BBA - Molecular and Cell Biology of Lipids (v.1686, #3)

Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma by Barbara Sias; Francine Ferrato; Maria-Teresa Pellicer-Rubio; Yvonick Forgerit; Philippe Guillouet; Bernard Leboeuf; Frédéric Carrière (169-180).
The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids.This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2).The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.
Keywords: Goat; Lipase; Lipolysis; Pancreatic lipase-related protein; Phospholipase; Reproduction; Seminal plasma; Hormonal regulation;

Specific formation of arachidonic acid and eicosapentaenoic acid by a front-end Δ5-desaturase from Phytophthora megasperma by Ellen Hornung; Martina Korfei; Christian Pernstich; Annett Struss; Helmut Kindl; Martin Fulda; Ivo Feussner (181-189).
The biosynthesis of arachidonic acid (20:4Δ5Z,8Z,11Z,14Z ) from linoleic acid in plants by transgenic means requires the sequential and specific action of two desaturation reactions and one elongation reaction. Here, we describe the isolation of a specific acyl-lipid-desaturase catalyzing the formation of the double bond at position 5 from a cDNA library from Phytophthora megasperma. The isolated full-length cDNA harbors a sequence of 1740 bp encoding a protein of 477 amino acids with a calculated molecular weight of 53.5 kDa. The desaturase sequence contained a predicted N-terminal cytochrome b 5-like domain, as well as three histidine-rich domains. For functional identification, the cDNA was expressed in Saccharomyces cerevisiae, and the formation of newly formed fatty acids was analyzed. The expression of the heterologous enzyme resulted in the formation of arachidonic acid after di-homo-γ-linolenic acid supplementation and in the formation of eicosapentaenoic acid synthesis from ω3-arachidonic acid. Results presented here on the substrate specificity identify this expressed protein as a classical Δ5-acyl-lipid-desaturase, capable of specifically introducing a double bond at the Δ5 position solely in 20-carbon-atom chain length fatty acids containing a double bond at position Δ8. Detailed analysis of the different lipid species showed a preferential occurrence of the desaturation reaction for fatty acids esterified to phosphatidylcholine.
Keywords: Acyl-group desaturase; Front end desaturase; Di-homo-γ-linolenic acid; ω3-arachidonic acid; Saccharomyces cerevisiae;

Apolipoprotein E delivery by peritoneal implantation of encapsulated recombinant cells improves the hyperlipidaemic profile in apoE-deficient mice by Aristides D. Tagalakis; Ivan A. Diakonov; Ian R. Graham; Karen A. Heald; Julian D. Harris; Jane V. Mulcahy; George Dickson; James S. Owen (190-199).
Plasma apolipoprotein E (apoE) is a 34-kDa polymorphic protein which has atheroprotective actions by clearing remnant lipoproteins and sequestering excess cellular cholesterol. Low or dysfunctional apoE is a risk factor for hyperlipidaemia and atherosclerosis, and for restenosis after angioplasty. Here, in short-term studies designed to establish proof-of-principle, we investigate whether encapsulated recombinant Chinese hamster ovary (CHO) cells can secrete wild-type apoE3 protein in vitro and then determine whether peritoneal implantation of the microcapsules into apoE-deficient (apoE−/−) mice reduces their hypercholesterolaemia.Recombinant CHO-E3 cells were encapsulated into either alginate poly-l-lysine or alginate polyethyleneimine/polybrene microspheres. After verifying stability and apoE3 secretion, the beads were then implanted into the peritoneal cavity of apoE−/− mice; levels of plasma apoE3, cholesterol and lipoproteins were monitored for up to 14 days post-implantation.Encapsulated CHO-E3 cells continued to secrete apoE3 protein throughout a 60-day study period in vitro, though levels declined after 14 days. This cell-derived apoE3 was biologically active. When conditioned medium from encapsulated CHO-E3 cells was incubated with cultured cells pre-labelled with [3H]-cholesterol, efflux of cholesterol was two to four times greater than with normal medium (at 8 h, for example, 7.4±0.3% vs. 2.4±0.2% of cellular cholesterol; P<0.001). Moreover, when secreted apoE3 was injected intraperitoneally into apoE−/− mice, apoE3 was detected in plasma and the hyperlipidaemia improved. Similarly, when alginate polyethyleneimine/polybrene capsules were implanted into the peritoneum of apoE−/− mice, apoE3 was secreted into plasma and at 7 days total cholesterol was reduced, while atheroprotective high-density lipoprotein (HDL) increased. In a second study, apoE was detectable in plasma of five mice treated with alginate poly-l-lysine beads, 4 and 7 days post-implantation, though not at day 14. Furthermore, their hypercholesterolaemia was reduced, while HDL was clearly elevated in all mice at days 4 and 7 (from 18.4±6.2% of total lipoproteins to 31.1±6.8% at 7 days; P<0.001); however, these had rebounded by day 14, possibly due to the emergence of anti-apoE antibodies.We conclude that microencapsulated apoE-secreting cells have the potential to ameliorate the hyperlipidaemia of apoE deficiency, but that the technology must be improved to become a feasible therapeutic to treat atherosclerosis.
Keywords: Atherosclerosis; Cholesterol; Gene transfer; Hypercholesterolaemia; Lipoprotein;

Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation by Marija Heffer-Lauc; Gordan Lauc; Leonardo Nimrichter; Susan E. Fromholt; Ronald L. Schnaar (200-208).
Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with ≥0.25% Triton X-100 at 4 °C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 °C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue.
Keywords: Microdomain; Brain; Myelin; Ganglioside; Triton X-100;

Effects of hydrophobic and hydrophilic bile salts on gallstone growth and dissolution in model biles by Niels G. Venneman; Marieke van Kammen; Willem Renooij; Gerard P. vanBerge-Henegouwen; Karel J. van Erpecum (209-219).
Cholesterol crystallization is a prerequisite for gallstone formation and growth, whereas dissolution of crystallized cholesterol forms the basis of nonsurgical therapy. Crystallization has been studied in detail, but dissolution mechanisms and effects of gallstones are largely unknown.We evaluated gallstone growth or dissolution, cholesterol crystallization and lipid distribution into various phases, in model biles with low or intermediate phospholipid contents (crystal-containing left two-phase or central three-phase zones), and with high phospholipid or low cholesterol contents (crystal-free right two-phase or bottom one-phase zones).In model biles with added gallstones plotting in left two-phase and central three-phase zones, gallstone masses increased, whereas crystallization in the aqueous phase was less than without gallstones (P<0.001). In biles plotting in the right two-phase zone, gallstone masses decreased, depending on bile salt hydrophobicity (TUDC>TC>TCDC: P<0.001). In biles plotting in the bottom one-phase zone containing TC or TCDC, gallstone masses increased. In contrast, gallstone masses decreased in case of TUDC with preferential distribution of cholesterol into emerging vesicles.Our findings suggest competition between gallstone surface and surrounding aqueous phase for precipitation of cholesterol in crystal-containing zones. Different gallstone dissolution mechanisms may exist for TUDC and TCDC.
Keywords: Bile salt hydrophobicity; Cholesterol; Crystallization; Gallstone dissolution; Gallstone growth;

We studied the incorporation of 14C-labeled fatty acids and glycerol into different classes of glycerolipids in an in vitro system containing liver microsomes from growing Wistar rats fed a calcium-deficient (CaD; 0.5 g/kg) diet for a 60-day period. Desaturase activities and incorporation of the elongation-desaturation metabolites into specific neutral and polar glycerolipids were also studied and correlated with the activities of various enzymes involved in complex lipid metabolism (acyl-CoA synthase, acyl-CoA hydrolase, DAG-acyltransferase, DAG-kinase, lysophospatidate-acyl-CoA transferase, phosphatidate-phosphohydrolase and phospholipase A2). Low calcium condition led to a significant increase in the incorporation (relative amounts and specific activities) of both labeled fatty acids and glycerol with a preferential increase of labeling in neutral lipids rather than in phospholipids. Acyl-CoA synthetase, diacylglycerol acyltransferase and diacylglycerol-3-P acyltransferase activities were increased in low calcium microsomes while diacylglycerol kinase, phospholipase A2 and palmitoyl-, stearoyl-, linoleyl-, α-linolenyl, and eicosatrienoyl-desaturases were decreased. The modifications observed in the interlipid and lipid/protein relationships, enzyme activities, and pattern of incorporation of labeled precursors into each glycerolipid class, suggest that decreased intake of calcium should be considered as a harmful risk factor for the development of cardiovascular diseases.
Keywords: Calcium deficiency; Glycerolipid metabolism; Neutral lipid; Phospholipid; Rat liver microsome;

