BBA - Molecular and Cell Biology of Lipids (v.1632, #1-3)

In mammals and Saccharomyces cerevisiae, sphingolipids have been a subject of intensive research triggered by the interest in their structural diversity and in mammalian pathophysiology as well as in the availability of yeast mutants and suppressor strains. More recently, sphingolipids have attracted additional interest, because they are emerging as an important class of messenger molecules linked to many different cellular functions. In plants, sphingolipids show structural features differing from those found in animals and fungi, and much less is known about their biosynthesis and function. This review focuses on the sphingolipid modifications found in plants and on recent advances in the functional characterization of genes gaining new insight into plant sphingolipid biosynthesis. Recent studies indicate that plant sphingolipids may be also involved in signal transduction, membrane stability, host–pathogen interactions and stress responses.
Keywords: Cerebroside; Cytochrome b 5; Desaturase; Hydroxylase; Long-chain base; Sphingolipid;

The first step in the biosynthesis of sphingolipids is the condensation of serine and palmitoyl CoA, a reaction catalyzed by serine palmitoyltransferase (SPT) to produce 3-ketodihydrosphingosine (KDS). This review focuses on recent advances in the biochemistry and molecular biology of SPT. SPT belongs to a family of pyridoxal 5′-phosphate (PLP)-dependent α-oxoamine synthases (POAS). Mammalian SPT is a heterodimer of 53-kDa LCB1 and 63-kDa LCB2 subunits, both of which are bound to the endoplasmic reticulum (ER) most likely with the type I topology, whereas other members of the POAS family are soluble homodimer enzymes. LCB2 appears to be unstable unless it is associated with LCB1. Potent inhibitors of SPT structurally resemble an intermediate in a probable multistep reaction mechanism for SPT. Although SPT is a housekeeping enzyme, its activity is regulated transcriptionally and post-transcriptionally, and its up-regulation is suggested to play a role in apoptosis induced by certain types of stress. Specific missense mutations in the human LCB1 gene cause hereditary sensory neuropathy type I, an autosomal dominantly inherited disease, and these mutations confer dominant-negative effects on SPT activity.
Keywords: Serine palmitoyltransferase; Sphingolipid; Sphingosine; Ceramide; Pyridoxal phosphate; Hereditary sensory neuropathy;

Effects of plasma apolipoproteins on lipoprotein lipase-mediated lipolysis of small and large lipid emulsions by Mayumi Yamamoto; Shin-ya Morita; Michiko Kumon; Misa Kawabe; Kazuchika Nishitsuji; Hiroyuki Saito; Aline Vertut-Doı̈; Minoru Nakano; Tetsurou Handa (31-39).
Large (ca. 120 nm) and small (ca. 35 nm) emulsions consisting of triolein (TO) and phosphatidylcholine (PC) were prepared as the primary protein-free models of chylomicrons and their remnants, respectively. Lipoprotein lipase (LPL)-mediated lipolysis of emulsion TO was retarded in chylomicron-free human plasma compared with the hydrolysis activated by isolated apolipoprotein C-II (apoC-II). In 30% plasma, free fatty acid (FFA) release rate was higher for large emulsions than for small ones, while both emulsions were hydrolyzed at similar rates in the presence of isolated apoC-II. Isolated apolipoprotein C-III (apoC-III) or apolipoprotein E (apoE) worked as LPL-inhibitor of the lipolysis activated by apoC-II. It was also observed that apolipoprotein A-I (apoA-I) showed distinct inhibitory effects on the lipolysis of large and small emulsions: more effective inhibition for small emulsions. Kinetic analyses showed that K m app and V max app for the lipolysis of emulsions were lower in the presence of 30% plasma than isolated apoC-II. ApoA-I also markedly decreased K m app and V max app for LPL-catalyzed hydrolysis of both emulsions. In chylomicron-free serum, the density of bound apoA-I at small emulsion surfaces was about three fold greater than large emulsion surfaces, but the binding densities of apoC-II, apoC-III and apoE were less for small emulsion surfaces than for large ones, suggesting that apoA-I preferentially binds to small particles and displaces other exchangeable apolipoproteins from particle surfaces. These results indicate that, in addition to the well known inhibitory effects of apoC-III and apoE, apoA-I in plasma regulates the lipolysis of triglyceride (TG)-rich emulsions and lipoproteins in a size-dependent manner.
Keywords: Lipoprotein lipase; Particle size; Apolipoprotein C-II; Apolipoprotein A-I; Apolipoprotein C-III; Apolipoprotein E;

The metabolism of 25-hydroxyvitamin D3 was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D3 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D3 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1α,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3 and 25,27-dihydroxyvitamin D3. An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D3 metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1α- and 27-hydroxylase activities towards 25-hydroxyvitamin D3 as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D3 metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D3 into 1α,25-dihydroxyvitamin D3, 25,27-dihydroxyvitamin D3 and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D3 metabolites such as 1α,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D3. The product had the mass spectrometic properties expected for 4β,25-dihydroxyvitamin D3. Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D3 into 1α,25-dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3. It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D3.
Keywords: Cytochrome P450; 1α-Hydroxylation; 26-Hydroxylation; 27-Hydroxylation; Vitamin D hydroxylase;

