conclusions: Possible conformational changes on binding of FA molecules to HSA have been observed through MD simulations. High- and low-affinity FA-binding sites on HSA have been identified based on binding free energy calculations. The relationship between the warfarin binding affinity of HSA and FA molecules has been clarified based on the results of simulations of multi-site FA binding that cannot be experimentally observed., General Significance: Molecular simulation approaches have great potentials to provide detailed biophysical insights into HSA as well as the effects of the binding of FAs or other ligands to HSA. This article is part of a Special Issue entitled Serum Albumin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Serum albumin is a major transport protein in mammals and is known to have at least seven binding sites for long-chain fatty acids (FAs)., Scope of Review: We have devised a new electron paramagnetic resonance (EPR) spectroscopic approach to gain information on the functional structure of serum albumin in solution in a "coarse-grained" manner from the ligands' point of view. Our approach is based on using spin labeled (paramagnetic) stearic acids self-assembled with albumin and subsequent nanoscale distance measurements between the FAs using double electron-electron resonance spectroscopy (DEER). Simple continuous wave (CW) EPR spectroscopy, which allows for quantification of bound ligands, complements our studies., Major Conclusions: Based on DEER nanoscale distance measurements, the functional solution structure of human serum albumin (HSA) has remarkably been found to have a much more symmetric distribution of entry points to the FA binding sites than expected from the crystal structure, indicating increased surface flexibility and plasticity for HSA in solution. In contrast, for bovine serum albumin (BSA), the entry point topology is in good agreement with that expected from the crystal structure of HSA. Changes in the solution structures between albumins can hence be revealed and extended to more albumins to detect functional differences at the nanoscale. Going beyond fundamental structural studies, our research platform is also excellently suited for general studies of protein-solvent interactions, temperature effects and ligand binding., General Significance: We discuss how our research platform helps illuminate protein dynamics and function and can be used to characterize albumin-based hybrid materials. This article is part of a Special Issue entitled Serum Albumin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Serum albumin is the major protein component of blood plasma and is responsible for the circulatory transport of a range of small molecules that include fatty acids, hormones, metal ions and drugs. Studies examining the ligand-binding properties of albumin make up a large proportion of the literature. However, many of these studies do not address the fact that albumin carries multiple ligands (including metal ions) simultaneously in vivo. Thus the binding of a particular ligand may influence both the affinity and dynamics of albumin interactions with another., Scope of Review: Here we review the Zn(2+) and fatty acid transport properties of albumin and highlight an important interplay that exists between them. Also the impact of this dynamic interaction upon the distribution of plasma Zn(2+), its effect upon cellular Zn(2+) uptake and its importance in the diagnosis of myocardial ischemia are considered., Major Conclusions: We previously identified the major binding site for Zn(2+) on albumin. Furthermore, we revealed that Zn(2+)-binding at this site and fatty acid-binding at the FA2 site are interdependent. This suggests that the binding of fatty acids to albumin may serve as an allosteric switch to modulate Zn(2+)-binding to albumin in blood plasma., General Significance: Fatty acid levels in the blood are dynamic and chronic elevation of plasma fatty acid levels is associated with some metabolic disorders such as cardiovascular disease and diabetes. Since the binding of Zn(2+) to albumin is important for the control of circulatory/cellular Zn(2+) dynamics, this relationship is likely to have important physiological and pathological implications. This article is part of a Special Issue entitled Serum Albumin., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is a potent antitumor drug that we rationally designed to regulate the membrane lipid composition and structure. The lipid modifications caused by 2OHOA treatments induce important signaling changes that end up with cell death (Terés et al., 2012 [1]). One of these regulatory effects is restoration of sphingomyelin levels, which are markedly lower in cancer cells compared to normal cells (Barceló-Coblijn et al., 2011 [2]). In this study, we report another important regulatory effect of 2OHOA on cancer cell membrane composition: a large increase in 2OHOA levels, accounting for ~15% of the fatty acids present in membrane phospholipids, in human glioma (SF767 and U118) and lung cancer (A549) cells. Concomitantly, we observed marked reductions in oleic acid levels and inhibition of stearoyl-CoA desaturase. The impact of these changes on the biophysical properties of the lipid bilayer was evaluated in liposomes reconstituted from cancer cell membrane lipid extracts. Thus, 