Stability and aggregation studies of non-sonicated arsonolipid-containing vesicles

In this study, we investigated non-sonicated arsonolipid-containing liposomes (arsonoliposomes) in terms of the influence of lipid composition on their stability, assessed as membrane integrity and physical stability [size]. Vesicles consisting of plain arsonolipids or mixtures of arsonolipids with cholesterol [Chol] or with distearoyl-phospatidylcholine [DSPC] were studied. Membrane integrity was evaluated by measuring the retention of incorporated 5-(6)carboxyfluorescein [CF] during incubation of the vesicles in Tris buffer, pH 7.4. Photon correlation spectroscopy was used to investigate the time-dependent aggregation of arsonoliposomes in the absence and presence of Ca(2+)ions. Vesicles composed of plain C18 (acyl fatty chain) arsonolipids were found to be unstable, with only 15% of the initially incorporated CF remaining in the vesicles after 24 hours. The addition of Chol to the membrane (1:1 mol/mol) significantly increased the stability of arsonoliposomes, while the addition of DSPC to the lipid bilayer (1:1 mol/mol) increased vesicle stability to a lower extent. The results of particle size analysis showed that non-sonicated arsonoliposomes consisting of plain arsonolipid Ars/Stearic are highly and rapidly aggregated, while calcium-induced aggregation is also significant, but slower. Aggregation could not be always explained on the basis of zeta potential changes, indicating that the process is complex.     

Arsonoliposomes: effect of lipid composition on their stability and morphology

The influence of the lipid composition of arsonoliposomes on their membrane integrity was investigated to evaluate whether it is possible to combine their action with drugs that can be encapsulated in their aqueous interior. This was investigated by measuring the retention of vesicle-encapsulated calcein (100 mM) during incubation, in the absence and presence of serum proteins. Liposomes containing various concentrations of arsonolipid (with the palmitoyl side chain) as well as egg-lecithin (phosphatidylcholine, PC) and cholesterol (lipid/chol 2:1 mol:mol) were prepared. In some experiments, PC was replaced by the synthetic phospholipid DSPC. All PC/arsonoliposomes tested are stable after 24 h of incubation in buffer at 37 degrees C. After incubation in the presence of serum proteins, arsonoliposomes that contain low amounts of arsonolipid (up to 5 mol% of the lipid content without cholesterol) are stable, whereas increased release of calcein is observed when vesicle arsonolipid concentration is raised (from 5 to 15 mol%). Further increase of arsonolipid content results in immediate decrease of calcein latency while the remaining calcein is rapidly released during incubation. DSPC/arsonoliposomes are comparably more stable, and membrane integrity is independent of the vesicle arsonolipid content, in the range investigated (15-40 mol% of the lipid content without cholesterol). Thereby, we conclude that more stable arsonoliposomes that incorporate high arsonolipid concentrations may be produced when PC is replaced by DSPC. The latter arsonoliposomes provide a system that may be used for combining arsonolipid activity with the activity of other drugs.     

Arsonoliposomes: Effect of Lipid Composition on Their Stability and Morphology

The influence of the lipid composition of arsonoliposomes on their membrane integrity was investigated to evaluate whether it is possible to combine their action with drugs that can be encapsulated in their aqueous interior. This was investigated by measuring the retention of vesicle-encapsulated calcein (100 mM) during incubation, in the absence and presence of serum proteins. Liposomes containing various concentrations of arsonolipid (with the palmitoyl side chain) as well as egg-lecithin (phosphatidylcholine, PC) and cholesterol (lipid/chol 2:1 mol:mol) were prepared. In some experiments, PC was replaced by the synthetic phospholipid DSPC. All PC/arsonoliposomes tested are stable after 24 h of incubation in buffer at 37°C. After incubation in the presence of serum proteins, arsonoliposomes that contain low amounts of arsonolipid (up to 5 mol% of the lipid content without cholesterol) are stable, whereas increased release of calcein is observed when vesicle arsonolipid concentration is raised (from 5 to 15 mol%). Further increase of arsonolipid content results in immediate decrease of calcein latency while the remaining calcein is rapidly released during incubation. DSPC/arsonoliposomes are comparably more stable, and membrane integrity is independent of the vesicle arsonolipid content, in the range investigated (15–40 mol% of the lipid content without cholesterol). Thereby, we conclude that more stable arsonoliposomes that incorporate high arsonolipid concentrations may be produced when PC is replaced by DSPC. The latter arsonoliposomes provide a system that may be used for combining arsonolipid activity with the activity of other drugs.     