Non-bullous congenital ichthyosiform erythroderma (NCIE) is one of the main clinical forms of ichthyosis. Genetic studies indicated that 12R-lipoxygenase (12R-LOX) or epidermal lipoxygenase-3 (eLOX3) was mutated in six families affected by NCIE [F. Jobard, C. Lefèvre, A. Karaduman, C. Blanchet-Bardon, S. Emre, J. Weissenbach, M. Özgüc, M. Lathrop, J.F. Prud'homme, J. Fischer, Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1, Hum. Mol. Genet. 11 (2002) 107–113.], but the impact of these mutations on LOX function has not been defined. To explore this, we overexpressed the wild-type or mutated enzymes in E. coli and COS7 cells and then analyzed the essential catalytic properties. We showed recently that human eLOX3 is a hydroperoxide isomerase (hepoxilin synthase) that converts the product of 12R-LOX, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE) to a specific epoxyalcohol. Using incubations with [14C]-labeled substrates and HPLC analyses, we found that the naturally occurring mutations totally eliminate the lipoxygenase activity of 12R-LOX and the hydroperoxide isomerase activity of eLOX3. We further demonstrate that the 12R-LOX/eLOX3-derived 8R-hydroxy-11R,12R-epoxide is converted by an epoxide hydrolase in COS7 cells and in human keratinocytes to a single isomer of 8,11,12-trihydroxyeicosa-5,9,14-trienoic acid. Taken together, the results support the hypothesis that 12R-LOX, eLOX3, and perhaps an epoxide hydrolase function together in the normal process of skin differentiation, and that the loss of function mutations are the basis of the LOX-dependent form of NCIE.
Keywords: Lipoxygenase; Ichthyosis; Mutation; 12R-LOX; Epoxyalcohol; Hepoxilin; Epoxide hydrolase; Skin; Keratocyte;

Perinatal essential fatty acid deficiency influences body weight and bone parameters in adult male rats by M. Korotkova; C. Ohlsson; B. Gabrielsson; L.Å. Hanson; B. Strandvik (248-254).
Fetal and postnatal nutrition have long-term effects on the risk for development of diseases late in life in humans and animals. The aim of the present study was to investigate the effect of dietary deficiency of essential fatty acids (EFA) in the perinatal period on later body weight and bone mass. During late gestation and throughout lactation, rats were fed a control or an EFA-deficient (EFAD) diet. At 3 weeks of age the offspring were weaned onto an ordinary chow and followed until adult age. The mean body weight of adult rats receiving the EFAD diet during the perinatal period was significantly increased from 12 weeks of age compared to the controls (P<0.05). Analysis by peripheral quantitative computerized tomography (pQCT) at 44 weeks of age showed that the trabecular volumetric bone mineral density (BMD) of the femur was significantly decreased (P<0.05) but the cortical bone mineral content, cortical area, and cortical thickness were increased (P<0.05) in the EFAD group of rats. The length of the femur was not affected. In conclusion, neonatal EFA deficiency was in adult rats associated with increased body weight and significant changes in both cortical and trabecular bone. The results indicate that regulatory mechanisms related to bone mass seemed to be programmed by EFA in the perinatal period. The nature of this modulation needs to be identified.
Keywords: Maternal diet; Programming; IGF-1; Leptin; Bone mineral density;