2OHOA increased the packing of ordered domains and decreased the global order of the membrane. The present results further support and extend the knowledge about the mechanism of action for 2OHOA, based on the regulation of the membrane lipid composition and structure and subsequent modulation of membrane protein-associated signaling., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Small multidrug resistance (SMR) protein family members confer bacterial resistance to toxic antiseptics and are believed to function as dual topology oligomers. If dual topology is essential for SMR activity, then the topology bias should change as bacterial membrane lipid compositions alter to maintain a "neutral" topology bias. To test this hypothesis, a bioinformatic analysis of bacterial SMR protein sequences was performed to determine a membrane protein topology based on charged amino acid residues within loops, and termini regions according to the positive inside rule. Three bacterial lipid membrane parameters were examined, providing the proportion of polar lipid head group charges at the membrane surface (PLH), the relative hydrophobic fatty acid length (FAL), and the proportion of fatty acid unsaturation (FAU). Our analysis indicates that individual SMR pairs, and to a lesser extent SMR singleton topology biases, are significantly correlated to increasing PLH, FAL and FAU differences validating the hypothesis. Correlations between the topology biases of SMR proteins identified in Gram+ compared to Gram- species and each lipid parameter demonstrated a linear inverse relationship., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Lipid composition and fatty acid analysis of the major classes of membrane phospholipids were determined during myogenic differentiation of L6 skeletal muscle cells. The cholesterol to glycerophospholipids ratio decreased during differentiation, both in total (TM) and detergent-resistant membranes (DRM). Analyses of the membrane lipids showed that differentiation had a major impact on the molecular composition of glycerophospholipids. A significant decrease in the concentration of saturated fatty acids was detected in glycerophospholipid classes, and to a lesser extent in sphingolipids, while the concentration of 16:1n-7, 18:1n-7 and 18:1n-9 increased. At the same time, the concentration of long polyunsaturated fatty acid chains decreased in TM and DRM glycerophospholipids, resulting in a lower saturated to unsaturated fatty acid ratio in myotubes as compared to myoblasts. Interestingly, the observed n-3/n-6 ratio was lower in differentiated cell membranes. PUFA supplementation of L6 cells led to an increase in myogenic differentiation correlated to an incorporation of added PUFAs in TM and DRM glycerophospholipids. As expected after n-3 PUFA supplementation, the n-3/n-6 ratio was clearly increased in TM and, surprisingly, this was also the case in isolated DRM. n-3 and n-6 PUFAs significantly and time-dependently increased the phosphorylation of kinase p70S6K1 during myogenic differentiation, revealing the activation of the upstream kinase mTORC1, a major regulator of cell cycle and protein translation. In contrast, PUFAs did not affect the phosphorylation of the kinase Akt, another pivotal regulator of cell metabolism. These results suggest that PUFA supplementation modified the membrane lipid composition and affected the differentiation of L6 cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Perfluorooctane sulfonate (PFOS) was produced by various industries and was widely used in diverse consumer products. Human sample analysis indicated PFOS contamination in body fluids. Animal studies revealed that PFOS tends to accumulate in livers and is able to induce hepatomegaly. However the underlying mechanism of PFOS-elicited hepatotoxicity has not yet been fully addressed. The objective of this study is to identify the cellular target of PFOS and to reveal the mechanisms of PFOS-induced toxicity., Methods: In this study, mature 8-week old male CD-1 mice were administered 0, 1, 5 or 10 mg/kg/day PFOS for 3, 7, 14 or 21 days. Histological analysis of liver sections, and biochemical/molecular analysis of biomarkers for hepatic lipid metabolism were assessed., Results: PFOS-induced steatosis was observed in a time- and dose-dependent manner. The gene expression levels of fatty acid translocase (FAT/CD36) and lipoprotein lipase (Lpl) were significantly increased by 10 and/or 5 mg/kg PFOS. Serum levels of very-low density lipoprotein were decreased by 14 days of PFOS exposure (p<0.05). The rate of mitochondrial β-oxidation was also found to be significantly reduced, leading to the restriction of fatty acid oxidation for energy production., Conclusion: Taken together, the disturbance of lipid metabolism leads to the accumulation of excessive fatty acids and triglycerides in hepatocytes., General Significance: Since PFOS-elicited pathological manifestation resembles one of the most common human liver diseases-nonalcoholic fatty liver disease, environmental exposure to PFOS may attribute to the disease progression., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The direct interaction of the antibiotic primycin with the plasma membrane was investigated by employing the well-characterized ergosterol-producing, amphotericin B-sensitive parental Candida albicans