Regulation of yeast phospholipid biosynthetic gene expression in response to inositol involves two superimposed mechanisms.

Transcription of phospholipid biosynthetic genes in the yeast Saccharomyces cerevisiae is maximally derepressed when cells are grown in the absence of inositol and repressed when the cells are grown in its presence. We have previously suggested that this response to inositol may be dictated by regulating transcription of the cognate activator gene, INO2. However, it was also known that cells which harbor a mutant opi1 allele express constitutively derepressed levels of target genes (INO1 and CHO1), implicating the OPI1 negative regulatory gene in the response to inositol. These observations suggested that the response to inositol may involve both regulation of INO2 transcription as well as OPI1-mediated repression. We investigated these possibilities by examining the effect of inositol on target gene expression in a strain containing the INO2 gene under control of the GAL1 promoter. In this strain, transcription of the INO2 gene was regulated in response to galactose but was insensitive to inositol. The expression of the INO1 and CHO1 target genes was still responsive to inositol even though expression of the INO2 gene was unresponsive. However, the level of expression of the INO1 and CHO1 target genes correlated with the level of INO2 transcription. Furthermore, the effect of inositol on target gene expression was eliminated by deleting the OPI1 gene in the GAL1-INO2-containing strain. These data suggest that the OPI1 gene product is the primary target (sensor) of the inositol response and that derepression of INO2 transcription determines the degree of expression of the target genes.

Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Low concentrations of diacylglycerol promote the binding of apolipophorin III to a phospholipid bilayer: a surface plasmon resonance spectroscopy study.

The binding of the exchangeable apolipoprotein apolipophorin III (apoLp-III) to an egg phosphatidylcholine bilayer as a function of the concentration of diacylglycerol (DG) in the bilayer was studied by surface plasmon resonance spectroscopy. At a DG concentration of 2 mol % in the bilayer, the binding of apoLp-III reached saturation. Under saturating conditions, apoLp-III forms a closely packed monolayer approximately 55 A thick, in which each molecule of protein occupies approximately 500 A2 at the membrane surface. These dimensions are consistent with the molecular size of the apoLp-III molecule determined by x-ray crystallography, if apoLp-III binds to the bilayer with the long axis of the apoLp-III normal to the membrane surface. In the absence of protein, the overall structure of the lipid bilayer was not significantly changed up to 2.5 mol% DG. However, at 4 and 6 mol % DG, the presence of nonbilayer structures was observed. The addition of apoLp-III to a membrane containing 6 mol % DG promoted the formation of large lipid-protein complexes. These data support a two-step sequential binding mechanism for binding of apoLp-III to a lipid surface. The first step is a recognition process, consisting of the adsorption of apoLp-III to a nascent hydrophobic defect in the phospholipid bilayer caused by the presence of DG. This recognition process might depend on the presence of a hydrophobic sensor located at one of the ends of the long axis of the apoLp-III molecule but would be consolidated through H-bond and electrostatic interactions. Once primary binding is achieved, subsequent enlargement of the hydrophobic defect in the lipid surface would trigger the unfolding of the apolipoprotein and binding via the amphipathic alpha-helices. This two-step sequential binding mechanism could be a general mechanism for all exchangeable apolipoproteins. A possible physiological role of the ability of apoLp-III to bind to lipid structures in two orientations is also proposed.

CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication.

Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs.

Pseudo-ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-film hydration method

Pseudo-ternary phase diagrams of the polar lipids Quil A, cholesterol (Chol) and phosphatidylcholine (PC) in aqueous mixtures prepared by the lipid film hydration method (where dried lipid film of phospholipids and cholesterol are hydrated by an aqueous solution of Quil A) were investigated in terms of the types of particulate structures formed therein. Negative staining transmission electron microscopy and polarized light microscopy were used to characterize the colloidal and coarse dispersed particles present in the systems. Pseudo-ternary phase diagrams were established for lipid mixtures hydrated in water and in Tris buffer (pH 7.4). The effect of equilibration time was also studied with respect to systems hydrated in water where the samples were stored for 2 months at 4degreesC. Depending on the mass ratio of Quil A, Chol and PC in the systems, various colloidal particles including ISCOM matrices, liposomes, ring-like micelles and worm-like micelles were observed. Other colloidal particles were also observed as minor structures in the presence of these predominant colloids including helices, layered structures and lamellae (hexagonal pattern of ring-like micelles). In terms of the conditions which appeared to promote the formation of ISCOM matrices, the area of the phase diagrams associated with systems containing these structures increased in the order: hydrated in water/short equilibration period < hydrated in buffer/short equilibration period < hydrated in water/prolonged equilibration period. ISCOM matrices appeared to form over time from samples, which initially contained a high concentration of ring-like micelles suggesting that these colloidal structures may be precursors to ISCOM matrix formation. Helices were also frequently found in samples containing ISCOM matrices as a minor colloidal structure. Equilibration time and presence of buffer salts also promoted the formation of liposomes in systems not containing Quil A. These parameters however, did not appear to significantly affect the occurrence and predominance of other structures present in the pseudo-binary systems containing Quil A. Pseudo-ternary phase diagrams of PC, Chol and Quil A are important to identify combinations which will produce different colloidal structures, particularly ISCOM matrices, by the method of lipid film hydration. Colloidal structures comprising these three components are readily prepared by hydration of dried lipid films and may have application in vaccine delivery where the functionality of ISCOMs has clearly been demonstrated. (C) 2003 Elsevier B.V. All rights reserved.