Preparation and properties of arsonolipid containing liposomes

Arsonolipids are analogs of phosphonolipids which have a chemically versatile head group. In preliminary cell culture studies, liposomes composed solely of arsonolipids or of phosholipid-arsonolipid mixtures, demonstrate a specific toxicity against cancer cells (Gortzi et al., unpublished results). The possibility of using such formulations as an alternative of arsenic trioxide with or without combination of other cytostatic agents (encapsulated in their aqueous interior) prompted the investigation of their physicochemical characteristics. Herein we compared the characteristics of arsonolipid containing vesicles with different lipid compositions. Experimental results and morphological observations reveal that non-sonicated formulations have different structures and stability (when both membrane integrity and aggregation are taken into account) depending on the acyl chain length of the arsonolipid. When phospholipids and especially cholesterol are included in their membranes almost all arsonolipids studied produce more stable vesicles. An interesting aspect of these arsonolipid containing vesicles is also their negative surface charge, which may be modulated by mixing phospholipids with arsonolipids. Sonicated vesicles have smaller sizes and profoundly higher stability, especially when containing cholesterol and phosphatidylcholine mixed with arsonolipids. The only exception is that of the arsonolipid with the C(12) acyl chain which was observed to produce long tubes which break down to cubes by sonication. In conclusion, these initial studies demonstrate that sonicated vesicles composed of arsonolipid and phospholipid mixtures mixed with cholesterol posses the stability required to be used as an arsonolipid delivery system. In addition, although cryo-electron microscopy demonstrated that the sonicated vesicles are elliptical in shape, their encapsulation efficiency is not significantly lower than sonicated phospholipid liposomes. Thereby, these vesicles may be also used for the delivery of other drug molecules which can be sufficiently retained in their aqueous interior.     

Incorporation of PEG-lipids in arsonoliposomes results in formation of highly stable arsenic-containing vesicles

We investigated the effect of pegylation on the physical stability, morphology and membrane integrity of arsonoliposomes. Arsonoliposomes composed of distearoylglycerophosphocholine (DSPC), cholesterol (Chol) and the palmitoyl side chain arsonolipid (with concentrations ranging from 0 mol% [DSPC/Chol vesicles] to 53 mol% of total lipid) containing either 4 or 8 mol% DPPE-PEG2000 or DSPE-PEG2000, were prepared by sonication. Arsonoliposome membrane integrity was evaluated by measuring the retention of encapsulated calcein in vesicles (during incubation in buffer or fetal calf serum [FCS]) while physical stability was evaluated by measuring vesicle dispersion turbidity (during incubation in water or CaCl(2)). Vesicle morphology was studied by cryo-electron microscopy. Experimental results show that: (i) PEG-lipids are incorporated in arsonoliposomes (as confirmed by the vesicle zeta potential modulation), (ii) pegylation of arsonoliposomes prevents their aggregation and fusion in the presence of calcium ions and (iii) when 8 mol% of PEG-DSPE is incorporated in arsonoliposomes based on their arsonolipid content, two groups of pegylated vesicles are formed: low content arsonoliposomes (<20 mol% arsonolipid) which are highly leaky and high content arsonoliposomes (>27 mol% arsonolipid) which are highly stable (70% calcein retention after 24h incubation in fetal calf serum [FCS]). In addition to high membrane integrity, the high content pegylated arsonoliposomes are morphologically perfect round-shaped vesicles without the sharp edges typically observed with non-pegylated DSPC-containing arsonoliposomes.     