strain 33erg(+) and its ergosterol-less amphotericin B-resistant plasma membrane mutant erg-2. The growth inhibition concentration in shaken liquid medium was 64 μgml(-1) for 33erg(+) and 128 μgml(-1) for erg-2, suggesting that the plasma membrane composition influences the mode of action of primycin. To determine the primycin-induced changes in the plasma membrane dynamic, electron paramagnetic resonance (EPR) spectroscopy methods were used, the spin-labeled fatty acid 5-(4,4-dimethyloxazolidine-N-oxyl)stearic acid) being applied for the in vivo measurements. The phase transition temperatures of untreated strain 33erg(+) and its mutant erg-2 were 12.5°C and 11°C, respectively. After 128 μgml(-1) primycin treatment, these values increased to 17.5°C and 16°C, revealing a significant reduction in the phospholipid flexibility. Saturation transfer EPR measurements demonstrated that, the rotational correlation times of the spin label molecule for the control samples of 33erg(+) and erg-2 were 60 ns and 100 ns. These correlation times gradually decreased on the addition of increasing primycin concentrations, reaching 8 μs and 1 μs. The results indicate the plasma membrane "rigidizing" effect of primycin, a feature that may stem from its ability to undergo complex formation with membrane constituent fatty acid molecules, causing alterations in the structures of phospholipids in the hydrophobic surface near the fatty acid chain region., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography-mass spectrometry (GC/MS) and subsequently liquid chromatography-mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: L-3-Hydroxy fatty acids are unusual metabolites and rarely occur in significant quantities in normal human physiology. Genetic defects of both long-chain and medium-/short-chain mitochondrial L-3 hydroxyacyl coenzyme A dehydrogenases (LCHAD, M/SCHAD) have been identified as significant metabolic diseases in humans often with severe clinical phenotypes and pathophysiology that appears to differ from other defects of straight chain fatty acid oxidation. It is felt that accumulation of these atypical fatty acid species may play a role in this pathology. We have therefore developed an assay to measure these compounds in body fluids, and tissue culture medium to help in the diagnosis of these disorders and to better study the effects of 3-hydroxy fatty acid accumulation., Methods: We have developed a stable isotope dilution, selected ion-monitoring gas chromatography-mass spectrometric assay for the measurement of all 3-hydroxy fatty acids from chain lengths C6 to C18 using 1,2 (13)C-labeled internal standards for all species. Authentic patient samples were utilized to develop reference intervals for control subjects, for those associated with patient samples confirmed at the molecular level to have either LCHAD or M/SCHAD deficiency and for patients who did not have disease but were fasting or on diets high in medium-chain fatty acids. Likewise, skin fibroblasts were obtained from patients with confirmed disease for additional study. Samples were also obtained from the hadh (M/SCHAD) knockout mouse., Results: The measurement of 3-hydroxy fatty acids in patient plasma is a valuable tool in the identification of defects of both enzymes. Severe starvation, prolonged fasting and increased medium-chain triglycerides in the diet produce a profile that is similar to that seen in M/SCHAD deficiency, making this a more difficult condition to diagnose but these biomarkers provide an important clue to the diagnosis, particularly in non-fasted, diet-controlled patients. Fibroblast studies in LCHAD deficiency demonstrate that long-chain 3-hydroxy fatty acid accumulation can be observed in cultured tissues. Incubation of cultured fibroblasts from LCHAD deficient patients with labeled fatty acids demonstrated a process of chain lengthening that has not previously been recognized., Conclusions: The measurement of body fluid and cultured cell 3-hydroxy fatty acids provides both diagnostic and pathogenic information regarding these genetic diseases of fatty acid oxidation in the mitochondrion. Presently, the measurement of medium- and short-chain species provides a major metabolic biomarker for the recognition of M/SCHAD deficiency., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Historically considered to be simple membrane components serving as structural elements and energy storing entities, fatty acids are now increasingly recognized as potent signaling molecules involved in many metabolic processes. Quantitative determination of fatty acids and exploration of fatty acid profiles have become common place in lipid analysis. We present here a reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids are achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography-mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples. ., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The bilayer properties and interactions with cholesterol of N-acyl hydroxylated sphingomyelins (SM) were examined, and results were compared to nonhydroxylated chain-matched SM. The natural OH(D)-enantiomer of hydroxylated SM (with 16:0 or 22:0 acyl chain lengths) analogs was synthesized. Measuring steady-state diphenylhexatriene anisotropy, we observed that pure 2OH-SM bilayers always showed higher (5-10 °C) gel-liquid transition temperatures (T(m)) compared to their nonhydroxylated chain-matched analogs. Bilayers made from 3OH(D)-palmitoyl SM, however, had lower T(m) (5 °C) than palmitoyl SM. These data show that hydroxylation in a position-dependent manner directly affected SM interactions and gel state stability. From the c-laurdan emission spectra, we could observe that 2OH-palmitoyl SM bilayers showed a redshift in the emission compared to nonhydroxylated palmitoyl SM bilayers, whereas the opposite was true for c-laurdan emission in 3OH-palmitoyl SM bilayers. All hydroxylated SM analogs were able to form sterol-enriched ordered domains in a fluid phospholipid bilayer. 