Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism最新文献

Gangliosides shed by tumor cells are immunosuppressive molecules, but the mechanisms of shedding are poorly understood. We therefore conducted a comprehensive study of shedding to identify the natural forms of shed gangliosides. By chemical detection and mass spectrometric analysis of the gangliosides of YAC-1 murine lymphoma cells, we first confirmed that all major ganglioside species are released. Then, by the combination of metabolic and cell surface radiolabeling, we further demonstrated that gangliosides are released directly from the cell plasma membrane, i.e. by shedding. Ultracentrifugation separated the conditioned medium of metabolically radiolabeled cells cultured in either serum-free or serum-containing medium into: (1) a pellet of 100–200 nm membrane vesicles (visualized by electron microscopy) containing nearly one-third of total shed gangliosides; and (2) the supernatant, which contained soluble gangliosides (two-thirds of the total shed gangliosides). Although the ganglioside concentration in the conditioned medium (6–14×10⁻⁸ M) was above the critical micelle concentration of purified YAC-1 gangliosides (<1×10⁻⁸ M), by gel filtration >90% of the soluble gangliosides were found in monomeric form (MW <2 kDa) and only <10% in micelles (130 kDa). Ultrafiltration of fresh conditioned medium likewise showed the existence of monomers, and the findings were confirmed in human Daoy medulloblastoma and mouse MEB4 melanoma cells. Thus, in their natural states, shed tumor cell gangliosides exist in three forms: membrane vesicles, micelles, and monomers.

Oxidation of unsaturated phosphatidylcholine (PC) produces fragmented phospholipids which have similar bioactivities as the platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-PC). Since a large number of molecular species are produced upon PC oxidation, the active ingredients have not been identified. We synthesized several short-chain PCs which are known to be characteristic PC oxidation products to test their PAF-like activity. The synthetic PCs contained palmitoyl or hexadecyl residues (both C₁₆) in sn-1 position, and propionyl (C₃), valeroyl (C₅), succinyl (C₄ with ω-carboxyl), glutaroyl (C₅ with ω-carboxyl), or suberoyl (C₈ with ω-carboxyl) residues in sn-2 position. Biological activity was measured by: (1) increase of intracellular calcium in human monocytes; (2) [³H]serotonin release from rabbit platelets; and (3) aggregation of human platelets. Specificity of the cellular response was tested by inhibition with the PAF-receptor antagonists BN 52021 and WEB 2086. Synthetic PC oxidation products activated both monocytes and platelets in a PAF-specific manner. The effective concentration varied with respect to assay system and chemical structure. In general, 1-hexadecyl-PCs were more effective than 1-palmitoyl-PCs, while increasing chain length in sn-2 position lowered biological activity. However, several 1-palmitoyl-PCs activated monocytes in concentrations between 10⁻⁸ and 10⁻⁶ M. In contrast, platelets were less susceptible to 1-palmitoyl-PCs. No significant difference was found between 2-valeroyl-PC (C₅ with ω-methyl) and 2-glutaroyl-PC (C₅ with ω-carboxyl). The data suggest that typical products of PC oxidation, containing propionyl, succinyl, or glutaroyl residues in sn-2 position, display PAF-like activity at micromolar concentrations.

The phospholipase gene phl was identified from Vibrio mimicus (ATCC33653) and sequenced. The entire open reading frame (ORF) was composed of 1410 nucleotides and encoding 470 amino acids. The phl was placed upstream of hemolysin gene (vmhA) with opposite direction of transcription. From the BLAST search program, the deduced amino acids sequence showed 74.4% identity with phospholipase gene (lec) from V. cholerae El Tor. The entire ORF of phospholipase gene was amplified by PCR and inserted into an Escherichia coli expression vector, pET22b(+) and introduced E. coli BL21(DE3). SDS–PAGE demonstrated that a protein corresponding to the phospholipase was overexpressed and migrated at a molecular mass of 53 kDa.

