Posters 1-9

1 Membrane Structure and Function

2 Cardiovascular Disease

3 Bone Health

4 Cell Cycle, Apoptosis and Cancer

5 Eicosanoids

6 Fatty Acid Synthesis and Metabolism

7 Cell Signaling, Transcription Factors, Gene Expression

8 Infant Nutrition

9 Maternal and Fetal Nutrition

1 Membrane Structure and Function

(1-1) DIFFERENCES BETWEEN MEMBRANES WITH DOCOSAHEXAENOIC (22:6n3) AND DOCOSAPENTAENOIC (22:5n6) HYDROCARBON CHAINS
Nadukkudy V. Eldho1, Scott E. Feller2, Stephanie Tristram-Nagle3, Klaus Gawrisch1 1Laboratory of Membrane Biochemistry and Biophysics, NIAAA, Rockville, USA; 2Wabash College, Crawfordsville, USA;3Carnegie Mellon Univ., Pittsburgh, USA

Recent NMR experiments support a view of polyunsaturated fatty acids (PUFA) as highly flexible molecules existingin a multitude of conformations with rapid conformational transitions. The underlying cause for this flexibility are extremely low potential barriers for rotation about the C-C bonds between the double bonds permitting PUFA torapidly change conformation without energetic penalties. The PUFA segments near the carbonyl group move with motional correlation times of the order of 1 ns. The correlation times decrease and the motional amplitudes increasefrom double bond to double bond, reaching correlation times of the order of 10 ps at the methyl terminal end of the PUFA chain. However there are significant differences in motional properties and distribution of PUFA along the bilayer normal between the ω-3 docosahexaenoic acid and the ω-6 docosapentaenoic acid. The DHA tends to have higher density near the lipid water interface compared to the DPA. The difference is reflected as a difference in chain order parameters of saturated stearic acid that is paired with DHA or DPA in phosphatidylcholines, but also in DHA and DPA order parameters, motional correlation times along the chain, in the electron density profiles of lipid bilayers obtained in x-ray diffraction experiments, and in molecular simulations. We speculate that these differences in the distribution of lipid hydrocarbon chains alter lateral pressure density profiles of membranes important for function of integral membrane proteins.

(1-2) EFFECT OF THE PHOSPHATIDYLETHANOLAMINE (PE) HEADGROUP ON RHODOPSIN ACTIVATION
A. Pacifico, E. Hayakawa*, D. C. Mitchell, and B. J. Litman. Lab of Membrane Biochemistry & Biophysics, NIAAA, NIH,
Rockville, MD, USA. *Keio Univ., Tropical Medicine and Parasitology, Sch. of Med. 35, Shinanomachi, Shinjuku-ku,Tokyo, Japan

The phosphatidylethanolamine (PE) headgroup is key for optimal activity of some proteins, such as PKC. We studied the effect of the PE headgroup on rhodopsin (Rho) activation in mixed di18:1PE (DOPE)/ di18:1PC (DOPC) vesicles. Vesicles containing Rho (lipid/protein = 85/1) in 0 mol%, 30 mol% or 50 mol% DOPE were examined. The 50 mol% PE sample was determined by phosphorous NMR to be in the lamellar phase across the temperature range studied (10 - 45°C). The level of formation of the G protein activating conformation of rhodopsin, MII, increased going from 0 to 30 mol% DOPE, but decreased when DOPE was increased to 50 mol%. The effect of PE on membrane phospholipid acyl chain order was probed by time-resolved fluorescence anisotropy decay. The fluorescence measurements showed that the phospholipid acyl chain order was lower in 30mol% DOPE relative to the 50 mol% DOPE sample. A comparison of 30 and 50 mol% DOPE samples shows that a higher level of MII formation correlated with a lower level of phospholipid acyl chain order. The effect of DOPE on MII formation can be explained in terms of a combination of preferential interaction between PE and Rho and the effect of the PE headgroup on phospholipid acyl chain order.

