BBA - Molecular and Cell Biology of Lipids (v.1531, #1-2)

The use of Pseudomonas acyl-CoA synthetase to form acyl-CoAs from dicarboxylic fatty acids by Kenneth G Milne; Angela Mehlert; Michael A.J Ferguson (1-3).
Pseudomonas acyl-CoA synthetase is shown to act on saturated dicarboxylic acids with a chain length of C10 or greater to produce conjugates containing a single CoA unit. The synthetase can, therefore, be used to generate novel acyl-CoA analogues for studies on proteins that utilise, bind to, or are modulated by acyl-CoAs.
Keywords: Acyl-CoA; Dicarboxylic; Synthetase;

Structural models of human apolipoprotein A-I: a critical analysis and review by Christie G. Brouillette; G.M. Anantharamaiah; Jeffrey A. Engler; David W. Borhani (4-46).
Human apolipoprotein (apo) A-I has been the subject of intense investigation because of its well-documented anti-atherogenic properties. About 70% of the protein found in high density lipoprotein complexes is apo A-I, a molecule that contains a series of highly homologous amphiphatic α-helices. A number of significant experimental observations have allowed increasing sophisticated structural models for both the lipid-bound and the lipid-free forms of the apo A-I molecule to be tested critically. It seems clear, for example, that interactions between amphipathic domains in apo A-I may be crucial to understanding the dynamic nature of the molecule and the pathways by which the lipid-free molecule binds to lipid, both in a discoidal and a spherical particle. The state of the art of these structural studies is discussed and placed in context with current models and concepts of the physiological role of apo A-I and high-density lipoprotein in atherosclerosis and lipid metabolism.
Keywords: Apolipoprotein A-I mutant; High density lipoprotein; Exchangeable apolipoprotein; Apolipoprotein E; Apolipophorin III; Molten globule; HDL model; Lipid binding; Protein folding;

Oil-bodies as substrates for lipolytic enzymes by Frédéric Beisson; Natalie Ferté; Ségolène Bruley; Robert Voultoury; Robert Verger; Vincent Arondel (47-58).
Plant seeds store triacylglycerols (TAGs) in intracellular organelles called oil-bodies or oleosomes, which consist of oil droplets covered by a coat of phospholipids and proteins. During seed germination, the TAGs of oil-bodies hydrolysed by lipases sustain the growth of the seedlings. The mechanism whereby lipases gain access to their substrate in these organelles is largely unknown. One of the questions that arises is whether the protein/phospholipid coat of oil-bodies prevents the access of lipase to the oil core. We have investigated the susceptibility of almond oil-bodies to in vitro lipolysis by various purified lipases with a broad range of biochemical properties. We have found that all the enzymes assayed were capable of releasing on their own free fatty acids from the TAG of oil-bodies. Depending on the lipase, the specific activity measured on oil-bodies using the pH-stat technique was found to range from 18 to 38% of the specific activity measured on almond oil emulsified by gum arabic. Some of these lipases are known to have a dual lipase/phospholipase activity. However, no correlation was found to exist between the ability of a lipase to readily and efficiently hydrolyse the TAG content of oil-bodies and the presence of a phospholipase activity. Kinetic studies indicate that oil-bodies behave as a substrate as other proteolipid organelles such as milk fat globules. Finally we have shown that a purified water-soluble plant lipase on its own can easily hydrolyse oil-bodies in vitro. Our results suggest that the lipolysis of oil-bodies in seedlings might occur without any pre-hydrolysis of the protein coat.
Keywords: Almond seed; Oil-body; Oleosin; Lipase; Colipase; Lag time;

Lysophosphatidic acid (LPA) is a phospholipid growth mediator found in serum at 2–20 μM. In many cell types, including human airway smooth muscle (HASM) cells, LPA-induced proliferation occurs at 10–100 μM LPA. At these concentrations LPA forms Ca2+ precipitates. The potential involvement of Ca2+ and Ca2+-LPA precipitates in LPA-induced HASM cell mitogenesis was investigated. In the absence of extracellular Ca2+, 10 and 30 μM LPA stimulated HASM cell mitogenesis. However, with 100 μM LPA in the absence of extracellular Ca2+, HASM cells exhibited a profound shape change and loss of viability, determined to be apoptosis by both DNA staining and assessment of cytosolic nucleosomal reactivity. A bioassay based on the adenosine 3′:5′-cyclic monophosphate response of C62B rat glioma cells was used to measure the bioactivity of LPA solutions prepared in Ca2+-free and Ca2+-containing medium. After 24 h, a 100 μM LPA solution in Ca2+-free medium contained markedly greater bioactivity than a 100 μM LPA solution made in Ca2+-containing medium. In summary, formation of Ca2+-LPA precipitates decreases the amount of biologically active LPA in solution, and high concentrations of bioactive LPA achieved in Ca2+-free but not in Ca2+-containing medium induce apoptosis of HASM cells.
Keywords: Airway smooth muscle cell; Lysophosphatidic acid; Extracellular Ca2+; Apoptosis; Proliferation;

