The formation of phosphatidylcholine oxidation products by stimulated phagocytes

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. The formation of monohydroperoxides (814 m/z) and bis-hydroperoxides (846 m/z) of PAPC was observed. However, the major oxidized product occurred at 828 m/z, and was identified as 1-palmitoyl-2-(5,6-epoxyisoprostane E-2)-sn-glycero-3-phosphocholine. These products were also formed in incubations where the neutrophils were replaced by mononuclear cells, and the amounts produced per million cells were similar. These results show that following oxidative attack by phagocytes stimulated by PMA, intact phospholipid oxidation products can be detected. The identification of an epoxyisoprostane phospholipid as the major product of phagocyte-induced phospholipid oxidation is novel, and in view of its inflammatory properties has implications for phagocyte involvement in atherogenesis.

CD14-dependent clearance of apoptotic cells by human macrophages:the role of phosphatidyiserine

Apoptotic-cell clearance is dependent on several macrophage surface molecules, including CD14. Phosphatidylserine (PS) becomes externalised during apoptosis and participates in the clearance process through its ability to bind to a novel receptor, PS-R. CD14 has the proven ability to bind phospholipids and may function as an alternative receptor for the externallsed PS of apoptotic cells. Here we demonstrate that CD14 does not function preferentially as a PS receptor in apoptotic-cell clearance. Compared with phosphatidylcholine and phosphatidylethanolamine, PS was the least active phospholipid binding to human monocyte-derived macrophages and showed no specificity for soluble or membrane-anchored CD14. Significantly, PS-containing liposomes a e to inhibit CD14-dependent uptake of apoptotic cells by macrophages. PS exposure was, however, found to be insufficient for either CD14-dependent or CD14-independent apoptotic-cell uptake by phagocytes. The additional features that enable apoptotic-cell clearance are derived from mechanisms that can be divorced temporally from those responsible for the morphological features of apoptosis.

Phagocyte oxidation of phospholipids: comparison of hydroperoxide, chlorohydrin and epoxyisoprostane formation by LC-MS

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by isolated human polymorphonuclear and mononuclear leukocytes, to provide a model of lipid oxidation in the absence of competing protein. PMA-stimulated cells were incubated with phospholipid vesicles contammg dipalmitoyl phosphatidylcholine (DPPC), palmitoyl-arachidonoyl phosphatidylcholine (PAPC), and stearoyl-oleoyl phosphatidylcholine (SOPC), before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry. In this system, oxidized phosphatidylcholines elute earlier than the native lipids owing to their decreased hydrophobicity, and can be identified according to their molecular mass. The formation of monohydroperoxides of P APC was observed routinely, together with low levels of hydroxides, but no chlorohydrin derivatives of P APC or SOPC were detected. However, the major oxidized product occurred at 828 m/z, and was identified as I-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine. These results show that phagocytes triggered by PMA cause oxidative damage to lipids predominantly by free radical mechanisms, and that electrophilic addition involving HOCl is not a major mechanism of attack. The contribution of myeloperoxidase and metal ions to the oxidation process is currently being investigated, and preliminary data suggest that myeloperoxidase-derived oxidants are responsible for the epoxyisoprostane phospholipid formation. The identification of an epoxyisoprostane phospholipid as the major product following phagocyte-induced phospholipid oxidation is novel and has implications for phagocyte involvement in atherogenesis.

Polymer-lipid interactions:biomimetic self-assembly behaviour and surface properties of poly(styrene-alt-maleic acid) with diacylphosphatidylcholines

Abstract Various lubricating body fluids at tissue interfaces are composed mainly of combinations of phospholipids and amphipathic apoproteins. The challenge in producing synthetic replacements for them is not replacing the phospholipid, which is readily available in synthetic form, but replacing the apoprotein component, more specifically, its unique biophysical properties rather than its chemistry. The potential of amphiphilic reactive hypercoiling behaviour of poly(styrene-alt-maleic acid) (PSMA) was studied in combination with two diacylphosphatidylcholines (PC) of different chain lengths in aqueous solution. The surface properties of the mixtures were characterized by conventional Langmuir-Wilhelmy balance (surface pressure under compression) and the du Noüy tensiometer (surface tension of the non-compressed mixtures). Surface tension values and 31P NMR demonstrated that self-assembly of polymer-phospholipid mixtures were pH and concentration-dependent. Finally, the particle size and zeta potential measurements of this self-assembly showed that it can form negatively charged nanosized structures that might find use as drug or lipids release systems on interfaces such as the tear film or lung interfacial layers. The structural reorganization was sensitive to the alkyl chain length of the PC.