(1-3) CURVATURE STRESS IN MEMBRANES WITH POLYUNSATURATED PHOSPHATIDYLETHANOLAMINES
Walter E. Teague1, Horia I. Petrache2, Nola Fuller3, R. Peter Rand3, Klaus Gawrisch1,1Laboratory of Membrane Biochemistry and Biophysics, NIAAA, NIH, Rockville, USA 2Laboratory of Physical and Structural Biology, NIH/NICHD, Bethesda, MD, USA,3Brock University, St. Catharines, ON, Canada

Neural membranes contain very high concentrations of phosphatidylethanolmines (PE) with polyunsaturated hydrocarbon chains. Lipid monolayers of PE have a propensity to curl, resulting in formation of nonlamellar phases. Lipid bilayers containing PE are under considerable curvature elastic stress that may influence the function of integral and peripheral membrane proteins like ion channels and membrane receptors. We determined membrane elastic properties of lipid monolayers composed of 18:0-22:6n-3 PE and 18:0-22:5n-6 PE. The purified lipids in water form an inverse hexagonal phase at temperatures above 25oC. Curvature elastic properties were determined by combined solid state NMR and x-ray diffraction experiments with controlled application of osmotic stress. The results show that both polyunsaturated PEs form monolayers with a high degree of spontaneous curvature. We observed differences between 18:0-22:6n-3 PE and 18:0-22:5n-6 PE consistent with an influence on chain packing from the loss of a single double bond. This work supports the idea that lipid bilayers rich in polyunsaturated PEs are under considerable curvature elastic stress. The release of even a fraction of this stress, at the lipid protein interface, may ease conversion of membrane receptor proteins such as rhodopsin to the activated state.

(1-4) SULFATIDE PROMOTED OLIGOMERIZATION OF COBRA CARDIOTOXIN IN LIPID RAFTDOMAINS RESPONSIVE FOR ITS CELL EFFECTS
Chia-Hui Wang1, Po-Long Wu1, Wei-Ning Huang1, Shao-Chen Lee1, Robert Monette2, Paul Morley2, Nongnuj Tanphaichitr3, Wen-guey Wu1 1Department of Life Sciences, National Tsinghua University, Hsinchu, Taiwan,2Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada 3Obsterrics and Gynecology Department, Ottawa Health Research Institute, Ottawa, Ontario, Canada

Cobra cardiotoxins (CTXs) are structurally related snake venom b-sheet polypeptides that cause systolic cardiac arrest and severe tissue necrosis of the bitten victim. These toxins are also called cytotoxins because of the general ability to perturb membrane structure in both excitable and non-excitable cells with ambiguous sites of action inbiological membranes. We had demonstrated before that negative charged lipids potentially induced CTXs oligomerization in vitro. However, binding abilities of CTXs toward other anionic ligands, such as sulfatedcarbohydrates, implied sulfated glycolipids might also be potential targets on outer leaflet of plasma membrane. Herein, we showed important role of sulfatide in CTXs’ action on the microdomain of plasma membrane accompaniedwith several cellular effects. In model membrane studies, sulfatide induced CTXs oligomerization and higher level of vesicle leakage than other negative charged phospholipids was through size-defined pore formation on membrane.We also demonstrated that CTXs acted on lipid raft domain in vivo. The colocalization of CTXs with sulfatide was observed by both immunoblotting and TLC on detergent-resistant fractions in sucrose gradient flotation. Confocalsection also suggested the location of CTXs at GM1/sulfatide-enriched region. CTXs-induced cell responses and cytotoxicity were correlated with sulfatide-presented raft domain. The pretreatment with anti-sulfatide antibodies orexogenous sulfatide incorporation suggested its involvement in calcium influx and CTX-induced cytotoxicity. In further, Rac-related cytoskeleton rearrangements as well as calcium release from internal store induced by CTXs were observed. In summary, we suggested the action of CTXs on sulfatide-enriched region induced multiple effects including oligomerization on plasma membrane, calcium influx associated cytotoxicity, induction of calcium release from internal store and possible regulatory signaling through cytoskeleton rearrangement.