Pigment gallstones have been reported to be closely associated with biliary tract infection. We previously reported that addition of unconjugated bilirubin (UCB), which is deconjugated by β-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of β-glucuronidase on the processes of pigment gallstone formation and cholesterol crystallization. Supersaturated MB (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 10.0 g/dl and a cholesterol saturation index (CSI) of 2.0) and native rat bile were mixed at a ratio of 3:1. Then, mixed bile was incubated with or without β-glucuronidase and changes of the following parameters were investigated over time: (1) the UCB/total bilirubin ratio; (2) cholesterol crystal formation; (3) the precipitate weight and the cholesterol concentration in the precipitate and supernatant; and (4) the lipid distribution of vesicles in the supernatant. Compared with β-glucuronidase-free bile, (1) β-glucuronidase-containing bile showed a significant increase of the UCB/total bilirubin ratio, (2) as well as a significantly longer nucleation time (96±17.0 vs. 114±20.0) and fewer cholesterol crystals. (3) The precipitate weight and the cholesterol concentration in the precipitate were significantly increased, while the cholesterol concentration in supernatant was decreased. (4) When mixed bile was incubated with β-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without β-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with β-glucuronidase, while cholesterol concentration was decreased in the supernatant (especially in the vesicles). This means that bile vesicles were more stable and it was more difficult for cholesterol crystals to form. Thus, the presence of β-glucuronidase may inhibit the formation of pure cholesterol stones even in the presence of cholesterol supersaturation.
Keywords: Cholesterol crystal; Unconjugated bilirubin; Bile lithogenicity;

Long-chain fatty acyl-CoA esters induce lipase activation in the absence of a water–lipid interface by M.Carmen Bañó; Herminia González-Navarro; Concepción Abad (55-61).
In most lipases a mobile element or lid domain covers the catalytic site of the enzyme and the lid opening event, which usually proceed at a lipid–water interface, is required to form the catalytically competent lipase. We report here a noticeable increase in activity of two fungal lipases assayed in aqueous solution in absence of any interface when adding submicellar concentrations of amphipathic physiological molecules like long-chain acyl-CoAs. The catalytic activity was dramatically dependent on the acyl chain length of the amphiphile and could be related with a lid-opening process. Our data support that lipase activation can be triggered in the absence of a well-defined interface, and stresses the notion that other non-aggregated amphipathic constituents of the local microenvironment can act as putative regulators of lipase activity.
Keywords: Lipase activation; Conformational change; Long-chain acyl-CoA ester;

Participation of phospholipase D and α/β-protein kinase C in growth factor-induced signalling in C3H10T1/2 fibroblasts by Vidar A.T. Thorsen; Marta Vorland; Bodil Bjørndal; Ove Bruland; Holm Holmsen; Johan R. Lillehaug (62-71).
We have studied phospholipase D (PLD) activation in relation to protein kinase C (PKC) and the involvement of PLD in extracellularly regulated kinase 1 (MAPK) (ERK1) activation and c-fos mRNA expression in C3H/10T1/2 (Cl8) fibroblasts. In these cells, the PLD activity was significantly increased by porcine platelet-derived growth factor (PDGF-BB), phorbol 12-myristate 13-acetate (PMA), and epidermal growth factor (EGF). PLD activation by PDGF-BB and PMA, but not EGF, was inhibited in Cl8 cells expressing the HAβC2-1 peptide (Cl8 HAβC2-1 cells), with a sequence (βC2-1) shown to bind receptor for activated C kinase 1 (RACK1) and inhibit c-PKC-mediated cell functions [Science 268 (1995) 247]. A role of α-PKC in PLD activation is further underscored by co-immunoprecipitation of α-PKC with PLD1 and PLD2 in non-stimulated as well as PMA- and PDGF-BB-stimulated Cl8 cells. However, only PKC in PLD1 precipitates was activated by these agonists, while the PKC in the PLD2 precipitates was constitutively activated. The c-fos mRNA levels in Cl8 cells increased more than 30-fold in response to either PDGF-BB, EGF, or PMA. Approximately 60% inhibition of this increase in c-fos mRNA levels was observed in Cl8 HAβC2-1 cells. Formation of phosphatidylbutanol (PtdBut) at the expense of phosphatidic acid (PtdH) in the presence of n-butanol inhibited ERK1 activation and c-fos mRNA expression in PDGF-BB-treated Cl8 cells. ERK activation by PMA was unaffected by n-butanol in Cl8 cells but almost abolished by n-butanol in Cl8 HAβC2-1 cells, showing that ERK activation by PMA is heavily dependent on PKC and PLD1. In contrast, ERK activation by EGF in both cell types was not sensitive to n-butanol. These results indicate (1) a role of a functional interaction between the RACK1 scaffolding protein and a αPKC-PLD complex for achieving full PLD activity in PDGF-BB- and PMA-stimulated Cl8 cells; (2) PLD-mediated PtdH formation is needed for optimal ERK1 activation by PDGF-BB and maximal increase in c-fos mRNA expression. These findings place PLD as an important component in PDGF-BB- and PMA-stimulated intracellular signalling leading to gene activation in Cl8 cells, while EGF does not require PLD.
Keywords: Phospholipase D; RACK1; PKC; ERK1; c-fos; PDGF-BB; C3H/10T1/2 fibroblasts;