Phase behavior of galactocerebrosides from bovine brain

Bovine brain cerebrosides (BOV-CER) were separated by high-performance liquid chromatography into cerebroside fractions with a single acyl chain type or with a relatively homogeneous acyl chain distribution. The thermal behavior of these isolated cerebroside fractions was studied by differential scanning calorimetry. Nonhydroxy (n-acyl) fatty acid cerebrosides (NFA-CER) possessing a saturated acyl chain (C16:0, C18:0, C24:0) exhibit their major order-disorder transition temperature TM at 83 degrees C, independent of chain length. NFA-CER possessing primarily unsaturated acyl chains (C24:1) exhibits TM at 70 degrees C. 2-Hydroxy fatty acid cerebrosides (HFA-CER), which possess a saturated hydroxyacyl chain (C18:0h, C24:0h), exhibit TM at 70-72 degrees C. Thus, naturally occurring cerebrosides exhibit high TM's that do not depend significantly on acyl chain length and that depend only to a small degree on unsaturation and the presence of a 2-hydroxy branch in the amide-linked chain. Isolated NFA-CER's each exhibit metastable polymorphism of the type previously described for unfractionated NFA-CER [Curatolo, W. (1982) Biochemistry 21, 1761]. Polymorphism in HFA-CER is complex, with a different type of thermal behavior observed for each isolated acyl chain fraction studied. On prolonged storage at low temperature, unfractionated HFA-CER and unfractionated BOV-CER reach a highly ordered gel state similar to that which is readily reached by NFA-CER's. These results indicate that all cerebrosides exhibit metastable polymorphism. However, the kinetic barriers to reaching the stable gel state are greater for HFA-CER and BOV-CER than for NFA-CER.     

Capability of arsonolipids to transport divalent cations: a Pressman cell study

The ability of the newly synthesized arsonolipids (2,3-diacyloxyprophlarsonic acids) to transport cations was studied using the Pressman cell. Experimental results demonstrate that arsonolipids are much more efficient carriers of Ca(2+) and Mg(2+) than natural phosphatidic acid in the Pressman cell experiments. The ability of arsonolipids to transfer Ca(2+) is affected by the lipid side chain length in the order: C(12)>C(14) approximately C(16). Ca(2+) is transferred faster than Mg(2+), suggesting that the latter is more tightly bound to the arsonolipids. The transfer kinetic curves are parabolic for C(12), while initially linear with a tendency to reach a steady state for C(14) and C(16), when the pH in the donor compartment was 8.3. The transport kinetics for both ions studied were best fitted by an equation derived from saturation kinetics that apply in reversible chemical reactions. The ion transfer rates increased as the pH in the donor compartment decreased.     

Elo1p-dependent carboxy-terminal elongation of C14:1Delta(9) to C16:1Delta(11) fatty acids in Saccharomyces cerevisiae

Saccharomyces cerevisiae medium-chain acyl elongase (ELO1) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media. Here we report that wild-type cells cultivated in myristoleic acid (C14:1Delta(9))-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Delta(11) fatty acid by gas chromatography-mass spectroscopy. Synthesis of C16:1Delta(11) was dependent on a functional ELO1 gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (alpha-elongation). In wild-type cells, the C16:1Delta(11) elongation product accounted for approximately 12% of the total fatty acids. This increased to 18% in cells that lacked a functional acyl chain desaturase (ole1Delta mutants) and hence were fully dependent on uptake and elongation of C14:1. The observation that ole1Delta mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient. Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes. In particular, the relative content of the two major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, was strongly dependent on the chain length of the supplemented fatty acid. Moreover, analysis of the acyl chain composition of individual phospholipid classes in cells supplemented with C14:1 revealed that the relative degree of acyl chain saturation characteristic for each phospholipid class appeared to be conserved, despite the gross alteration in the cellular acyl chain pool. Comparison of the distribution of fatty acids that were taken up and elongated (C16:1Delta(11)) to those that were endogenously synthesized by fatty acid synthetase and then desaturated by Ole1p (C16:1Delta(9)) in individual phospholipid classes finally suggested the presence of two different pools of diacylglycerol species. These results will be discussed in terms of biosynthesis of different phospholipid classes via either the de novo or the Kennedy pathway.     