A comparison of pseudo-ternary diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by lipid-film hydration and dialysis

Pseudo-ternary diagrams for Quil A, phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) and cholesterol were established in order to identify combinations that result in the formation of immune-stimulating complex (ISCOM) matrices and other colloidal structures produced by these three components in aqueous systems following lipid-film hydration or dialysis (methods that can be used to produce ISCOMs). In addition, the effect of equilibration time (1 month at 4degreesC) on the structures formed by the various combinations of the three components was investigated. Depending on the ratio of Quil A, cholesterol and phospholipid, different colloidal particles, including ISCOM matrices, liposomes and ring-like micelles, were found irrespective of the preparation method used. In contrast, worm-like micelles were only observed in systems prepared by lipid-film hydration. For samples prepared by dialysis, ISCOM matrices were predominantly found near the Quil A apex of the pseudo-ternary diagram (> 50% Quil A). On the other hand, for samples prepared by lipid-film hydration, ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram (> 50% phospholipid). The regions in the pseudo-ternary diagrams in which ISCOM matrices were observed increased following an extended equilibration time, particularly for samples prepared by lipid-film hydration. Differences were also observed between pseudoternary diagrams prepared using either PE or PC as phospholipids.

Surface-active phospholipid (surfactant) in equine tendon and tendon sheath fluid

AIM: To investigate the presence of surface-active phospholipid (SAPL, or surfactant) in equine tendon and tendon sheath fluid. METHODS: The left front flexor tendon and sheath were removed from five Thoroughbred horses. Phospholipid was extracted from tendon sheath fluid using Folch reagent and quantified using spectroscopy. Transmission electron microscopy (TEM) was used to observe the tendon surfaces. RESULTS: The presence of phospholipid (90.6 (SD 4.3) mu g/ml) in tendon sheath fluid, plus the appearance of oligolamellar layers and lamellar bodies on the tendon surface were indicative of SAPL. CONCLUSIONS: Evidence of SAPL was found in equine tendon, and may have a similar lubricating function as reported for synovial joints. CLINICAL RELEVANCE: These findings may have important implications for normal tendon function and possible therapeutic adjuncts for tendon and tendon sheath injuries.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Molecular dynamics study of MscL interactions with a curved lipid bilayer

Mechanosensitivity is a ubiquitous sensory mechanism found in living organisms. The simplest known mechanotransducing mechanism is found in bacteria in the form of the mechanosensitive membrane channel of large conductance, MscL. This channel has been studied extensively using a variety of methods at a functional and structural level. The channel is gated by membrane tension in the lipid bilayer alone. It serves as a safety valve protecting bacterial cells against hypoosmotic shock. MscL of Escherichia coli embedded in bilayers composed of asymmetric amounts of single-tailed and double-tailed lipids has been shown to gate spontaneously, even in the absence of membrane tension. To gain insight into the effect of the lipid membrane composition and geometry on MscL structure, a fully solvated, all-atom model of MscL in a stress-free curved bilayer composed of double- and single-tailed lipids was studied using a 9.5-ns molecular dynamics simulation. The bilayer was modeled as a domed structure accommodating the asymmetric composition of the monolayers. During the course of the simulation a spontaneous restructuring of the periplasmic loops occurred, leading to interactions between one of the loops and phospholipid headgroups. Previous experimental studies of the role of the loops agree with the observation that opening starts with a restructuring of the periplasmic loop, suggesting an effect of the curved bilayer. Because of limited resources, only one simulation of the large system was performed. However, the results obtained suggest that through the geometry and composition of the bilayer the protein structure can be affected even on short timescales.