(1-5) EFFECTS OF THE HYPOTHENSIVE DRUG, 2-HYDROXYOLEIC ACID, ON THE STRUCTURAL PROPERTIES OF MODEL MEMBRANES
F.Barceló, J. Prades, J. Frau, S.S. Funari*, C. Scotta, M. Vidal, and P.V. Escribá,Laboratory of Molecular and Cellular Biomedicine, Institut Universitari d’Investigacions en Ciències de la Salut, University of the Balearic Islands, Palma de Mallorca, Spain. *Max-Planck Institute for Colloids and Interfaces, Hamburg, Germany

It has been demonstrated that short-term administration of the synthetic fatty acid 2-hydroxyoleic (2OHOA), decreased blood pressure in rats by modulating G-protein-mediated cellular signaling (R. Alemany et al., Hypertension 43(2004)249). If the membrane structure is involved in cell signaling processes, then structural alterations of cell membranes can regulate the behavior of the membrane and may influence pathological situations. 2OHOA is a structural derivative of oleic acid (OA). The hypotensive activity of 2OHOA are most likely to originate in its effects on the structural properties of the plasma membrane. Thus, we set out to elucidate the structural bases underlying the biological effect of 2OHOA by analyzing its interaction with model lipid membranes using DSC, NMR, X-ray diffraction and molecular dynamic modelization. 2OHOA alters the thermotropic behavior of 1,2-dielaidoyl-snglycero- 3-phosphoethanolamine, thereby promoting the formation of hexagonal phases (HII) despite stabilizing the lamellar phase. The molecular bases underlying these structural alterations were analyzed by comparing the effects produced by 2OHOA with those of OA. The effects of 2OHOA on the physical properties of model membranes, could account for the changes in the localization and activity of peripheral membrane proteins involved in the hypotensive properties of this drug.

(1-6) NMR EVIDENCE FOR RAFTS IN BIOMEMBRANES
Klaus Gawrisch, Ivan V. Polozov,Laboratory of Membrane Biochemistry and Biophysics, NIAAA, NIH, Rockville, USA

We developed 1H magic angle spinning (MAS) NMR into a tool for detection of liquid ordered domains (rafts) in biological membranes. There is evidence that rafts are liquid ordered, cholesterol-rich domains surrounded by a liquid disordered lipid matrix. We established that liquid ordered domains have distinctly different 1H MAS NMR spectra that are discernible from the spectra of their liquid disordered environment. By dipolar echo 1H NMR spectroscopy we determined that the spectral differences are caused by a strong reduction in the rate of gauche/trans isomerization of lipid hydrocarbon chains. In spectra recorded as a function of temperature, the onset of liquid-ordered domain (raft) formation is seen as sudden linebroadening, independent of raft size. Formation of large rafts (diameter >0.3 µm) in a liquid disordered environment resulted in signal superposition of resonance signals from both environments. Experiments require only 10 µL of membrane material without the need for introduction of labels. The method is suitable for investigation of cell membrane preparations. In experiments on rod outer segment discs extracted from raising doubts about the validity of detergent extraction methods for raft detection. The size of rafts may be studied directly by 1H MAS NMR with application of pulsed field gradients. An example of such experiments on a model lipid system will be shown.

(1-7) MOLECULAR INTERACTIONS OF TRANS FATTY ACID-CONTAINING PHOSPHOLIPIDSWITH MEMBRANE RECEPTOR AND CHOLESTEROL
Shui-Lin Niu and Burton J. Litman,Laboratory of Membrane Biochemistry & Biophysics, NIAAA, National Institutes of Health, Rockville, Maryland, USA

Trans fatty acids (TFAs) are incorporated into our food chain mainly due to the process of partial hydrogenation of unsaturated oils. The average American is estimated to consume 5.3 grams or more of TFAs every day. The intake of TFAs results in the elevation of LDL cholesterol and increased risks for coronary heart disease. In this study, we examined the difference in molecular interactions with membrane receptor and cholesterol by the replacement of cis fatty acid (CFAs) with TFAs in membrane phospholipids. Our study found that TFA-phospholipids exhibited 50-80% higher cholesterol affinity than the corresponding CFA-phospholipids. TFA-phospholipid increased membrane acyl chain packing order relative to CFA-phospholipid reflected by the higher gel-to-fluid phase transition temperature and the enhanced thermal transition temperature of rhodopsin, a G protein coupled membrane receptor. The increased membrane packing by TFA-phospholipid correlated with reduced rhodopsin activation, measured by the extent of metarhodopsin II formation, which is consistent with previous findings on membrane acyl chain packing and rhodopsin activation. The combination of enhanced cholesterol affinity and reduced membrane receptor activation by the replacement of CFAs with TFAs in membrane phospholipids may have important roles in cholesterol homeostasis, which could attribute to the elevation of LDL cholesterol by the intake of TFAs.