2-Hydroxylation appeared to increase domain thermostability compared to nonhydroxylated SM, whereas 3-hydroxylation appeared to decrease domain stability. When sterol affinity to bilayers containing SM analogs was determined (cholestatrienol partitioning), the affinity for hydroxylated SM analog bilayers was clearly reduced compared to the nonhydroxylated SM bilayers. Our results with hydroxylated SM analogs clearly show that hydroxylation affects interlipid interactions in a position-dependent manner., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Three families of non-reducing trisaccharide fatty acid monoesters bearing C₁₀ to C₁₈ acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to μM. The new compounds are capable of dissolving phospholipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed β-dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Many studies using genetic and proteomic approaches have revealed phenotypic differences between planktonic and sessile bacteria but the mechanisms of biofilm formation and the switch between the two growth modes are not well understood yet. In this study, we focused on inner membrane lipidome modifications when Pseudomonas aeruginosa cells were grown as biofilm. Lipid analyses were performed by Electrospray Ionization Mass Spectrometry. Results showed a drastic decrease of the uneven-numbered chain phospholipids and a slight increase of long chain PEs in sessile organisms as compared with planktonic counterparts, suggesting a better lipid stability in the bilayer and a decrease in membrane fluidity. The impact of sessile growth on lipid domains was then investigated by Brewster Angle Microscopy (BAM) and Atomic Force Microscopy (AFM). Observations showed that inner membrane lipids of P. aeruginosa formed domains when the pressure was close to physiological conditions and that these domains were larger for lipids extracted from biofilm bacteria. This is coherent with the mass spectrometry analyses., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate-and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5mM detergent. Between 3.2 and 10mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs; the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111)., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Undigested food is fermented in the colon by the microbiota and gives rise to various microbial metabolites. Short-chain fatty acids (SCFA), including acetic, propionic and butyric acid, are the principal metabolites produced. However, most of the literature focuses on butyrate and to a lesser extent on acetate; consequently, potential effects of propionic acid (PA) on physiology and pathology have long been underestimated. It has been demonstrated that PA lowers fatty acids content in liver and plasma, reduces food intake, exerts immunosuppressive actions and probably improves tissue insulin sensitivity. Thus increased production of PA by the microbiota might be considered beneficial in the context of prevention of obesity and diabetes type 2. The molecular mechanisms by which PA may exert this plethora of physiological effects are slowly being elucidated and include intestinal cyclooxygenase enzyme, the G-protein coupled receptors 41 and 43 and activation of the peroxisome proliferator-activated receptor γ, in turn inhibiting the sentinel transcription factor NF-κB and thus increasing the threshold for inflammatory responses in general. Taken together, PA emerges as a major mediator in the link between nutrition, gut microbiota and physiology., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The SH4 domain of Fyn, a member of the Src family of tyrosine kinases, though rich in polar amino acid residues, anchors to the cytosolic face of membranes upon fatty acylation. In order to probe the requirement of specific fatty acylation at the N-terminus and at the side-chain of this domain for membrane-association, we have studied the interaction of peptides corresponding to the polar segment of the SH4 domain of Fyn and its mono- and dually fatty acylated analogs with model membranes. While the polar segment without covalently linked fatty acids (KDKEATKLTEW-amide) does not interact with lipid vesicles, peptides with one covalently linked fatty acid at the N-terminus or in the side-chain, associate with zwitterionic and anionic lipids to varying degrees. The interaction of dually acylated peptides (Myr-GK(epsilon-myr)KDKEATKLTEW-amide and Myr-GC(S-pal)KDKEATKLTEW-amide) with lipids depends on the linkage between fatty acyl side-chain and peptide backbone. The peptide chain associates with membranes only when the side-chain acylation is via an amide bond