Phase behavior of a phospholipid/fatty acid/water mixture studied in atomic detail

Molecular dynamics simulations have been used to study the phase behavior of a dipalmitoylphosphatidylcholine (DPPC)/palmitic acid (PA)/water 1:2:20 mixture in atomic detail. Starting from a random solution of DPPC and PA in water, the system adopts either a gel phase at temperatures below similar to 330 K or an inverted hexagonal phase above similar to 330 K in good agreement with experiment. It has also been possible to observe the direct transformation from a gel to an inverted hexagonal phase at elevated temperature (similar to 390 K). During this transformation, a metastable fluid lamellar intermediate is observed. Interlamellar connections or stalks form spontaneously on a nanosecond time scale and subsequently elongate, leading to the formation of an inverted hexagonal phase. This work opens the possibility of studying in detail how the formation of nonlamellar phases is affected by lipid composition and (fusion) peptides and, thus, is an important step toward understanding related biological processes, such as membrane fusion.

Solubilisation of drugs within liposomal bilayers:alternatives to cholesterol as a membrane stabilising agent

Objectives The aim of this work was to investigate the effect of cholesterol on the bilayer loading of drugs and their subsequent release and to investigate fatty alcohols as an alternative bilayer stabiliser to cholesterol. Methods The loading and release rates of four low solubility drugs (diazepam, ibuprofen, midazolam and propofol) incorporated within the bilayer of multilamellar liposomes which contained a range of cholesterol (0–33 mol/mol%) or a fatty alcohol (tetradecanol, hexadecanol and octadecanol) were investigated. The molecular packing of these various systems was also investigated in Langmuir monolayer studies. Key findings Loading and release of drugs within the liposome bilayer was shown to be influenced by their cholesterol content: increasing cholesterol content was shown to reduce drug incorporation and inclusion of cholesterol in the bilayer changed the release profile of propofol from zero-order, for phosphatidyl choline only liposomes, to a first-order model when 11 to 33 total molar % of cholesterol was present in the formulation. At higher bilayer concentrations substitution of cholesterol with tetradecanol was shown to have less of a detrimental impact on bilayer drug loading. However, the presence of cholesterol within the liposome bilayer was shown to reduce drug release compared with fatty alcohols. Monolayer studies undertaken showed that effective mean area per molecule for a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) : cholesterol mixture deviated by 9% from the predicted area compared with 5% with a similar DSPC : tetradecanol mixture. This evidence, combined with cholesterol being a much more bulky structure, indicated that the condensing influence of tetradecanol was less compared with cholesterol, thus supporting the reduced impact of tetradecanol on drug loading and drug retention. Conclusions Liposomes can be effectively formulated using fatty alcohols as an alternative bilayer stabiliser to cholesterol. The general similarities in the characteristics of liposomes containing fatty alcohols or cholesterol suggest a common behavioural influence for both compounds within the bilayer.

αα'-trehalose 6,6'-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying

Trehalose is a well known protector of biostructures like liposomes and proteins during freeze-drying, but still today there is a big debate regarding its mechanism of action. In previous experiments we have shown that trehalose is able to protect a non-phospholipid-based liposomal adjuvant (designated CAF01) composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6-dibehenate (TDB) during freeze-drying [D. Christensen, C. Foged, I. Rosenkrands, H.M. Nielsen, P. Andersen, E.M. Agger, Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying, Biochim. Biophys. Acta, Biomembr. 1768 (2007) 2120-2129]. Furthermore it was seen that TDB is required for the stabilizing effect of trehalose. Herein, we show using the Langmuir-Blodgett technique that a high concentration of TDB present at the water-lipid interface results in a surface pressure around 67 mN/m as compared to that of pure DDA which is approximately 47 mN/m in the compressed state. This indicates that the attractive forces between the trehalose head group of TDB and water are greater than those between the quaternary ammonium head group of DDA and water. Furthermore, addition of trehalose to a DDA monolayer containing small amounts of TDB also increases the surface pressure, which is not observed in the absence of TDB. This suggests that even small amounts of trehalose groups on TDB present at the water-lipid interface associate free trehalose to the liposome surface, presumably by hydrogen bonding between the trehalose head groups of TDB and the free trehalose molecules. Hence, for CAF01 the TDB component not only stabilizes the cationic liposomes and enhances the immune response but also facilitates the cryo-/lyoprotection by trehalose through direct interaction with the head group of TDB. Furthermore the results indicate that direct interaction with liposome surfaces is necessary for trehalose to enable protection during freeze-drying.