(1-8) MODIFICATION OF BILAYER ELASTICITY BY CHOLESTEROL AND ITS PRECURSORS
Horia I. Petrache, Daniel Harries, V. Adrian Parsegian,Laboratory of Physical and Structural Biology, NICHD, National Institutes of Health, Bethesda, MD, USA

Cholesterol and its metabolic and evolutionary precursors have been shown to affect physical properties of lipidbilayers. These effects are customarily interpreted in terms of material properties such as lateral (area) compressibility and bending elasticity. Here we present X-ray measurements of sterol effects on lamellar and inverted hexagonal phases of disaturated and monounsaturated phosphatidylcholines. Measurements of hexagonal structures and their response to osmotic stress give us direct information on the monolayer curvature as well as bending energies. While monolayer (intrinsic) curvature is not explicit in the lamellar geometry, measurements of interlamellar spacing and fluctuations give information on bilayer elasticity. By measuring the response of multilamellar structures to osmotic stress, we show how bending rigidities differ for the different sterol- containing bilayers. Cholesterol has the strongest effect, making most rigid bilayers. For further comparison between different sterols, by using non-polar solvents such as tetradecane, a transition from lamellar to inverted hexagonal can be induced and its response to modification of lateral stress, a structural feature relevant to interactions within biological membranes.

(1-9) MODES OF LONG CHAIN FATTY ACID TRANSPORT ACROSS MEMBRANES
Peter Pohl, Uwe Peterson, Sapar M. Saparov, Andrey V. Krylov, Elena E. Pohl§ ; Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany; §Charite, Humboldt Universität zu Berlin, Berlin, Germany

Long-chain fatty acids (FA) may translocate across lipid bilayers as anions as well as after proton or cation binding. To clarify the contribution of the different transport modes, a defined system was developed for simultaneous monitoring of FA movement and membrane conductance. Planar membranes were formed asymmetrically, i.e. only one of the two membrane leaflets contained FA anions. The resulting interleaflet surface potential difference was used to monitor FA flip flop. FA transfer between both leaflets occurred immediately after membrane formation. Neither flip-flop of saturated (stearic acid) nor unsaturated (arachidonic acid) FAs was accompanied by a significant increase in membrane conductivity. Nevertheless, FAs were able to induce transmembrane cation transport. Using scanning ion sensitive microelectrodes, we measured an electrically silent cation permeability of about 2.5x10-7 cm s-1. Also the rate of FA anion translocation (2x10-6 s-1) was found to be too small to contribute significantly to the electroneutral flip flop of protonated fatty acids.

(1-10) EXPRESSION AND ACTIVITY OF TWO RECOMBINANT ISOFORMS OF RAT NMT1
V. Rioux, M. Brochet, F. Pedrono, S. Daval, D. Catheline, M. Bouriel, H. Guillou, and P. Legrand,Laboratoire de Biochimie ENSA-INRA Rennes, FRANCE

Protein N-myristoylation refers to the covalent attachment of myristic acid, by an amide linkage, to the NH2-terminal glycine residue of eukaryotic and viral proteins. Myristoyl-CoA: protein N-myristoyltransferase (NMT) is the enzyme catalyzing this stable acylation. Since several myristoylated proteins are involved in cell signaling, the activity of NMT may have an important effect on the level of myristoylation of these proteins and therefore on cell regulation and metabolism. Biochemical studies have shown the presence of multiple distinct protein forms of native mammalian NMTs in vivo and 2 NMT genes, named type I and II, have been identified. Tissue specific expression and specific catalytic activity of these isoforms may be important for regulating the level of protein myristoylation, but has not been studied yet. In the present work, the longest cDNA (accession #NM_148891) encoding rat NMT1 protein was isolated. It contains 4 potential in-frame start codons. The longest putative cDNA contains 1491 nucleotides and the shortest 1251 nucleotides. After cloning these two putative cDNAs into a plasmid allowing transfection into COS-7 cells, the expression and activity of the two recombinant proteins were studied. The results show that both proteins possess apparent molecular weights different from the predicted molecular weights derived from their respective cDNA, while their activity could be similar. Further characterization of NMT enzymes could contribute to an improved understanding of the nutritional effect ofmyristic acid in the cells.

