Radioactive Lipids Research Articles

Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.

Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.

Binding of Low Density Lipoproteins to Lipoprotein Lipase Is Dependent on Lipids but Not on Apolipoprotein B

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.

Formation of lipid reserves in fat body and eggs of the yellow fever mosquito, Aedes aegypti

We examined the accumulation of lipids in adult females of the mosquito, Aedes aegypti. Females emerged with about 100 μg lipid in the fat body. With access to sugar water lipids increased over seven days to 300 μg. After a blood meal on day five, sugar-fed females accumulated 120–140 μg of lipids in their ovaries within 2 days. At the same time the lipid content of the fat body decreased by 100 μg, indicating transfer of lipids from fat body to oocytes. Experiments in which fat body lipids were prelabelled support this conclusion. Label was transferred to oocytes: in mature oocytes the specific radioactivity of lipids was 80% of the specific radioactivity of prelabeled fat body lipids. Components of blood meals are also used to synthesize oocyte lipids. Fat bodies of females starved for four days had only 27 μg of lipids left. When these females were given a blood meal, they matured oocytes, although the number of ooyctes was reduced and ovaries contained only half the amount of lipids found in ovaries of females which had first fed on sugar water. Fat body lipids of these females had only slightly increased to 36 μg. This demonstrates that female Ae. aegypti use sugar to synthesize lipids, but they can also use components of blood for this purpose.

Advances in the development of microdiets for gilthead seabream, Sparus aurata: a review

The performance of microdiets (MDs) for larvae of marine fish is frequently improved when they are co-fed with Artemia. This suggests that nutritional factors in the live food are positively influencing the ingestion, digestion and assimilation of the MD. This paper reviews recent advances in MD development on the gilthead seabream with special emphasis on studies that isolated, identified and tested these live food factors in MD with the aim of improving their performance. MD ingestion rates in gilthead seabream larvae increased up to 120% when the fish were exposed to the visual and chemical stimuli of various concentrations of Artemia nauplii. The free amino acids (FAA) alanine, glycine and arginine and the compound betaine were identified from the Artemia rearing medium as metabolites, which stimulated this larval response. Similarly, MD supplemented with phospholipids (PL), particularly phosphatidylcholine (PC), stimulated feeding activity and was consumed up to 45% better in young larval seabream. Moreover, dietary PC appears to have in parallel and/or in tandem a postprandial enhancing effect on lipoprotein synthesis, resulting in improved transport of dietary lipids from the mucosa of the digestive tract to the body tissues. Live food may also contribute exogenous enzymes to the digestion process or provide factors that stimulate larval pancreatic secretions or activate gut zymogens. Seabream larvae ingesting MD supplemented with porcine pancreatic extract (0.05% DW diet) showed a 30% increase in assimilation and demonstrated significantly ( P<0.05) improved growth. Older seabream larvae showed 6.75 times more radioactivity in tissue lipids when fed 14C-triacylglycerol (TAG)-labeled MD supplemented with porcine lipase, while younger larvae demonstrated no improved assimilation. Factors in live Artemia may influence digestion by stimulating an endocrine response. This was shown when Artemia consumed by seabream larvae elicited a 300% increase in the level of the digestive hormone bombesin compared to levels in larvae given only a MD. On the other hand, liposomes containing the FAA methionine ingested by halibut juveniles elicited higher levels of the digestive hormone cholecystokinin (CCK) compared to juveniles ingesting liposomes containing physiological saline or fish extract. These studies suggested that mobilizing the native endocrine factors associated with the feeding and digestive processes could improve MD performance in gilthead seabream and other species by maximizing its utilization.