Effect of incorporating cholesterol into DDA:TDB liposomal adjuvants on Bilayer properties, biodistribution, and immune responses

Cholesterol is an abundant component of mammalian cell membranes and has been extensively studied as an artificial membrane stabilizer in a wide range of phospholipid liposome systems. In this study, the aim was to investigate the role of cholesterol in cationic liposomal adjuvant system based on dimethyldioctadecylammonium (DDA) and trehalose 6,6'-dibehenate (TDB) which has been shown as a strong adjuvant system for vaccines against a wide range of diseases. Packaging of cholesterol within DDA:TDB liposomes was investigated using differential scanning calorimetery and surface pressure-area isotherms of lipid monolayers; incorporation of cholesterol into liposomal membranes promoted the formation of a liquid-condensed monolayer and removed the main phase transition temperature of the system, resulting in an increased bilayer fluidity and reduced antigen retention in vitro. In vivo biodistribution studies found that this increase in membrane fluidity did not alter deposition of liposomes and antigen at the site of injection. In terms of immune responses, early (12 days after immunization) IgG responses were reduced by inclusion of cholesterol; thereafter there were no differences in antibody (IgG, IgG1, IgG2b) responses promoted by DDA:TDB liposomes with and without cholesterol. However, significantly higher levels of IFN-gamma were induced by DDA:TDB liposomes, and liposome uptake by macrophages in vitro was also shown to be higher for DDA:TDB liposomes compared to their cholesterol-containing counterparts, suggesting that small changes in bilayer mechanics can impact both cellular interactions and immune responses. © 2013 American Chemical Society.

Biomimetic synthetic lubricants (Biosurfactants)

Poly(styrene-co-maleic anhydride) (PSMA) based copolymers are known to undergo conformational transition in response to environmental stimuli. This smart behaviour makes it possible to mimic the behaviour of native apoproteins. The primary aim of this study was to develop a better understanding of the structure-property relationships of various PSMA-based copolymers sought. The work undertaken in this thesis has revealed that the responsive behaviour of PSMA-based copolymers can be tailored by varying the molecular weight, hydrophobic (styrene) and hydrophilic (maleic acid) balance, and more so in the presence of additional hydrophobic, mono-partial ester moieties. Novel hydrophilic and hydrophobic synthetic surfactant protein analogues have successfully been prepared. These novel lipid solubilising agents possess a broad range of HLB (hydrophilic-lipophilic balance) values that have been estimated. NMR spectroscopy was utilised to confirm the structures for PSMA-based copolymers sought and proved useful in furthering understanding of the structure-property relationships of PSMA-based copolymers. The association of PSMA with the polar phospholipid, 2-dilauryl-sn-glycero-3- phosphocholine (DLPC) produces polymer-lipid complexes analogous to lipoprotein assemblies present in the blood plasma. NMR analysis reveals that the PSMA-based copolymers are not perfectly alternating. Regio-irregular structures, atactic and random monomer sequence distribution have been identified for all materials studied. Novel lipid solubilising agents (polyanionic surfactants) have successfully been synthesised from a broad range of PSMA-based copolymers with desired estimated HLB values that interact with polar phospholipids (DLPC/DPPC) uniquely. Very low static and dynamic surface tensions have been observed via the du Noϋy ring method and Langmuir techniques and correlate well with the estimated HLB values. Synthetic protein-lipid analogues have been successfully synthesised, that mimic the unique surface properties of native biological lubricants without the use of solvents. The novel PSMA-DLPC complexes have successfully been combined with hyaluronan (hyaluronic acid, HA). Today, the employment of HA is economically feasible, because it is readily available from bacterial fermentation processes in a thermally stable form - HyaCare®. The work undertaken in this thesis highlights the usage of HA in biolubrication applications and how this can be optimised and thus justified by carefully selecting the biological source, concentration, molecular weight, purity and most importantly by combining it with compatible boundary lubricating agents (polar phospholipids). Experimental evidence supports the belief that the combined HA and PSMA-DLPC complexes provide a balance of rheological, biotribological and surface properties that are composition dependent, and show competitive advantage as novel synthetic biological lubricants (biosurfactants).