Protein palmitoylation in vitro was studied using bovine rhodopsin as the substrate and a partially purified acylating enzymatic activity (PAT) from placental membranes. PAT incorporates fatty acid into rhodopsin with higher efficiency (10 times higher initial rate), as compared to autoacylation. The activity is sensitive to heat and trypsin, indicating a protein-mediated enzymatic process and requires the native conformation of rhodopsin. The presence of deacylated, free cysteine residues in dark-adapted rhodopsin increases palmitoylation via PAT. The sites for non-enzymatic and enzymatic palmitoylation could not be distinguished by peptide mapping. The reversible palmitoylation described here will provide a tool for the study of the role of palmitoylation in photoreceptor function.

Our recent finding of the co-localization of 11-cis retinyl esters and 11-cis retinyl ester hydrolase (11-cis REH) activity in bovine retinal pigment epithelium (RPE) plasma membrane (PM) has led us to explore the possibility that the PM may provide 11-cis retinal for rhodopsin regeneration. In the RPE, visual chromophore is synthesized via a membrane associated 11-cis retinol dehydrogenase (11-cis RD). Accordingly, bovine RPE membranes enriched with either endoplasmic reticulum (ER) or plasma membrane (PM) enzyme markers were prepared and assayed for visual cycle enzyme activities. Pronounced 11-cis RD activity was associated with both ER- and PM-enriched membrane fractions. In contrast, 11-cis REH activity was mostly recovered in PM-enriched fractions while LRAT activity was found only in ER-enriched membranes. The finding that both 11-cis retinol and 11-cis retinal can be produced at the PM of the bovine RPE strongly suggests that 11-cis retinyl esters at this subcellular locale serve as a precursor of visual chromophore for pigment regeneration.

Studies focused on the intake of different dietary fats have shown changes in membrane lipid composition and, as a result, alterations in membrane physical properties. These changes affect erythrocyte morphology, receptor activity and oxygen transport, among others. Here, we compare the effects of diets exclusively differing in the type of fat (olive oil rich in monounsaturates, sunflower oil rich in n–6 polyunsaturates and fish oil rich in n–3 polyunsaturates) on fatty acid composition of plasma and erythrocyte membranes and erythrocyte morphology under scanning electron microscopy in rats. Monounsaturates are highest in animals fed olive oil diets; as are linoleic and arachidonic acids in sunflower oil-fed animals and n–3 PUFAs in fish oil-fed animals. The lowest levels of arachidonic acid are found in fish oil-fed animals and so are n–3 PUFAs in sunflower oil-fed animals. Our results show that sunflower oil-fed animals present lower discocyte, the major cell shape related to tissue oxygen supply, and higher codocyte percentages than olive oil- and fish oil-fed groups. Echinocyte percentage is higher in fish oil-fed animals with respect to the other two groups. The collective data indicate that olive oil elevates monounsaturates and the number of discocytes, pointing out a possible beneficial aspect of this dietary fat.

The micellization of sodium cholate (NaC) was studied at 298.2 K by aqueous solubility at different pH values. Using a stepwise association model of cholate anions without the sodium counterion, the aggregation number ($\text{n}$) of the cholate micelle was evaluated and found to increase with the total concentration, indicating that the mass action model worked quite well. The $\text{n}$ value at 60 mM was found equal to 16. The membrane potential measurement of sodium ion with a cation exchange membrane was made in order to confirm the low counterion binding to micelle. The solubilization of alkylbenzenes (benzene, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, n-pentylbenzene, n-hexylbenzene) and polycyclic aromatic compounds (naphthalene, anthracene, pyrene) into the aqueous micellar solution of sodium cholate was carried out. Solubilizate concentrations at equilibrium were determined spectrophotometrically at 298.2 K. The first stepwise association constants ($\text{K}{}_1$) between solubilizate monomer and vacant micelle were evaluated from the equilibrium concentrations and found to increase with increasing hydrophobicity of the solubilizate molecules. From the Gibbs energy change for solubilization at the different mean aggregation numbers and from molecular structure of the solubilizates, the function of sodium cholate micelle for solubilization was discussed and was compared with data from conventional aliphatic micelles.