Oil biosynthesis in microsomal membrane preparations from Mortierella alpina

Microsomal membrane preparations from Mortierella alpina catalysed the conversion of sn-glycerol 3-phosphate and [(14)C]oleoyl-CoA to radioactive phosphatidic acid, diacylglycerol and triacylglycerol. Experiments with lysophosphatidic acid and [(14)C]oleoyl-CoA gave a similar pattern of radioactivity in the complex lipids. The specific activity of lysophosphatidate acyltransferase was almost eight times greater than sn-glycerol-3-phosphate acyltransferase, indicating that the first acylation step was limiting in oil assembly in the microsomal membranes. Little conversion of radioactive oleate into phosphatidylcholine occurred, suggesting that triacylglycerol assembly and its relationship to phosphatidylcholine metabolism differed to that found in oilseeds.

Platelet-activating Factor (PAF)-dependent Transacetylase and Its Relationship with PAF Acetylhydrolases

Platelet-activating Factor (PAF)-dependent Transacetylase and Its Relationship with PAF Acetylhydrolases

Changes in cell phospholipid metabolism in vitro in the presence of HEMA and its degradation products

Objectives: Diacylglycerol-kinase (DAG-kinase) is an enzyme that phosphorylates diacylglycerol (DAG) to phosphatidic acid (PA), which serves as a precursor to phosphoglycerides involved in cell signaling or as cell membrane structural components. DAG-kinase can be inhibited by diacylethylene glycols (DAEG). We hypothesize that 2-hydroxyethyl methacrylate (HEMA) may alter phosphorylation of DAG to PA following intracellular formation of DAEG. Methods: Cultured rabbit kidney (RK 13) epithelial cells were treated with HEMA, EG, or known inhibitors of DAG-kinase for 24 h, then exposed to [ 32 P] O 4 − in the presence of a synthetic diacylglycerol for 2 h. Other cultures were radiolabeled with [ 3H]-oleic acid for 24 h, then exposed to HEMA for an additional 24 h. The cells were harvested and the lipids extracted. Radioactive lipids were separated by thin layer chromatography, located by autoradiography, and quantitated as cpm/ug protein. Cell cultures treated with HEMA were homogenized and the DAG-kinase activity was assayed and expressed as cpm/ug protein. Data were analyzed by one-way ANOVA and Newman–Keuls Multiple Comparison Test. Results: Cultures exposed to HEMA or known DAG-kinase inhibitors exhibited reduced incorporation of radioactivity in the PA fraction compared to control cultures. Direct assays of DAG-kinase activity from cells exposed to HEMA demonstrated decreased enzyme activity. Evaluation of cell phospholipid synthesis showed altered formation of phosphatidylethanolamine and phosphatidylcholine. Significance: Results suggest that HEMA impairs formation of PA, possibly by acylation of EG released by hydrolysis of the HEMA and resultant production of the inhibitor DAEG. The decreased availability of PA may alter PA-dependent cell structural lipid pathways and lipid-dependent signaling pathways, altering cell growth.

A mutual inhibitory effect on absorption of sphingomyelin and cholesterol

Several studies have shown that there is a strong physical interaction between cholesterol and sphingomyelin (SM). The critical factor is thought to be the high degree of saturation in the very long acyl chains of SM. In this study we examined the effects of SM on cholesterol absorption in the rat and compared them with those of phosphatidylcholine (PC). Cholesterol absorption was studied by use of the dual-isotope plasma ratio method. We also studied the effect of sterols on the fecal excretion of undigested SM and its metabolites after a single oral meal of 3H-dihydrosphingosine-labeled SM. When cholesterol was given dissolved in soybean oil, without addition of SM or other phospholipids, absorption was 68 ± 12% in the rat intestine. As a general feature the absorption was less efficient from the cholesterol/phospholipid dispersions. In dispersions with cholesterol and SM, the lowest cholesterol absorption (9 ± 2%) was seen with a cholesterol:SM molar ratio of 1:1. With dispersions of cholesterol and different PC substrates the absorption of cholesterol was lower with saturated PC (16 ± 8%) than with soybean-PC (22 ± 4%) or dioleoyl PC (23 ± 8%). Uptake of SM in the rat intestine was reduced by sterols. For example, percentage recovery of 3H radioactivity in fecal lipids was 38 ± 8% when SM was given with cholesterol and 16 ± 3% without any sterol. One third of the radioactivity in feces was present as ceramide. Sitostanol had the same effect on uptake of SM as cholesterol. This study shows that when rats are fed mixtures of SM and cholesterol the intestinal uptake of both lipids is decreased. By feeding mixtures of SM and sterols the exposure of the colon to ceramide can be increased.