Lysophosphatidylcholine derived from deer antler extract suppresses hyphal transition in Candida albicans through MAP kinase pathway by Juyoung Min; Youn-Jin Lee; Young-Ah Kim; Hyun-Sook Park; So-Yeop Han; Gil-Ja Jhon; Wonja Choi (77-89).
A family of 2-lysophosphatidylcholines (lyso-PCs) was isolated from deer antler extract, guided exclusively by hyphal transition inhibitory activity in Candida albicans. Structural determination of the isolated lyso-PCs by spectroscopic methods, including infrared spectroscopy, 1H nuclear magnetic resonance (NMR), 13C NMR, 2D correlation spectroscopy NMR, fast atom bombardment mass spectrometry and tandem mass spectrometry, confirmed that the natural products were composed of at least four different lyso-PCs varying in fatty acid moiety at the sn-1 position of the glycerol backbone. The major lyso-PCs were confirmed as 1-stearoyl-, 1-oleoyl-, 1-linoleoyl- and 1-palmitoyl-2-lyso-sn-glycero-3-phosphatidylcholines. Lyso-PC specifically suppressed the morphogenic transition from yeast to hyphae in C. albicans, without affecting the growth of either yeast or hyphae. Lyso-PC exerted hyphal transition that suppressed activity in the broad spectrum of the Candida species, such as C. albicans, Candida krusei, Candida guilliermondii and Candida parapsilosis. Northern analysis indicated that the suppression was mediated through the mitogen-activated protein kinase pathway.
Keywords: 2-Lysophosphatidylcholine; Deer antler extract; Hyphal transition; Mitogen-activated protein kinase pathway; Candida albicans;

In order to clarify the pathological outcome of congenital diaphragmatic hernia (CDH), we devised an animal model of CDH by administration of 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) to pregnant rats, and determined the level and distribution of lung surfactant using the monoclonal antibody toward sphingomyelin and disaturated phosphatidylcholine (disat-PC). In control rats, the concentration of disat-PC was found to increase greatly from 16 to 18 days of gestation. Intragastric administration of nitrofen to pregnant rats at day 9 of gestation resulted in CDH in 42.7% of fetuses delivered after 20 days of gestation. In nitrofen-treated fetuses, the concentration of disat-PC in the lungs was lower than those in control fetuses, and surfactant apoprotein SP-A was similarly reduced in nitrofen-treated fetuses. However, the concentration of disat-PC in nitrofen-treated fetuses was higher than that in control fetuses at 18 days of gestation, indicating a synthetic potential of surfactant in nitrofen-treated fetuses comparable to that at the late stage of normal gestation. Immunohistochemical study with the antibody revealed that surfactant phospholipid was mainly in the form of intracellular granules in nitrofen-treated fetuses, probably causing the hypoplastic lungs and then CDH, in contrast to the uniform distribution on the pulmonary alveolar surface in control fetuses.
Keywords: Amniotic fluid; Congenital diaphragmatic hernia; Disaturated phosphatidylcholine; Pulmonary alveoli; Sphingomyelin; Thin layer chromatography-immunostaining;