Our previous study (Biochim. Biophys. Acta 1393 (1998) 336–348, this issue) found that dietary hydroperoxides of trilinoleoylglycerol were broken down, releasing linoleic acid hydroperoxides (LA-OOH) in the stomach without reaching the intestines. The present paper describes the catabolic fate of LA-OOH in rat gastrointestines, in an attempt to elucidate those products which can be absorbed into the body. At an intragastric dose of 6.5 or 18 μmol, LA-OOH was not transported to the intestines as determined by HPLC. At large doses (200 or 800 μmol), much greater than that in the daily diet, there was partial leakage of LA-OOH to the intestines. The periodical fate was analyzed with 17.2 μmol [U-¹⁴C]LA-OOH chemically and radiochemically. Exemplifying the product composition at 30 min after treatment (as percentage of dosed amount), 27% unchanged LA-OOH, 9.7% epoxyketones, 3.5% hydroxyls (LA-OH), 2.4% decomposed aldehydes, and 13% unknown products were found in the gastric lumen. Another 25% was incorporated in the gastric tissue, and the other 6.4% occurred in the intestinal lumen and tissue as decomposed aldehyde. The LA-OH further decomposed to aldehydes with time in the stomach. When an aldehyde mixture was prepared and dosed, significant increases in hexanal and 4-hydroxynonenal were detected in the liver 15 h later. These results show that the dietary LA-OOH is decomposed to aldehydes in the stomach and that aldehydes are partly absorbed into the body.

Selective uptake of high-density lipoprotein (HDL)-associated cholesteryl esters (CE), i.e. lipid uptake independent of HDL particle uptake, delivers CE to the liver and steroidogenic tissues in vivo and in vitro. From human plasma HDL, two major subpopulations of particles can be isolated: one contains both apolipoprotein (apo) A-I and apo A-II (designated LpA-I:A-II) as dominant protein components, whereas in the other apo A-II is absent (LpA-I). In this study, selective CE uptake from LpA-I and LpA-I:A-II by cultured cells was investigated. LpA-I and LpA-I:A-II were isolated by immunoaffinity chromatography from human plasma high-density lipoprotein3 (HDL₃, d=1.125–1.21 g/ml) and both particles were radiolabeled in the protein (¹²⁵I) as well as in the CE moiety ([³H]cholesteryl oleyl ether ([³H]CEt)). Several control experiments validated the labeling methodology applied. To investigate selective CE uptake, human Hep G2 hepatoma cells, human hepatocytes in primary culture and human skin fibroblasts were incubated in medium containing doubly radiolabeled LpA-I or LpA-I:A-II particles. Thereafter cellular tracer content was determined. For each cell type the rate of apparent lipoprotein particle uptake according to the lipid tracer ([³H]CEt) was in substantial excess over that due to the protein tracer (¹²⁵I), demonstrating selective CE uptake from LpA-I as well as from LpA-I:A-II. This difference in uptake between [³H]CEt and ¹²⁵I, i.e. the rate of apparent selective CE uptake, was significantly higher for LpA-I compared to LpA-I:A-II, and this was dose- as well as time-dependent. Thus in human hepatic cells and fibroblasts, CE are selectively taken up to a higher extent from LpA-I compared to LpA-I:A-II. These results may suggest that LpA-I particles of the human plasma HDL fraction may be those lipoproteins which more efficiently deliver CE to the liver via the selective uptake pathway whereas LpA-I:A-II may play a less important role.

Pretreatment of mouse peritoneal macrophages with interleukin-13 (IL-13) potentiates the mobilization of arachidonic acid (AA) and the production of HETEs but does not affect the production of cyclooxygenase metabolites triggered by the suboptimal concentration of an inflammatory agonist (opsonized-zymosan). Cycloheximide suppresses these effects of IL-13 suggesting that de novo protein synthesis is involved. Indeed, IL-13 induces a time-dependent increase in the levels of cytolosic PLA2 (cPLA2) protein and mRNA. This study demonstrates a new pathway for IL-13 to modulate the inflammatory process in macrophages via modifications of cPLA2 expression and subsequent AA mobilization.