Alzheimer's Abeta1-40 peptide modulates lipid synthesis in neuronal cultures and intact rat fetal brain under normoxic and oxidative stress conditions.

The effect of amyloid beta (Abeta), the major constituent of the Alzheimer's (AD) brain on lipid metabolism was investigated in cultured nerve cells and in a fetal rat brain model. Differentiated (NGF) and undifferentiated PC12 cells or primary cerebral cell cultures were incubated with [14C]acetate in the absence or presence of Abeta1-40. Incorporation of label into lipid species was determined after lipid extraction and TLC separation. Phosphatidylcholine (PC) and phosphatidylserine (PS) synthesis was increased by Abeta1-40, in a dose dependent manner, an effect which was more pronounced in differentiated PC12 cells. A significant proportion of radioactivity (5-6%) was released into the medium with a radioactivity distribution similar to that of the cellular lipids. Cholesterol and PC were the highest labeled medium lipids. Increasing Abeta1-40 concentration up to 0.1 microg/ml in cerebral cells but not in PC12 cells, caused a relative increase (1.5 fold) in release of PS, while that of PE decreased. Stimulation of PS release may possibly be associated with apoptotic cell death. Abeta1-40 peptide (5 microg) was administered intraperitoneally into rat fetuses (18 days gestation) along with [14C]acetate (2 microCi/fetus). After 24 h, the maternal-fetal blood supply was occluded for 20 min (ischemia) followed by 15 min reperfusion. Fetuses were killed and liver and brain tissue subjected to lipid extraction and radioactivity determination after TLC. Abeta1-40 peptide increased synthesis of different classes of lipids up to 20-40% in brain tissue compared to controls. Labeling of liver lipids was decreased by Abeta1-40 by 20-30%. A general decrease in synthesis of lipids was observed after ischemia/reperfusion. Our data suggest that Abeta1-40 peptide regulates normal lipid biosynthesis but under ischemia it compromises it. The latter finding may confirm the oxidative stress etiology in AD and suggests that Abeta1-40 modulation of lipid metabolism may have Alzheimer's pathological relevance, particularly at high peptide concentrations.

Lipid conversions during enrichment of Artemia

Artemia nauplii were enriched for 24 h with radiolabelled fatty acid ethyl esters and then starved for a subsequent period of 24 h. Analyses of the distribution of radioactivity in lipids from samples taken at the end of the enrichment period and after the subsequent starvation showed that the ethyl esters were readily converted into other lipid classes, mainly triacylglycerols, during assimilation by the nauplii. The proportions of radioactivity recovered in free fatty acids and phospholipids increased during the starvation period indicating the mobilisation of fatty acids from triacylglycerols for use in catabolism and in the formation of biomembrane lipids. The distribution pattern of radioactivity from [U– 14 C ]22:6 n−3 in the fatty acids of the nauplii demonstrates that Artemia are capable of retroconverting 22:6 n−3 to 20:5 n−3.

Alterations in cell lipid metabolism by glycol methacrylate (HEMA)