and not via a thioester bond. Our investigations indicate that acylation is essential for membrane targeting and unacylated polar stretch of the SH4 domain does not have a role in membrane-anchoring. Side-chain acylation via a thioester bond not only provides membrane anchorage but also directs the peptide chain away from the bilayer which might be important to enable the full length protein to interact with other signaling partners., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We investigated the effect of a non-mammalian omega-3 desaturase in a mouse hepatocarcinogenesis model. Mice containing double mutations (DM) in c-myc and TGF-alpha (transforming growth factor-alpha), leading to liver neoplasia, were crossed with mice containing omega-3 desaturase. MRI analysis of triple mutant (TM) mice showed the absence of neoplasia at all time points for 92% of mice in the study. Pathological changes of TM (TGFalpha/c-myc/fat-1) mouse liver tissue was similar to control mouse liver tissue. Magnetic resonance spectroscopy (MRS) measurements of unsaturated fatty acids found a significant difference (p<0.005) between DM and TM transgenic (Tg) mice at 34 and 40 weeks of age. HPLC analysis of mouse liver tissue revealed markedly decreased levels of omega-6 fatty acids in TM mice when compared to DM (TGFalpha/c-myc) and control (CD1) mice. Mass spectrometry (MS) analysis indicated significantly decreased 16:0/20:4 and 18:1/20:4 and elevated 16:0/22:6 fatty acyl groups in both GPCho and GPEtn, and elevated 16:0/20:5, 18:0/18:2, 18:0/18:1 and 18:0/22:6 in GPCho, within TM mice compared to DM mice. Total fatty acid analysis indicated a significant decrease in 18:1n9 in TM mice compared to DM mice. Western blot analysis of liver tissue showed a significant (p<0.05) decrease in NF-kappaB (nuclear factor-kappaB) levels at 40 weeks of age in TM mice compared to DM mice. Microarray analysis of TM versus DM mice livers at 40 weeks revealed alterations in genes involved in cell cycle regulation, cell-to-cell signaling, p53 signaling, and arachidonic acid (20:4) metabolism. Endogenous omega-3 fatty acids were found to prevent HCC development in mice., (Copyright @ 2010 Elsevier B.V. All rights reserved.)

Infection with human immunodeficiency virus (HIV) and treatment with HIV-protease inhibitor (PI)-based highly active antiretroviral therapies (HAART) is associated with dysregulated fatty acid and lipid metabolism. Enhanced lipolysis, increased circulating fatty acid levels, and hepatic and intramuscular lipid accumulation appear to contribute to insulin resistance in HIV-infected people treated with PI-based HAART. However, it is unclear whether currently prescribed HIV-PIs directly alter skeletal muscle fatty acid transport, oxidation, and storage. We find that ritonavir (r, 5micromol/l) plus 20micromol/l of atazanavir (ATV), lopinavir (LPV), or darunavir (DRV) reduce palmitate oxidation(16-21%) in differentiated C2C12 myotubes. Palmitate oxidation was increased following exposure to high fatty acid media but this effect was blunted when myotubes were pre-exposed to the HIV-PIs. However, LPV/r and DRV/r, but not ATV/r suppressed palmitate uptake into myotubes. We found no effect of the HIV-PIs on FATP1, FATP4, or FABPpm but both CD36/FAT and carnitine palmitoyltransferase 1 (CPT1) were reduced by all three regimens though ATV/r caused only a small decrease in CPT1, relative to LPV/r or DRV/r. In contrast, sterol regulatory element binding protein-1 was increased by all 3 HIV-PIs. These findings suggest that HIV-PIs suppress fatty acid oxidation in murine skeletal muscle cells and that this may be related to decreases in cytosolic- and mitochondrial-associated fatty acid transporters. HIV-PIs may also directly impair fatty acid handling and partitioning in skeletal muscle, and this may contribute to the cluster of metabolic complications that occur in people living with HIV., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties., (Copyright 2010 Elsevier B.V. All rights reserved.)

Facilitation of fatty acid uptake in beta cells could potentially affect beta cell metabolism and secretory function; however such effects have not been clearly documented. CD36 facilitates uptake of fatty acids (FA) in muscle and adipose tissue and is likely to exert a similar effect in beta cells. We investigated the impact of over-expressing CD36 on fatty acid uptake and beta cell function by a Tet-on system in INS-1 cells. Doxycycline dose-dependently increased the CD36 protein with localization mainly in the cell membrane. Over-expression increased both specific uptake and efflux of oleate whereas intracellular glycerides were only marginally increased and incorporation of 14C-oleate or -palmitate into di- or triglycerides not affected. The normal potentiation of glucose-induced insulin secretion by acute addition of FA (50-100 micromol/l oleate and palmitate) was lost and the normal inhibitory effect of high glucose both on oleate oxidation and on the activity of carnitine palmitoyltransferase I was reduced. Over-expression did not induce apoptosis. We conclude that induction of the CD36 transporter increases uptake of FA, the consequences of which are blunting of the functional interplay between glucose and FA on insulin secretion and oxidative metabolism., (2009 Elsevier B.V. All rights reserved.)