Effects of aromatic residues at the ends of transmembrane alpha-helices on helix interactions with lipid bilayers

We have studied the effects of aromatic residues at the ends of peptides of the type Ac-KKGL(n)()WL(m)()KKA-amide on their interactions with lipid bilayers as a function of lipid fatty acyl chain length, physical phase, and charge. Peptide Ac-KKGFL(6)WL(8)FKKA-amide (F(2)L(14)) incorporated into bilayers of phosphatidylcholines containing monounsaturated fatty acyl chains of lengths C14-C24 at a peptide:lipid molar ratio of 1:100 in contrast to Ac-KKGL(7)WL(9)KKA-amide (L(16)) which did not incorporate at all into dierucoylphosphatidylcholine [di(C24:1)PC]; Ac-KKGYL(6)WL(8)YKKA-amide (Y(2)L(14)) incorporated partly into di(C24:1)PC. Lipid-binding constants relative to that for dioleoylphosphatidylcholine (C18:1)PC were obtained using a fluorescence quenching method. For Y(2)L(14) and F(2)L(14), relative lipid-binding constants increased with increasing fatty acyl chain length from C14 to C24; strongest binding did not occur at the point where the hydrophobic length of the peptide equalled the hydrophobic thickness of the bilayer. For Ac-KKGYL(9)WL(11)YKKA-amide (Y(2)L(20)), increasing chain length from C18 to C24 had little effect on relative binding constants. Anionic phospholipids bound more strongly than zwitterionic phospholipids to Y(2)L(14) and Y(2)L(20) but effects of charge were relatively small. In two phase (gel and liquid crystalline) mixtures, all the peptides partitioned more strongly into liquid crystalline than gel phase; effects were independent of the structure of the peptide or of the lipid (dipalmitoylphosphatidylcholine or bovine brain sphingomyelin). Addition of cholesterol had little effect on incorporation of the peptides into lipid bilayers. It is concluded that the presence of aromatic residues at the ends of transmembrane alpha-helices effectively buffers them against changes in bilayer thickness caused either by an increase in the chain length of the phospholipid or by the presence of cholesterol.     

The elongation of exogenous fatty acids and the control of phospholipid acyl chain length in Micrococcus cryophilus.

The synthesis of fatty acids de novo from acetate and the elongation of exogenous satuated fatty acids (C12-C18) by the psychrophilic bacterium Micrococcus cryophilus (A.T.C.C. 15174) grown at 1 or 20 degrees C was investigated. M. cryophilus normally contains only C16 and C18 acyl chains in its phospholipids, and the C18/C16 ratio is altered by changes in growth temperature. The bacterium was shown to regulate strictly its phospholipid acyl chain length and to be capable of directly elongating myristate and palmitate, and possibly laurate, to a mixture of C16 and C18 acyl chains. Retroconversion of stearate into palmitate also occurred. Fatty acid elongation could be distinguished from fatty acid synthesis de novo by the greater sensitivity of fatty acid elongation to inhibition by NaAsO2 under conditions when the supply of ATP and reduced nicotinamide nucleotides was not limiting. It is suggested that phospholipid acyl chain length may be controlled by a membrane-bound elongase enzyme, which interconverts C16 and C18 fatty acids via a C14 intermediate; the activity of the enzyme could be regulated by membrane lipid fluidity.     

The critical micelle concentrations of lysophosphatidic acid and sphingosylphosphorylcholine

The critical micelle concentrations (CMC) of lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) were measured by isothermal titration calorimetry. The CMC of LPA decreases with salt concentration and acyl chain length. In water at 25 °C, the CMC values of 1-acyl-2-lyso-sn-glycero-3-phosphatidic acid are 1.850, 0.540, 0.082, and 0.346 mM, respectively, when the acyl group is myristoyl, palmitoyl, stearoyl, and oleoyl. The CMC of SPC in 10 mM sodium phosphate buffer, pH 7.4, at 25 °C was 0.158 mM, and did not change with an increase in salt concentration.     

Optimization of receptor-G protein coupling by bilayer lipid composition II: formation of metarhodopsin II-transducin complex.

The visual transduction system was used as a model to investigate the effects of membrane lipid composition on receptor-G protein coupling. Rhodopsin was reconstituted into large, unilamellar phospholipid vesicles with varying acyl chain unsaturation, with and without cholesterol. The association constant (K(a)) for metarhodopsin II (MII) and transducin (G(t)) binding was determined by monitoring MII-G(t) complex formation spectrophotometrically. At 20 degrees C, in pH 7.5 isotonic buffer, the strongest MII-G(t) binding was observed in 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC), whereas the weakest binding was in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) with 30 mol% cholesterol. Increasing acyl chain unsaturation from 18:0,18:1PC to 18:0,22:6PC resulted in a 3-fold increase in K(a). The inclusion of 30 mol% cholesterol in the membrane reduced K(a) in both 18:0,22:6PC and 18:0,18:1PC. These findings demonstrate that membrane compositions can alter the signaling cascade by changing protein-protein interactions occurring predominantly in the hydrophilic region of the proteins, external to the lipid bilayer. These findings, if extended to other members of the superfamily of G protein-coupled receptors, suggest that a loss in efficiency of receptor-G protein binding is a contributing factor to the loss of cognitive skills, odor and spatial discrimination, and visual function associated with n-3 fatty acid deficiency.     