Components of dental resins such as dimethylaminoethyl methacrylate (DMAEMA) can alter cell lipid composition, presumably by esterase-mediated hydrolysis. The resulting dimethylethanolamine is incorporated into cell phospholipids, while the methacrylic acid may alter several metabolic pathways. We hypothesize that HEMA is cleaved in a similar manner and the released ethylene glycol is incorporated into cell lipids, yielding phosphatidylethylene glycol (PtEG), and the methacrylic acid alters other lipid pathways in a manner similar to that of methacrylic acid released from hydrolysis of DMAEMA. Cultures of hamster buccal pouch (HCP) and rabbit kidney (RK13) epithelial cells were exposed to subtoxic concentrations of HEMA in the presence of [14C]-acetate or [3H]-oleic acid. Other cultures were prelabeled with [14C ]-acetate followed by exposure to various concentrations of HEMA. Cell lipids were extracted by the method of Bligh and Dyer and separated by thin layer chromatography on silica gel K-6 plates or SG-81 silica gel loaded chromatography paper. The fate of the ethylene glycol was traced using [14C]-ethylene glycol. Radioactive lipids were located using autoradiography and known standard lipids and quantitated by liquid scintillation spectrometry. In the presence of HEMA several classes of lipids were altered. Among the neutral lipids, the most notable changes involved sterol precursors, triglycerides, fatty acids, and cholesterol esters, while phosphatidylcholine was affected among the phospholipids. The results differed quantitatively between the two cell types. Results also suggest that EG, including that released by hydrolysis of HEMA, is incorporated into cell phospholipids, producing PtEG. The changes in neutral lipid labeling may occur by alteration of lipid synthetic pathways utilizing acetyl Co-A as well as inhibition of enzymes involved in synthesis of cholesterol from sterol precursors and hydrolysis of cholesterol esters. Synthesis of PtEG may take place via phospholipase D-mediated headgroup exchange. Alterations in the cellular lipids may affect cell membrane properties and associated cell functions.

P0 Protein Mediated Targeting of Liposomes to Melanoma Cells with High Level of ICAM-1 Expression

The feasibility of targeting liposomes reconstituted with P0 protein (P0-liposomes) to melanoma cells with high level of intercellular adhesion molecule-1 (ICAM-1) expression was investigated. P0 protein an immunoglobulin superfamily (IgSF) cell adhesion molecule from peripheral nerve myelin, was used as a model targeting ligand. Liposome uptake by the cells was quantitated using radioactive lipids. The presence of intact P0 protein in the liposome bilayer increased the extent of interaction of liposomes with human M21 (7.80 fold) and A-375 (4.62 fold) melanoma cells compared to control liposomes of same lipid composition but no P0 protein, whereas with MeM 50-10 melanoma cells no significant increase was found (1.70 fold). The extent of binding of P0-liposomes to the melanoma cells correlated with the level of ICAM-1 expression on cells (r2 = 0.9996). M21 and A-375 cells express ICAM-1 (the percentage of stained cells, PSC, was 95% and 85%, respectively), whereas, MeM 50-10 cells do not. P0 protein also increased the interaction of liposomes with P0 protein expressing CHO-X2 cells (4.36 fold, as a positive control) compared to control liposomes. The indirect flow cytometry experiments using biotinylated P0 protein showed that P0 protein itself in solution also binds to M21 cells but does not bind to MeM 50-10 cells. Preincubation of M21 cells with anti-ICAM-1 monoclonal antibody decreased the binding of biotinylated P0 protein to the M21 cells by 35%. P0 protein or its binding domain(s) that mediate the targeting of liposomes through adhesive interactions may be useful for the development of novel types of drug delivery systems. This approach may have relevance in the treatment of metastatic cancers, inflammation and viral diseases, where cell adhesion proteins are over expressed.

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17α-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.

Astrocytes are mainly responsible for the polyunsaturated fatty acid enrichment in blood–brain barrier endothelial cells in vitro