The membrane-bound sensor kinase KdpD and the cytoplasmic response regulator KdpE regulate the expression of the kdpFABC operon coding for the high affinity potassium uptake system KdpFABC in Escherichia coli. The signal transduction cascade of this two component system is activated under K(+)-limiting conditions in the medium, but is less sensitive to high osmolality. In order to test whether K(+) limitation affects membrane phospholipid composition and whether this change affects kdpFABC expression, we analysed the phospholipid composition of E. coli under these conditions. Our measurements revealed that there is an increase in the cardiolipin (CL) content during the exponential growth phase at the expense of the zwitterionic phospholipid phosphatidylethanolamine. The higher anionic phospholipid content occurs along with an increase of transcriptional activity of the cls gene coding for CL synthase. Furthermore, in vivo studies with E. coli derivatives carrying mutations in genes coding for enzymes involved in phospholipid biosynthesis revealed that the increase in the anionic lipid composition enhances the expression rate of the kdpFABC operon. Finally, we show that kinase activity of KdpD is stimulated in its native membrane environment by fusion with liposomes of anionic, but reduced with liposomes of zwitterionic phospholipids.

The membrane lipid bilayer is one of the primary cellular components affected by variations in hydration level, which cause changes in lipid packing that may have detrimental effects on cell viability. In this study, Fourier transform infrared (FTIR) spectroscopy was used to quantify changes in the membrane phase behavior, as identified by membrane phase transition temperature (T(m)), of Escherichia coli during desiccation and rehydration. Extensive cell desiccation (1 week at 20%-40% RH) resulted in an increase in T(m) from 8.4+/-1.7 degrees C (in undried control samples) to 16.5+/-1.3 degrees C. Fatty acid methyl ester analysis (FAME) on desiccated samples showed an increase in the percent composition of saturated fatty acids (FAs) and a decrease in unsaturated FAs in comparison to undried control samples. However, rehydration of E. coli resulted in a gradual regression in T(m), which began approximately 1 day after initial rehydration and plateaued at 12.5+/-1.8 degrees C after approximately 2 days of rehydration. FAME analysis during progressive rehydration revealed an increase in the membrane percent composition of unsaturated FAs and a decrease in saturated FAs. Cell recovery analysis during rehydration supported the previous findings that showed that E. coli enter a viable but non-culturable (VBNC) state during desiccation and recover following prolonged rehydration. In addition, we found that the delay period of approximately 1 day of rehydration prior to membrane reconfiguration (i.e. decrease in T(m) and increase in membrane percent composition of unsaturated FAs) also preceded cell recovery. These results suggest that changes in membrane structure and state related to greater membrane fluidity may be associated with cell proliferation capabilities.

Nuclear receptors can be activated by chemicals, metabolites, hormones or environmental compounds to regulate gene expression. Bioassay-guided screening of mouse tissue extracts found that natural fatty acids of a certain carbon length and level of unsaturation could activate the mouse orphan nuclear receptor, testicular orphan receptor 4 (TR4). Subsequent experiments focused on gamma-linoleic acid, a compound identified during screening of mouse tissues that exerts regulatory activity in TR4 transactivation assays. gamma-linoleic acid positively modulates TR4 activity to promote the expression of downstream genes such as apolipoprotein E (ApoE) and phosphoenolpyruvate carboxykinase, and to activate a reporter carrying direct repeat 1 from the ApoE promoter. It also induced the interaction of TR4 with transcription coregulators such as RIP140 and PCAF. Comparisons of transactivation by TR4 and the metabolism-related peroxisome proliferator-activated nuclear receptors indicate that gamma-linoleic acid regulation is specific to TR4. The data suggest that TR4 might exert its physiological function by sensing certain lipids. Identifying these compounds could be useful for examining the physiological pathways in which TR4 and its target genes are involved.