Gene expression and molecular composition of phospholipids in rat brain in relation to dietary n−6 to n−3 fatty acid ratio by Gwendolyn Barceló-Coblijn; Klára Kitajka; László G. Puskás; Endre Hőgyes; Agnes Zvara; László Hackler; Tibor Farkas (72-79).
Rats were fed from conception till adulthood either with normal rat chow with a linoleic (LA) to linolenic acid (LNA) ratio of 8.2:1 or a rat chow supplemented with a mixture of perilla and soy bean oil giving a ratio of LA to LNA of 4.7:1. Fat content of the feed was 5%. Fatty acid and molecular species composition of ethanolamine phosphoglyceride was determined. Effect of this diet on gene expression was also studied. There was an accumulation of docosahexaenoic (DHA) and arachidonic acids (AA) in brains of the experimental animals. Changes in the ratio sn-1 saturated, sn-2 docosahexaenoic to sn-1 monounsaturated, sn-2 docosahexaenoic were observed. Twenty genes were found overexpressed in response to the 4.7:1 mixture diet and four were found down-regulated compared to normal rat chow. Among them were the genes related to energy household, lipid metabolism and respiration. The degree of up-regulation exceeded that observed with perilla with a ratio of LA to LNA 8.2:1 [Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2619]. It was concluded that brain sensitively reacts to the fatty acid composition of the diet. It was suggested that alteration in membrane architecture and function coupled with alterations in gene expression profiles may contribute to the observed beneficial impact of n−3 type polyunsaturated fatty acids on cognitive functions.
Keywords: Linoleic acid; Linolenic acid; Docosahexaenoic acid; Phosphatidylethanolamine; Molecular specie; Gene expression; Learning; Memory;

Bezafibrate is a dual ligand for PPARα and PPARβ: studies using null mice by Jeffrey M Peters; Toshifumi Aoyama; Amanda M Burns; Frank J Gonzalez (80-89).
Bezafibrate is a known activator of peroxisome proliferator-activated receptors (PPARs) that can activate both PPARα and PPARβ. To determine the role(s) of these receptors in mediating the biological effects of this chemical, the effect of bezafibrate was examined in PPARα-null and PPARβ-null mice. Wild-type, PPARα-null, or PPARβ-null mice were fed either a control diet or one containing 0.5% bezafibrate for 10 days. Bezafibrate feeding caused a significant increase in liver weight in wild-type and PPARβ-null mice compared to controls, while liver weight was unchanged in bezafibrate-fed PPARα-null mice. Gonadal adipose stores were significantly smaller in wild-type and PPARβ-null mice fed bezafibrate than in controls, and this effect was not found in similarly fed PPARα-null mice. Analysis of liver, white adipose tissue, and intestinal mRNAs showed that bezafibrate caused similar changes of mRNAs encoding lipid metabolizing enzymes in wild-type and PPARβ-null mice compared to controls. Interestingly, in PPARα-null mice, bezafibrate also induced several mRNAs previously thought to be solely controlled by PPARα, showing that the effects of this drug are not exclusively modulated by this PPAR isoform. Western blot analysis of liver protein was consistent with changes in mRNA expression showing that the alterations in mRNA expression correlate with protein expression in this tissue. Results from these studies demonstrate that the effect of bezafibrate is mediated in large part by PPARα, although some changes in gene expression are dependent on PPARβ. In contrast to other PPARα ligands such as WY-14,643, induction of some target genes by bezafibrate can also be modulated in the absence of a functional PPARα.
Keywords: Bezafibrate; Peroxisome proliferator-activated receptor; Gene expression;

Structure elucidation of phenolic compounds from red/white wine with antiatherogenic properties by Elizabeth Fragopoulou; Smaragdi Antonopoulou; Tzortzis Nomikos; Constantinos A. Demopoulos (90-99).
The oxidative modification of low-density lipoproteins (LDL) is supposed to play a critical role in atherogenesis. During this oxidation a potent inflammatory phospholipid mediator named platelet activating factor (PAF) is produced, and it is believed to be the key for the initiation of the inflammation and therefore for the process of atherogenesis. From many studies, it is established that wine has beneficial effects on health, including protection against cardiovascular diseases. According to our point of view, the cardioprotective effect of wine may be attributed partly to the existence of PAF antagonists in red or white wine and partly to the existence of antioxidants that reduce the oxidation of LDL and therefore the production of PAF. In this study, wine compounds that antagonize PAF were isolated and purified via chromatographic procedures, and determined structurally using chemical, enzymatic and spectroscopic methods.
Keywords: Wine; Platelet activating factor; Phenolic; Antiatherogenic; Electrospray mass spectrometry (ESMS);