To determine the respective roles of endothelial cells from brain capillaries and astrocytes in the conversion of circulating 18:2n–6 and 18:3n–3 into 20:4n–6 and 22:6n–3, respectively, a coculture of the two cell types mimicking the in vivo blood–brain barrier was used. During the culture period, endothelial cells cultured on an insert were set above the medium of a Petri dish containing or not a stabilized culture of astrocytes. Five days after confluence, labeled 18:2n–6 and 18:3n–3 (10 μm each) were added to the endothelial cells and incubated for 48 h. Analogous experiments were also performed by using each cell type cultured alone in the culture device. The distribution of radioactivity in lipids and fatty acids was studied in all the compartments of the culture device. Endothelial cells cultured alone weakly converted the precursor fatty acids into 20:4n–6 and 22:6n–3. When endothelial cells were cocultured with astrocytes, their content of polyunsaturated fatty acids increased dramatically. This effect was associated with the uptake of polyunsaturated fatty acids from the lower medium (astrocyte medium). These fatty acids were released by astrocytes after they were synthesized from the precursor fatty acids that passed through the endothelial cell monolayer into the lower medium. Polyunsaturated fatty acids were released by astrocytes as unesterified fatty acids and as phospholipids (mainly phosphatidylcholine and lysophosphatidylcholine) even when the medium was devoid of serum.▪ These results suggest that astrocytes could play a major role in the delivery of essential polyunsaturated fatty acids to the barrier itself and to the brain.—Bernoud, N., L. Fenart, C. Bénistant, J. F. Pageaux, M. P. Dehouck, P. Molière, M. Lagarde, R. Cecchelli, and J. Lecerf. Astrocytes are mainly responsible for the polyunsaturated fatty acid enrichment in blood–brain barrier endothelial cells in vitro.

Use of a photoactivatable lipid to probe the topology of PA63 of Bacillus anthracis in lipid membranes.

The protective antigen of Bacillus anthracis is a key protein that promotes the translocation of the enzymatic moieties of the two toxins of B. anthracis into the cell cytoplasm. The membrane topology of the active form of the protective antigen (PA63) was investigated by proteolysis of PA63 inserted into liposomes containing a photoactivatable, radioactive lipid, and characterization of the N-terminal moiety of the deeply-inserted (and therefore radiolabeled) peptides. A single sequence starting at residue Ala258 was identified. Fourier-transform infrared spectroscopy showed that the protected peptide was mainly adopting a beta-sheet structure whose orientation was compatible with a transmembrane organization.

MgATP has different inhibitory effects on the use of 1-acyl-lysophosphatidylcholine and lyso platelet-activating factor acceptors by neuronal nuclear acetyltransferase activities

The inhibitory effects of MgATP on neuronal nuclear acetyltransferase activities were studied using lyso platelet-activating factor (lyso-PAF, 1-alkyl- sn-glycero-3-phosphocholine) and lysophosphatidylcholine (lyso-PC, 1-acyl- sn-glycero-3-phosphocholine). The nuclear (N 1) acetylation of lyso-PC was more profoundly inhibited by MgATP. MgATP did not alter the apparent K m for acetyl-CoA in either acetylation reaction. The inhibitory effects of MgATP were not seen for other nucleotides or MgAMP-PCP. Kinase inhibitors such as staurosporine (1 μM), chelerythrine, and R59022 (diglyceride kinase inhibitor I) did not block the MgATP inhibition of either acetylation. However, the addition of phospholipids to the assays indicated a selective inhibitory effect for PIP (25–50 μM) in the nuclear acetylation of lyso-PAF. When N 1 was incubated with [γ- 33P]ATP, phosphatidic acid and PIP were the principal radioactive lipid products. While the extent of MgATP inhibition of lyso-PAF acetylation was similar at different concentrations of lyso-PAF, increasing lyso-PC concentrations greatly decreased the MgATP inhibition seen in lyso-PC acetylations. Nuclear envelopes prepared in the presence of PMSF, and fraction N 1 exposed to PMSF, did not show the inhibitory effect of MgATP on lyso-PC acetylation. PMSF (an inhibitor of certain phospholipase and lysophospholipase activities) did not reduce the MgATP inhibition of lyso-PAF acetylation. Arachidonoyl trifluoromethylketone, an inhibitor of cytosolic phospholipases A 2 and of lysophospholipase activity associated with cPLA 2, also blocked the inhibitory effect of MgATP on lyso-PC acetylation. Using radioactive lyso-PC substrate, fraction N 1 produced labeled free fatty acid and phosphatidylcholine. In the presence of acetyl-CoA, the production of radioactive phosphatidylcholine increased almost 6-fold when MgATP was also included in these incubations. In the presence of MgATP and acetyl-CoA, PMSF reduced the levels of radioactive free fatty acid and phosphatidylcholine derived from lyso-PC, while Triacsin C, an inhibitor of acyl CoA synthetase, decreased phosphatidylcholine labeling. These findings suggest that MgATP inhibition of lyso-PC acetylation results from a loss of lyso-PC substrate that is largely mediated by nuclear lysophospholipase, acyl-CoA synthetase and lyso-PC acylation. Thus the neuronal nuclear production of Acyl PAF may be regulated by paths that compete for the lyso-PC substrate. In contrast, the acetylation of lyso-PAF is inhibited by PIP, a product of nuclear PI kinase reactions.