Properties of the binding sites for the sn-1 and sn-2 acyl chains on the phosphatidylinositol transfer protein from bovine brain

We have studied the properties of the fatty acyl binding sites of the phosphatidylinositol transfer protein (PI-TP) from bovine brain, by measuring the binding and transfer of pyrenylacyl-containing phosphatidylinositol (PyrPI) species and pyrenylacyl-containing phosphatidylcholine (PyrPC) species as a function of the acyl chain length. The PyrPI species carried a pyrene-labeled acyl chain of variable length in the sn-2 position and either palmitic acid [C(16)], palmitoleic acid [C(16:1)], or stearic acid [C(18:1)] in the sn-1 position. Binding and transfer of the PI species increased in the order C(18) less than C(16) less than C(16:1), with a distinct preference for those species that carry a pyrenyloctanoyl [Pyr(8)] or a pyrenyldecanoyl [Pyr(10)] chain. The PyrPC species studied consisted of two sets of positional isomers: one set contained a pyrenylacyl chain of variable length and a C(16) chain, and the other set contained an unlabeled chain of variable length and a Pyr(10) chain. The binding and transfer experiments showed that PI-TP discriminates between positional isomers with a preference for the species with a pyrenylacyl chain in the sn-1 position. This discrimination is interpreted to indicate that separate binding sites exist for the sn-1 and sn-2 acyl chains. From the binding and transfer profiles it is apparent that the binding sites differ in their preference for a particular acyl chain length. The binding and transfer vs chain length profiles were quite similar for C(16)Pyr(x)PC and C(16)Pyr(x)PI species, suggesting that the sn-2 acyl chains of PI and PC share a common binding site in PI-TP.     

Identification of a 51-kilodalton polypeptide fatty acyl chain acceptor in soluble extracts from mouse cardiac tissue

We have identified a protein in the soluble fraction from mouse cardiac tissue extracts which is rapidly and selectively acylated by myristyl CoA. This protein was partially purified by anion-exchange chromatography and gel filtration, and the acylation reaction was measured using [3H]myristyl CoA as substrate, followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to resolve [3H]fatty acyl polypeptides. The [3H]acyl protein migrated as heterogeneous bands corresponding to relative masses (MrS) of 42,000-51,000 under nonreducing conditions or as a single polypeptide of Mr 51,000 in the presence of reducing agents. Fatty acyl chain incorporation into protein was very rapid and already maximum after 30 s of incubation, whereas no acylation was detected using heat-denatured samples or when the reaction was stopped immediately after initiation. Only the acyl CoA served as fatty acyl chain donor. No incorporation into protein occurred when myristyl CoA was substituted by myristic acid, ATP, and CoA. A time-dependent reduction in the level of [3H]fatty acyl polypeptide was observed upon addition of excess unlabeled myristyl CoA, indicating the ability of the labeled acyl moiety of the protein to turn over during incubation. The saturated C10:0, C14:0, and C16:0 acyl CoAs were more effective to chase the label from the [3H]acyl polypeptide than the C18:0 and C18:1 acyl CoAs. These results provide evidence for a 51-kilodalton polypeptide which serves as an acceptor for fatty acyl chains and could represent an important intermediate in fatty acyl chain transfer reactions in cardiac tissue.     

Fatty acid synthetase activity in Mycobacterium smegmatis. Characterization of the acyl carrier protein-dependent elongating system.