Association behavior of the synthetic protein-lipid complex analogues

Hypercoiling poly(styrene-ALT-maleic anhydride) (PSMA) is known to undergo conformational transition in response to environmental stimuli. This behavior allows it to associate with the phospholipid, 2-dilauryl-SN-glycero-3- phosphocholine (DLPC) to produce nanostructures analogous to lipoproteins. The complex represents a new bio-mimetic delivery vehicle with applications in the cosmetic and pharmaceutical industries. This study investigates, for the first time, the association behavior of PSMA and DLPC through the combination of different analytical techniques. The results indicate that the association is primarily driven by hydrophobic interactions and depends on various factors including the polymer/lipid ratio, the polymer molecular weight and the pH of the aqueous environment. The conformational transition of PSMA leads to the formation of discrete micellar complexes involving anisotropic-to-isotropic lipid phase transformation. As the number of hydrophobic moieties in the polymer is increased, the pH-dependent conformational transition of the polymer plays less important part in achieving this phase transition of the lipid. © (2012) Trans Tech Publications.

Targeted mass spectrometry methods for detecting oxidative post-translational modifications

Oxidative post-translational modifications (oxPTMs) can alter the function of proteins, and are important in the redox regulation of cell behaviour. The most informative technique to detect and locate oxPTMs within proteins is mass spectrometry (MS). However, proteomic MS data are usually searched against theoretical databases using statistical search engines, and the occurrence of unspecified or multiple modifications, or other unexpected features, can lead to failure to detect the modifications and erroneous identifications of oxPTMs. We have developed a new approach for mining data from accurate mass instruments that allows multiple modifications to be examined. Accurate mass extracted ion chromatograms (XIC) for specific reporter ions from peptides containing oxPTMs were generated from standard LC-MSMS data acquired on a rapid-scanning high-resolution mass spectrometer (ABSciex 5600 Triple TOF). The method was tested using proteins from human plasma or isolated LDL. A variety of modifications including chlorotyrosine, nitrotyrosine, kynurenine, oxidation of lysine, and oxidized phospholipid adducts were detected. For example, the use of a reporter ion at 184.074 Da/e, corresponding to phosphocholine, was used to identify for the first time intact oxidized phosphatidylcholine adducts on LDL. In all cases the modifications were confirmed by manual sequencing. ApoB-100 containing oxidized lipid adducts was detected even in healthy human samples, as well as LDL from patients with chronic kidney disease. The accurate mass XIC method gave a lower false positive rate than normal database searching using statistical search engines, and identified more oxidatively modified peptides. A major advantage was that additional modifications could be searched after data collection, and multiple modifications on a single peptide identified. The oxPTMs present on albumin and ApoB-100 have potential as indicators of oxidative damage in ageing or inflammatory diseases.

Optimisation of chromatographic separation of oxidised phospholipids and detection with targeted approaches provides greater coverage of the oxidised lipidome

Phospholipid oxidation by adventitious damage generates a wide variety of products with potentially novel biological activities that can modulate inflammatory processes associated with various diseases. To understand the biological importance of oxidised phospholipids (OxPL) and their potential role as disease biomarkers requires precise information about the abundance of these compounds in cells and tissues. There are many chemiluminescence and spectrophotometric assays available for detecting oxidised phospholipids, but they all have some limitations. Mass spectrometry coupled with liquid chromatography is a powerful and sensitive approach that can provide detailed information about the oxidative lipidome, but challenges still remain. The aim of this work is to develop improved methods for detection of OxPLs by optimisation of chromatographic separation through testing several reverse phase columns and solvent systems, and using targeted mass spectrometry approaches. Initial experiments were carried out using oxidation products generated in vitro to optimise the chromatography separation parameters and mass spectrometry parameters. We have evaluated the chromatographic separation of oxidised phosphatidylcholines (OxPCs) and oxidised phosphatidylethanolamines (OXPEs) using C8, C18 and C30 reverse phase, polystyrene – divinylbenzene based monolithic and mixed – mode hydrophilic interaction (HILIC) columns, interfaced with mass spectrometry. Our results suggest that the monolithic column was best able to separate short chain OxPCs and OxPEs from long chain oxidised and native PCs and PEs. However, variation in charge of polar head groups and extreme diversity of oxidised species make analysis of several classes of OxPLs within one analytical run impractical. We evaluated and optimised the chromatographic separation of OxPLs by serially coupling two columns: HILIC and monolith column that provided us the larger coverage of OxPL species in a single analytical run.