Veratridine-induced depolarization reduces the palmitoylation of brain and myelin glycerolipids.

In this study, we have investigated the effect of neuronal depolarization on the palmitoylation of myelin lipids. For this purpose, brain slices from 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various drugs. Veratridine (100 microM) reduced the incorporation of [3H]palmitate into all brain glycerolipids by 40-50%, whereas the labeling of sphingolipids was unaffected. Similar results were obtained by using [3H]glycerol as a precursor, demonstrating that veratridine also causes a reduction in the de novo synthesis of glycerolipids. Both tetrodotoxin (1 microM) and ouabain (1 mM) prevented the effect of veratridine, indicating that it is mediated through the opening of voltage-gated sodium channels and involves the stimulation of the Na+/ K+ pump. Decreased levels of both ATP, due to activation of the Na+,K+-ATPase, and the precursor palmitoyl-CoA were found in the veratridine-treated slices, thus explaining the reduction in lipid synthesis. Neuronal depolarization also decreased the synthesis of lipids present in the myelin fraction. The relatively high specific radioactivity of myelin lipids and the results from both repeated purification experiments and mixing experiments ruled out the possibility that the radioactive lipids present in myelin could derive from contamination with other subcellular fraction(s). Because neither mature oligodendrocytes nor myelin is known to express voltage-dependent Na+ channels, it is conceivable that the effect of veratridine on myelin glycerolipid metabolism occurs by an indirect mechanism such as an increase in the extracellular [K+]. However, the presence of 60 mM KCl in the medium did not affect the acylation of either brain or myelin lipids. These results raise questions as to the absence of sodium channels in myelinating oligodendrocytes and/or myelin.

Nutrition of Penaeus japonicus

This article provides an overview of the nutritional requirements of larval and juvenile Penaeus japonicus, referring to past studies briefly and focusing on recent studies on proteins, lipids, and other essential nutrients. Also, the quantitative requirements of nutrients in crustacean species were critically evaluated in relation to net nutrient intake in P. japonicus. Like fish and other vertebrates, larval and juvenile P. japonicus require 10 essential amino acids, essential fatty acids, vitamins, and minerals for their normal development, growth, survival, and reproduction. In contrast to fish and other vertebrates, however, both larval and juvenile prawns require dietary sources of sterols and some phospholipids as indispensable nutrients. Metabolic studies on juvenile P. japonicus using radioactive lipids suggested that dietary phospholipids take part in the transport of dietary lipids, especially cholesterol, through the hemolymph in their body. In nutritional studies, leaching or loss of dietary nutrients into the water is an obstacle to development of valid conclusions on nutrient requirements of P. japonicus, particularly the larvae.