The structure and thermotropic phase behaviour of aqueous dispersions of dipalmitoylphosphatidylcholine and glucosylceramide rich in C-24 fatty acyl residues was investigated by synchrotron X-ray diffraction methods. Binary mixtures comprised of molar ratios 2.5:100, 6.5:100, 12.6:100, 25:100, 40:100 and 50:100, glucolipid:phospholipid were examined in heating and cooling scans of 2 degrees /min between 25 and 85 degrees C. Small-angle reflections indicated coexisting lamellar structures over the entire temperature range investigated. Reversible thermotropic changes were observed in one lamellar structure that is consistent with transitions between gel, ripple and fluid lamellar phases of pure phospholipid. The temperature of these transitions, however, were progressively shifted up by about 5 degrees C in the mixture containing the highest proportion of glucolipid and coincided with a published endothermic peak observed in this mixture. A higher-temperature endotherm was associated with molecular rearrangements on transition of the gel phase phospholipid to the fluid phase. This rearrangement was associated with the appearance of identifiable transient intermediate structures in the small-angle scattering region. The glucolipid formed stoichiometric mixtures with the phospholipid at all temperatures investigated and there was no evidence of phase separation of pure glucolipid. Analysis of the wide-angle scattering profiles during an initial heating scan of a binary mixture comprised of 40:60 glucolipid:phospholipid was consistent with a phase transition of pure phospholipid at about 43 degrees C coexisting with a liquid-ordered phase formed from the two lipids. This was confirmed by analysis of the small-angle scattering peaks of this mixture recorded at 25 and 65 degrees C which showed that a glucolipid-rich phase coexisted with almost pure bilayers of phospholipid at both temperatures. The glucolipid-rich phase consisted of 45:55 mole ratio glucolipid:phospholipid at 25 degrees C with pure phospholipid in gel phase and 42:58 mole ratio at 65 degrees C when the phospholipid was in the fluid phase. The results are discussed with reference to the role of the length of the N-acyl substituent of the sphingolipids in formation of complexes with phospholipids.

Lung surfactant protein B (SP-B) is critical to minimizing surface tension in the alveoli. The C-terminus of SP-B, residues 59-80, has much of the surface activity of the full protein and serves as a template for the development of synthetic surfactant replacements. The molecular mechanisms responsible for its ability to restore lung compliance were investigated with circular dichroism, differential scanning calorimetry, and (31)P and (2)H solid-state NMR spectroscopy. SP-B(59-80) forms an amphipathic helix which alters lipid organization and acyl chain dynamics in fluid lamellar phase 4:1 DPPC:POPG and 3:1 POPC:POPG MLVs. At higher levels of SP-B(59-80) in the POPC:POPG lipid system a transition to a nonlamellar phase is observed while DPPC:POPG mixtures remain in a lamellar phase. Deuterium NMR shows an increase in acyl chain order in DPPC:POPG MLVs on addition of SP-B(59-80); in POPC:POPG MLVs, acyl chain order parameters decrease. Our results indicate SP-B(59-80) penetrates deeply into DPPC:POPG bilayers and binds more peripherally to POPC:POPG bilayers. Similar behavior has been observed for KL(4), a peptide mimetic of SP-B which was originally designed using SP-B(59-80) as a template and has been clinically demonstrated to be successful in treating respiratory distress syndrome. The ability of these helical peptides to differentially partition into lipid lamellae based on their degree of monounsaturation and subsequent changes in lipid dynamics suggest a mechanism for lipid organization and trafficking within the dynamic lung environment.

Comparative lipid analysis demonstrated reduced amount of PG (50%) and lower ratio of MGDG/DGDG in iron-stressed Synechococcus sp. PCC 7942 cells compared to cells grown under iron sufficient conditions. In parallel, the monoenoic (C:1) fatty acids in MGDG, DGDG and PG increased from 46.8%, 43.7% and 45.6%, respectively in control cells to 51.6%, 48.8% and 48.7%, respectively in iron-stressed cells. This suggests increased membrane dynamics, which may facilitate the diffusion of PQ and keep the PQ pool in relatively more oxidized state in iron-stressed compared to control cells. This was confirmed by chlorophyll fluorescence and thermoluminescence measurements. Analysis of carotenoid composition demonstrated that the induction of isiA (CP43') protein in response to iron stress is accompanied by significant increase of the relative abundance of all carotenoids. The quantity of carotenoids calculated on a Chl basis increased differentially with nostoxanthin, cryptoxanthin, zeaxanthin and beta-carotene showing 2.6-, 3.1-, 1.9- and 1.9-fold increases, respectively, while the relative amount of caloxanthin was increased only by 30%. HPLC analyses of the pigment composition of Chl-protein complexes separated by non-denaturating SDS-PAGE demonstrated even higher relative carotenoids content, especially of cryptoxanthin, in trimer and monomer PSI Chl-protein complexes co-migrating with CP43' from iron-stressed cells than in PSI complexes from control cells where CP43' is not present. This implies a carotenoid-binding role for the CP43' protein which supports our previous suggestion for effective energy quenching and photoprotective role of CP43' protein in cyanobacteria under iron stress.