Immunoadjuvant activity of interferon-γ-liposomes co-administered with influenza vaccines by M.L van Slooten; I Hayon; I Babai; Z Zakay-Rones; E Wagner; G Storm; E Kedar (99-110).
In an attempt to potentiate the relatively low immunogenicity of the currently used influenza vaccines, especially in high-risk groups, monovalent and divalent subunit vaccine preparations were co-administered with free or liposome-associated murine interferon γ (mIFNγ) as an adjuvant. Recombinant murine IFNγ was entrapped (50–70% efficiency) in two types of large multilamellar vesicles: mIFNγ-LIP A-‘conventional’ liposomes, and mIFNγ-LIP B- ‘surface-depleted’ liposomes, in which 60 and 8% of the associated cytokine was located at the external liposome membrane, respectively. Subunit preparations containing the viral surface proteins hemagglutinin and neuraminidase (HN) were injected once, i.p. (0.5 μg each), into BALB/c mice, alone and combined with free or liposomal mIFNγ (mIFNγ-LIP, 0.5 or 3.0 μg). Sera were tested 3–16 weeks post-vaccination by hemagglutination inhibition (HI), and by ELISA for IgG1 and IgG2a antibodies (Abs). In addition, protective immunity against intranasal viral infection was assayed at 11 and 17 weeks post-vaccination. The results showed that: (a) Vaccination with HN alone produces very low HI and IgG titers and does not afford any protection. (b) Although co-administration with free mIFNγ (particularly using 3.0 μg) markedly enhances HI titer as well as the IgG1 and IgG2a levels, protection is negligible (0–33%). (c) In most cases, mIFNγ-LIP is significantly more potent than free mIFNγ (2–40-fold increase in Ab titer), and the low dose (0.5 μg) is generally more efficient than the high dose. Up to 83% of the mice co-vaccinated with mIFNγ-LIP were protected against viral challenge. (d) Both the IgG2a level and the HI titer appear to be crucial for protection. (e) Although the two liposomal preparations differ in their cytokine release profile in vivo and in their bioactivity in vitro, their adjuvant activity is comparable.
Keywords: Liposome; Drug delivery system; Influenza vaccine; Interferon-γ;

The relationship between 1H-NMR mobile lipid intensity and cholesterol in two human tumor multidrug resistant cell lines (MCF-7 and LoVo) by Maria T Santini; Rocco Romano; Gabriella Rainaldi; Perla Filippini; Elena Bravo; Loredana Porcu; Andrea Motta; Annarica Calcabrini; Stefania Meschini; Pietro L Indovina; Giuseppe Arancia (111-131).
The high resolution proton nuclear magnetic resonance (1H-NMR) spectra of two different cell lines exhibiting multidrug resistance (MDR) as demonstrated by the expression of the well-known energy-driven, membrane-bound 170 kDa P-glycoprotein pump known as Pgp were investigated. In particular, the mobile lipid (ML) profile, and the growth and biochemical characteristics of MCF-7 (human mammary carcinoma) and LoVo (human colon adenocarcinoma) sensitive and resistant tumor cells were compared. The results indicate that both MCF-7 and LoVo resistant cells have a higher ML intensity than their respective sensitive counterparts. However, since sensitive and resistant cells of each pair grow in the same manner, variations in growth characteristics do not appear to be the cause of the ML changes as has been suggested by other authors in non-resistant tumor cells. In order to investigate further the origin of the ML changes, lipid analyses were conducted in sensitive and resistant cell types. The results of these experiments show that resistant cells of both cell types have a greater amount of esterified cholesterol and saturated cholesteryl ester and triglyceride fatty acid than their sensitive counterparts. From a thorough analysis of the data obtained in this paper utilizing numerous techniques including biological, biophysical and biochemical ones, it is hypothesized that cholesterol and triglyceride play a pivotal role in inducing changes in NMR ML signals. The importance of these lipid variations in MDR is discussed in view of the controversy regarding the origin of ML signals and the paramount role played by the Pgp pump in resistance.
Keywords: Multidrug resistance; 1H-NMR; Mobile lipid; Cholesterol; P-glycoprotein;

cDNAs encoding major plasma apolipoproteins (apo) were cloned from the eel Anguilla japonica liver and their nucleotide sequences determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that eel lipoproteins contain apolipoproteins of 28 kDa and 14 kDa as major components. Each of the two apolipoproteins showed two isoforms having different isoelectric points as demonstrated by two-dimensional electrophoresis. The two 28 kDa components had different N-terminal amino acid sequences, whereas the two 14 kDa components had an identical one. Then cDNA clones encoding these apolipoproteins were isolated from a cDNA library constructed from the eel liver. An acidic 28 kDa component (28 kDa-1) consisted of 259 amino acids including a putative signal peptide of 27 residues, whereas a basic 28 kDa component (28 kDa-2) was composed of 260 amino acids containing a putative signal peptide of 23 residues. The tandem repeating units, which are characteristic of apolipoproteins, for 28 kDa-1 showed 27.8% identity to that of porcine apoA-IV, although mammalian apoA-IV is about 40 kDa and much larger than 28 kDa-1. However, the repeating units of 28 kDa-2 showed 52.5% identity to that of Atlantic salmon apoA-I. The 14 kDa apolipoprotein consisted of 142 amino acids containing a putative signal peptide of 20 residues. It has a novel sequence differing from apolipoproteins of other vertebrates. The transcriptional expressions of 28 kDa-1, 28 kDa-2, and 14 kDa components were all restricted to the liver, except for the transcripts of 28 kDa-2 which were also slightly expressed in the intestine.
Keywords: Eel; Plasma; Lipoprotein; Apolipoprotein;