Mycobacterium smegmatis extracts contain two fatty acyl synthetase systems (Brindley, D.N., Matsumura, S. and Bloch, K. (1966) Nature 224, 666-669). One is the extensively studied multienzyme complex, (molecular weight 1.39 - 10(6)) which produces shorter C16 and C18) and longer (C24 and higher) fatty acids in a bimodal pattern. The second synthetase is acyl carrier-protein (ACP) dependent and elongates the CoA derivatives of C12 and longer chains. In contrast to the type I synthetase which also extends long fatty acyl chains, the ACP-dependent system produces homologous fatty acids up to 30 carbon atoms long in approximately equal proportions. Other properties which distinguish the ACP-dependent system from the multienzyme complex include the resistance to high concentrations of palmitoyl-CoA and to low ionic strength and the lack of stimulation by mycobacterial polysaccharides. The possibility that the two fatty acid synthetases are complimentary in their function is discussed.     

Phase diagrams of pseudo-binary phospholipid systems. I. Influence of the chain length differences on the miscibility properties of cephaline/cephaline/water systems

The miscibility properties of homologous cephalines (PEs) were studied by means of differential scanning calorimetry (DSC). The phase diagrams of 5 pseudo-binary cephaline/cephaline/water systems (50% water) are discussed. In the high temperature L alpha-phase, all the homologous cephalines of fatty acid chain length from C = 12 to C = 18 were completely miscible. On the other hand in the low temperature L beta-phase, a miscibility gap occurred in dependence on the differences of the acyl chain lengths. Further, a distinct succession of the phase diagram types was observed according to increasing chain length differences of the PEs: complete miscibility (systems di-(C12:O)-PE/di-(C14:O)-PE/H2O; di-(C14:O)-PE/di-C(16:O)-PE/H2O)----peritectic mixing behaviour (systems di-(C12:O)-PE/di-(C16:O)-PE/H2O; di-(C14:O)-PE/di-(C18:O)-PE/H2O)----eutectic mixing behaviour (system di-(C12:O)-PE/di-(C18:O)-PE/H2O). The change in the type of phase diagram from azeotropic to semi-azeotropic and from semi-azeotropic to eutectic is interpreted by means of the Landau theory.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

Presence of covalently attached fatty acids in rat apolipoprotein B via thiolester linkages

Thiolester linked lipids in rat apolipoprotein B (ApoB) were examined by incubating reduced and carboxymethylated ApoB in 6 M urea buffer with [14C]methylamine at pH 8.5, 30 degrees C. It was observed that [14C]methylamine was covalently incorporated into ApoB, and there was a [14C]methylamine modified product which was lipid in nature. After extraction with organic solvents, the [14C]methylamine labeled product showed its Rf on TLC to be similar to that of the synthetic N-methyl fatty acyl amide. After purification on TLC and transesterification with 3 N methanolic HC1, methyl esters of C16:0, C18:0 and C18:1 fatty acids were identified by gas-liquid chromatography. These results suggest that rat ApoB, similar to human ApoB, contained covalently linked fatty acids through the high energy, labile thiolester bonds.     

Phosphatidylcholine fluidity and structure affect lecithin:cholesterol acyltransferase activity

The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type (cis vs. trans), or position of the double bonds in 18 or 20 carbon sn-2 fatty acyl chains and recombined with [(3)H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn-2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn-2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon (r(2) = 0.61, n = 18) and 20 carbon (r(2) = 0.93, n = 5) sn-2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn-2 fatty acyl chain PC species and 90% of an inert PC ether matrix (sn-1 18:1, sn-2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated (r(2) = 0.71) with that of 100% PC rHDL containing the same 18 carbon sn-2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity. We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant.     

A high yield procedure for the preparation of arsonolipids (2,3-diacyloxypropylarsonic acids)

The crucial step in the preparation of the title arsonolipids starting from the dichloromethane-soluble dithioarsonite CH₂(OH)CH(OH)CH₂–As(SPh)₂ is to avoid an internal cyclization during the acylation which protects the primary –OH group from being acylated. This was to a large extent accomplished by using fatty acyl chloride in the presence of the weak base pyridine and controlling the temperature and rate of the acyl chloride addition, giving ～70% yields of arsonolipids. The presence of catalytic amounts of 4-dimethylaminopyridine boosted the yields to 82–85%. This yield is a great improvement over the yields (20–55%) previously achieved. The acylating systems (RCO)₂O or RCOCl and BF₃·Et₂O gave only moderate yields (25–60%) of arsonolipids.