The heat inactivation of Lactococcus lactis was studied by determination of cell counts, and by FT-IR spectroscopy recording the average structure of cell proteins. Cell counts were measured after incubation milk buffer or milk buffer with 1. 5 M sucrose, and FT-IR spectra were recorded in (2)H(2)O or (2)H(2)O with 1. 5 M sucrose in the range of 6-75 degrees Celsius. Sucrose protected L. lactis against heat inactivation. The cell counts differed by up to 6-log cycles after treatment in milk buffer as compared to milk buffer with sucrose. The (1)H/(2)H exchange in proteins, and secondary structure elements were detected by the analysis of amide I', amide II and amide II' bands. A reduced (1)H/(2)H exchange as well as a lower content of disordered structural elements was observed when sucrose was present. Conformational fluctuations of native proteins as indicated by the (1)H/(2)H exchange were apparent already at sublethal temperatures. The loss of viability of L. lactis occurred in the same temperature range as the loss of the protein secondary structure. These results demonstrate that sucrose protects L. lactis against heat inactivation, and that the increased heat stability of proteins in the presence of sucrose contributed to this enhanced heat resistance.

Electrophysiological characterisation of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into defined lipid bilayers. Here, we describe an automated technique for reconstitution of membrane proteins into lipid bilayer membranes, which substantially reduces both the reconstitution time and the amount of protein required for the membrane formation. The method allows the investigation of single protein channels as well as insertion of multiple copies (approximately 10(7)) into a single bilayer. Despite a comparatively large membrane area (up to 300 microm diameter), the high stability of the membrane permits the application of transmembrane voltages up to 300 mV. This feature is especially important for studies of inner membrane mitochondrial proteins, since they act at potentials up to approximately 200 mV under physiological conditions. It is a combination of these advantages that enables the detailed investigation of the minuscule single protein conductances typical for proton transporters. We have applied the new technique for the reconstitution and electrophysiological characterisation of human recombinant uncoupling protein 1, hUCP1, that has been overexpressed in E. coli and purified from inclusion bodies. We demonstrate that hUCP1 activity in the presence of fatty acids is comparable to the activity of UCP1 isolated from brown adipose tissue.

Conjugated linoleic acids (CLAs) are bioactive lipid compounds showing anti-atherogenic actions in cell culture experiments and animal models of atherosclerosis without exact knowledge about the underlying mechanisms. CLAs were recently reported to be further metabolized to bioactive conjugated metabolites indicating that these metabolites are possibly involved in mediating the anti-atherogenic actions of CLA. Regarding the lack of information with respect to the formation of CLA metabolites in the vascular endothelium, which is strongly involved in the process of atherosclerosis, the present study aimed to explore the potential formation of CLA metabolites in vascular endothelial cells. The results from the present study show for the first time that the CLA isomers cis-9, trans-11 CLA and trans-10, cis-12 CLA are metabolized within endothelial cells to beta-oxidation products such as CD16:2c7t9 and CD16:2t8c10 and elongation products such as CD20:2c11t13, CD20:2t12c14 as well as CD22:2c13t15 and CD22:2t14c16. Different CD16:2/CLA ratios observed between cells treated with different CLA isomers indicate that the metabolism of CLAs depends on the configuration of the conjugated double bonds. In conclusion, regarding the biological activity reported for CD20:2t12c14 and other metabolites of CLA, the present results indicate that metabolites of CLA are possibly also involved in mediating the anti-atherogenic actions of CLA.

After wounding, the moss Physcomitrella patens emits fatty acid derived volatiles like octenal, octenols and (2E)-nonenal. Flowering plants produce nonenal from C18-fatty acids via lipoxygenase and hydroperoxide lyase reactions, but the moss exploits the C20 precursor arachidonic acid for the formation of these oxylipins. We describe the isolation of the first cDNA (PpHPL) encoding a hydroperoxide lyase from a lower eukaryotic organism. The physiological pathway allocation and characterization of a downstream enal-isomerase gives a new picture for the formation of fatty acid derived volatiles from lower plants. Expression of a fusion protein with a yellow fluorescent protein in moss protoplasts showed that PpHPL was found in clusters in membranes of plastids. PpHPL can be classified as an unspecific hydroperoxide lyase having a substrate preference for 9-hydroperoxides of C18-fatty acids but also the predominant substrate 12-hydroperoxy arachidonic acid is accepted. Feeding experiments using arachidonic acid show an increase in the 12-hydroperoxide being metabolized to C8-aldehydes/alcohols and (3Z)-nonenal, which is rapidly isomerized to (2E)-nonenal. PpHPL knock out lines failed to emit (2E)-nonenal while formation of C8-volatiles was not affected indicating that in contrast to flowering plants, PpHPL is only involved in formation of a specific subset of volatiles.