t-Butyl hydroperoxide and oxidized low density lipoprotein enhance phospholipid hydrolysis in lipopolysaccharide-stimulated retinal pericytes by Gabriella Lupo; Carmelina D Anfuso; Nicola Ragusa; Robert P Strosznajder; Michal Walski; Mario Alberghina (143-155).
Free radicals induced by organic peroxides or oxidized low density lipoprotein (oxLDL) play a critical role in the development of atherosclerosis. In investigating this process, and the concomitant inflammatory response, the role of pericytes, cells supporting the endothelial ones in blood vessels, has received little attention. In this study we tested the hypothesis that tert-butyl hydroperoxide (t-BuOOH) and oxLDL, administered in sublethal doses to the culture medium of retinal pericytes, function as prooxidant signals to increase the stimulation of the peroxidation process induced by lipopolysaccharide (LPS). Confluent cell monolayers were exposed to t-BuOOH (25–400 μM), native LDL or oxLDL (3.4–340 nmol hydroperoxides/mg protein, 1–100 μM). LPS (1 μg/ml), t-BuOOH (200 μM), and oxLDL (100 μM), but not native LDL, incubated for 24 h with cells, markedly increased lipid peroxidation, cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release in a time- and dose-dependent manner. AACOCF3, a potent cPLA2 inhibitor, and the antioxidant α-tocopherol strongly inhibited the prooxidant-stimulated AA release. Long-term exposure to maximal concentrations of t-BuOOH (400 μM) or oxLDL (100 μM) had a sharp cytotoxic effect on the cells, described by morphological and biochemical indices. The presence of t-BuOOH or oxLDL at the same time, synergistically increased phospholipid hydrolysis induced by LPS alone. 400 μM t-BuOOH or 100 μM oxLDL had no significant effect on the stimulation of an apoptosis process estimated by DNA laddering and light and electron microscopy. The results indicate that (i) pericytes may be the target of extensive oxidative damage; (ii) activation of cPLA2 mediates AA liberation; (iii) as long-term regulatory signals, organic peroxide and specific constituents of oxLDL increase the pericyte ability to degrade membrane phospholipids mediated by LPS which was used, in the present study, to simulate in vitro an inflammatory burst in the retinal capillaries.
Keywords: Peroxidation; Pericyte; Phospholipid; Oxidized low density lipoprotein; Cytosolic phospholipase A2;

Previously we have shown that intraamniotic administration of ethyl docosahexaenoate (Et-DHA) to pregnant rats resulted in decreased lipid peroxidation in the fetal brain, under a variety of conditions (S. Glozman, P. Green, E. Yavin, J. Neurochem. 70 (1998) 2482–2491). In the present study we examine the potential mechanisms to explain this effect. This was done by a pharmacological approach, utilizing brain slice preparations from Et-DHA treated or control rats in the presence of various agents and examining the formation of products in the tissue slices or incubation medium. Et-DHA treated brains produced 2–3-fold more prostanoids (PN) than control brains, indicating cyclooxygenase (COX) activation. Indomethacin at 50 μM inhibited PN formation and also abolished Et-DHA induced decrease in lipid peroxides, as evident by the levels of thiobarbituric acid reactive substances (TBARS) released in the medium. The phospholipase A2 inhibitors quinacrine and p-bromophenacyl bromide added at 0.1 mM concentration each to either slices from controls or Et-DHA treated fetal brains, decreased TBARS production. Et-DHA treated brains released 2.2-fold more nitric oxide (NO) than control brains and NO synthase (NOS) inhibitors abolished this effect. Increasing the concentration of NO by the addition of an NO donor greatly decreased the concentration of the TBARS in the medium. These results suggest that at least some of the effect of Et-DHA on decreased lipid peroxidation may be explained by a shift of oxygen species utilization via enzymatically regulated, therefore metabolically controlled, COX and NOS activities.
Keywords: Arachidonic acid metabolism; Phospholipid; Cyclooxygenase; Phospholipase A2; Docosahexaenoic acid;