Results of an isotope tracer experiment with arctic deep-sea nematodes

A stable isotope (13C)-labeling experiment was performed to quantify the importance of bacterial carbon as a food source for an Arctic deep-sea nematode community. Bacterial functional groups were isotopically enriched with 13C-glucose, 13C-acetate, 13C- bicarbonate, and 13C-amino acids injected into sediments collected from 1280 m depth at 79uN, 6uE, west of Svalbard. Incorporation of the 13C label into bacterial phospholipid-derived fatty acids (PLFAs) and nematodes in the top 5 cm of the sediment was monitored over a 7-d period. The 13C dynamics of nematodes was fitted with a simple isotope turnover model to derive the importance of the different bacterial functional groups as carbon sources for the nematodes. The different substrates clearly labeled different bacterial groups as evidenced by differential labeling of the PLFA patterns. The deep-sea nematode community incorporated a very limited amount of the label, and the isotope turnover model showed that the dynamics of the isotope transfer could not be attributed to bacterivory. The low enrichment of nematodes suggests a limited passive uptake of injected 13C-labeled substrates. The lack of accumulation suggests that the injected 13C-labeled dissolved organic carbon compounds are not important as carbon sources for deep-sea nematodes. Since earlier studies with isotopically enriched algae also found limited uptake by nematodes, the food sources of deep-sea nematodes remain unclear.

The effects of dietary omega-3 fatty acids on brain function are sex, age and brain-region specific

Omega (n)-3 polyunsaturated fatty acids (PUFA) have beneficial effects in neuropsychiatric illnesses. The goals of this thesis were to determine the effects of feeding diets varying in n-3 PUFA on brain fatty acid composition, and neurotrophin and myelin-related gene expression of the brain in an age, sex, and region-specific manner. A diet high in n-3 PUFA altered phospholipid docosahexaenoic acid (DHA) and oleic acid composition in an age, sex, and region-specific manner. Diet had no effect on the mRNA expression of brain-derived neurotrophic factor (BDNF) and tropomyosin-receptor kinase-B (TrkB); however, stearoyl-CoA desaturase-1 (SCD1) and myelin basic protein (MBP) gene expression increased in offspring fed a diet high in n-3 PUFA in an age, sex, and region-specific manner. DHA treatment to ex vivo cerebral cortical cells showed an increase in BDNF, TrkB, SCD1, and MBP mRNA expression compared to control cells. The mRNA expression of BDNF and SCD1 was higher in DHA treated cells compared to arachidonic acid treated cells. Overall, the data presented in this thesis suggests that the potential benefits of n-3 PUFA on brain function are sex, age and brain-region specific.

Geochemistry, fatty acid content and phylogenetic affiliation of soil samples from Livingston Island, South Shetland Archipelago, Antarctica

Microorganisms inhabit very different soil habitats in the ice-free areas of Antarctica, playing a major role in nutrient cycling in cold environments. We studied the soil characteristics and the dominant bacterial composition from nine different soil profiles located on Livingston Island (maritime Antarctica). The total carbon (TC) and total nitrogen (TN) values were high for the vegetated soils, decreasing with depth, whereas the values for the mineral soils were generally low. Soil pH was more acidic for moss-covered soils and neutral to alkaline for mineral soils. Numbers of culturable heterotrophic bacteria were higher at vegetated sites, but significant numbers were also detectable in carbon-depleted soils. Patterns of denaturing gradient gel electrophoresis (DGGE) revealed a highly heterogeneous picture throughout the soil profiles. Subsequent sequencing of DGGE bands revealed in total 252 sequences that could be assigned to 114 operational taxonomic units, showing the dominance of members of the Bacteroidetes and Acidobacteria. The results of phospholipid fatty acid analysis showed a lack of unsaturated fatty acids for most of the samples. Samples with a prevalence of unsaturated over saturated fatty acids were restricted to several surface samples. Statistical analysis showed that the dominant soil bacterial community composition is most affected by TC and TN contents and soil physical factors such as grain size and moisture, but not pH. Keywords