Targeting Liposomes Through Immunoglobulin Superfamily Domains: P0 Protein as a Model

The objective of this study was to evaluate the potential of P0 protein, a cell adhesion molecule from peripheral nerve myelin, as a targeting ligand for liposomes. To evaluate binding characteristics and identify possible binding domains, cell-interaction studies were carried out with P0 protein reconstituted into liposomes (P0 liposomes) under various conditions. P0 liposomes with intact P0 protein were tested after endoglycosidase F treatment (cleaves the carbohydrate moiety) or trypsin digestion (removes the hydrophobic portion, residues 1-79, leaving the carbohydrate portion intact). The cellular uptake was quantitated using radioactive lipids in the liposome bilayer and a liposome-entrapped water soluble compound (inulin). The presence of intact P0 protein in the liposome bilayer increased the rate of interaction of liposomes 3-4 times with M21 melanoma and HTB-11 neuroblastoma cells (cells of neuroectodermal origin), two times with Caki-1 renal carcinoma cells, and marginally with 3T3 fibroblasts (mesodermal origin). This binding was inhibited by anti-chick P0 antibodies and Fab fragments. A control transmembrane glycoprotein, glycophorin A., when reconstituted in liposomes had no effect on the binding of liposomes with M21 cells. The results indicate that P0 protein plays a specific role in the binding of liposomes to cells. Removal of the N-asparagine linked carbohydrate from the P0 protein in the liposomes resulted in an increase of their association with M21 cells five times that of control liposomes (no protein) and two times that of the non-endoglycosidase F-treated P0 liposomes. To further characterize the binding domains of P0 reconstituted into liposomes, competition studies were carried out in the presence of synthetic P0-peptides. The competition studies indicated that both the extracellular (residues 90-96) and intracellular (residues 201-207) domain of P0 protein may be involved in the interaction with cell membranes. The results suggest that P0 protein is capable of mediating specific heterophilic interactions with various cell lines and targeting to cells of neuroectodermal origin may be achieved.

Stimulatory effect of progesterone and 5α-progesterone on lipid synthesis in hamster flank organs

In order to study lipid synthesis in female hamster flank organs during the estrous cycle, glands were obtained from different animals during different phases of the cycle. Lipid metabolism of [U- 14C]glucose was studied under in vitro conditions. The radioactive lipids formed, were extracted from the glands and quantified. An aliquot of the extract was submitted to TLC to isolate and identify the lipids formed. Lipid synthesis in the glands increased significantly during estrous compared with the diestrous phase, suggesting the influence of sex hormones on lipid metabolism in flank organs. The radioactive lipids isolated and identified were: phospholipids, cholesterol, glycerides, waxes, and cholesterol esters. The percentages of conversion from [U- 14C]glucose to cholesterol esters were minor in extracts from glands of diestrous animals as compared to estrous and proestrous animals, whereas glycerides and waxes increased significantly in metestrous and diestrous animals. To verify and evaluate the role of estrogen and progesterone in lipid synthesis, three different groups of gonadectomized female hamsters were injected daily with estradiol, progesterone, and 5α-progesterone. After treatments, glands were incubated with [U- 14C]glucose to determine the incorporation of radioactive glucose into lipids under culture conditions. The radioactive lipids synthesized were subsequently extracted and identified. The results showed that estrogen had no effect on in vitro [U- 14C]glucose incorporation into lipids by flank organs, compared to the vehicle, whereas progesterone and 5α-progesterone increased radioactive lipid synthesis by the glands significantly ( p < 0.05). The radioactive lipids isolated and identified from extracts of gonadectomized female hamster flank organs were: phospholipids, cholesterol, triglycerides, waxes, and cholesterol esters. Gonadectomy increased phospholipid synthesis and decreased that of monoglycerides, diglycerides, and waxes. The percentages of conversion from [U- 14C]glucose to cholesterol esters were higher after progesterone and 5α-progesterone treatments than in vehicle and estrogen-treatments. Our data indicate that progesterone and 5α-progesterone-treatments increased the in vitro [U- 14C]glucose incorporation into lipids in flank organs from gonadectomized female hamsters. Furthermore, the major lipid synthesized by glands under these stimuli were cholesterol fatty acids. Thus, progesterone and 5α-progesterone alter the consistency of sebum from gonadectomized female glands.