The cellular lipids of Romboutsia.

We have examined the lipids of three isolates, Romboutsia lituseburensis, Romboutsia ilealis, and Romboutsia sp. strain FRIFI, of the newly described genus Romboutsia by two-dimensional thin-layer chromatography (2D-TLC) and by liquid chromatography/mass spectrometry (LC/MS). We have found three phospholipids, phosphatidylglycerol (PG), cardiolipin and phosphatidic acid in all three species. A fourth phospholipid, lysyl-PG, was found in R. lituseburensis and strain FRIFI. Polyprenyl-phosphates were identified in the lipid extracts of all three species. Three glycolipids, mono-, di- and tri-hexosyldiacylglycerol, were common to all three species. An additional glycolipid, tetrahexosyl-diacylglycerol was identified in strain FRIFI. Acylated trihexosyldiacylglycerol and acyl-tetrahexosydiacylglycerol were also found in R. ilealis and strain FRIFI. Remarkably, no alk-1-enyl ether lipids (plasmalogens) were present in Romboutsia as distinct from bacteria of the related genus Clostridium in which these ether lipids are common. We have compared the lipidome of Romboutsia with that recently described for Clostridium difficile, which has plasmalogens, no lysyl-PG, and no tetrahexosyl-diacylglycerol. According to 16S rRNA gene sequencing, Romboutsia spp. and C. difficile are closely related (>95% sequence identity).

Phonon anharmonicity and negative thermal expansion in SnSe

The anharmonic phonon properties of SnSe in the Pnma phase were investigated with a combination of experiments and first-principles simulations. Using inelastic neutron scattering (INS) and nuclear resonant inelastic X-ray scattering (NRIXS), we have measured the phonon dispersions and density of states (DOS) and their temperature dependence, which revealed a strong, inhomogeneous shift and broadening of the spectrum on warming. First-principles simulations were performed to rationalize these measurements, and to explain the previously reported anisotropic thermal expansion, in particular the negative thermal expansion within the Sn-Se bilayers. Including the anisotropic strain dependence of the phonon free energy, in addition to the electronic ground state energy, is essential to reproduce the negative thermal expansion. From the phonon DOS obtained with INS and additional calorimetry measurements, we quantify the harmonic, dilational, and anharmonic components of the phonon entropy, heat capacity, and free energy. The origin of the anharmonic phonon thermodynamics is linked to the electronic structure.

Soil properties and microbial indicators of samples from Lake Wellman, Darwin Mountains, Antarctica

Four pedons on each of four drift sheets in the Lake Wellman area of the Darwin Mountains were sampled for chemical and microbial analyses. The four drifts, Hatherton, Britannia, Danum, and Isca, ranged from early Holocene (10 ka) to mid-Quaternary (c. 900 ka). The soil properties of weathering stage, salt stage, and depths of staining, visible salts, ghosts, and coherence increase with drift age. The landforms contain primarily high-centred polygons with windblown snow in the troughs. The soils are dominantly complexes of Typic Haplorthels and Typic Haploturbels. The soils were dry and alkaline with low levels of organic carbon, nitrogen and phosphorus. Electrical conductivity was high accompanied by high levels of water soluble anions and cations (especially calcium and sulphate in older soils). Soil microbial biomass, measured as phospholipid fatty acids, and numbers of culturable heterotrophic microbes, were low, with highest levels detected in less developed soils from the Hatherton drift. The microbial community structure of the Hatherton soil also differed from that of the Britannia, Danum and Isca soils. Ordination revealed the soil microbial community structure was influenced by soil development and organic carbon.