(1-11) THE Gβγ-DIMER DRIVES THE INTERACTION OF HETEROTRIMERIC Gi PROTEINS WITH NONLAMELLAR MEMBRANE STRUCTURES
Oliver Vögler, Jesús Casas and Pablo V. Escribá,Laboratory of Molecular and Cellular Biomedicine, Department of Biology, University of the Balearic Islands, Ctra. Valldemossa Km 7.5, Palma de Mallorca, Spain

Heterotrimeric G proteins are peripheral membrane proteins which propagate signals from cell surface receptors into the cell. Since the transmembranal regions of these receptors are capable to influence plasma membrane properties, protein-lipid interactions should play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins, and the entities formed upon receptor-mediated activation (Gαand Gβγ), to model membranes (liposomes). The model membranes used were composed of defined membrane lipids, capable of organizing into either lamellar or nonlamellar (hexagonal H_II) membrane structures. We demonstrated that although heterotrimeric Gi proteins and Gβγ-dimers can bind to lipid bilayers composed of phosphatidylcholine, their binding to membranes was significantly enhanced by the HII propensity of phosphatidylethanol-amine (898 ± 58% and 1575 ± 316%). Conversely, the binding of activated Gαi-subunits to liposomes was drastically impaired by HII-prone phospholipid (-73.9 ± 1.6%). This has important consequences in cell signaling. First, the binding characteristics of the Gβγ-dimer accounts for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gβγ-dimers and Gα-subunits with membrane lipids, in part explain their different cellular localization during signaling. These findings suggest an active role of membrane lipids in cell signaling through G protein-coupled receptors.

2. Cardiovascular Disease

(2-1) ANTIHYPERTENSIVE EFFECTS OF THE MONOUNSATURATED FATTY ACID 2- HYDROXYOLEIC ACID IN SPONTANEOUSLY HYPERTENSIVE RATS
Carolina Egea, Silvia Terés, Carmela Baamonde, Oliver Vögler, Pablo V. Escribá and Regina Alemany, Laboratory of Molecular and Cellular Biomedicine, Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain

We have recently demonstrated that the monounsaturated fatty acid 2-hydroxyoleic acid (2-OHOA) reduces blood pressure in Sprague-Dawley rats. In the present study, we investigated the cardiovascular effects of 2-OHOA in spontaneously hypertensive rats (SHR), an animal model of genetic hypertension, and their normotensive counterpart, WKY rats. Oral administration of 2-OHOA induced significant and sustained decreases of blood pressure in time- (1-7 days) and dose (100-900 mg/kg/12h)-dependent manners in SHR rats. Treatments with 2-OHOA (600 mg/kg/12h for 7 days) reduced systolic (-54 mm Hg, -27%) and diastolic (-51 mm Hg, -28%) blood pressure and the heart rate (-79 beats/min, -19%) in SHR but not in WKY rats. At the molecular level, 2-OHOA increased forskolinstimulated adenylyl cyclase (AC) activity in aorta membranes from SHR and WKY rats (increase of 32 ± 3% and 45 ± 19%, respectively), whereas decreased aortic cGMP levels by 77 ± 9% in WKY rats. Similarly, plasmatic cAMP levels were found increased by 59 ± 12% only in 2-OHOA-treated SHR rats. On the other hand, the densities of the aortic catalytic and regulatory (II?) protein kinase A (PKA) subunits were found up-regulated in 2-OHOA-treated SHR rats. Our results indicate that 2-OHOA treatment enhances the production of cAMP in plasma and aorta by activation of AC, leading to an upregulation of PKA and a strong reduction of blood pressure in SHR rats.

(2-2) EFFECTS OF A COMBINATION OF NUTRIENTS SUPPLE-MENTED IN MILK IN THE NUTRITIONAL MANAGEMENT OF PERIPHERAL VASCULAR DISEASE (PVD) PATIENTS
JJ Carrero*, M González-Santiago*, L Baró*, J Fonollá*, M Gómez†, LM Salmerón†, E Ros†, J Jiménez*, JJ Boza*and E López-Huertas* * PULEVA Biotech SA. 66, Camino Purchil, Granada, SPAIN. † Angiology Dept, San Cecilio Hospital, Granada, Spain

Introduction. n-3 PUFA, oleic acid and folic acid consumption has been associated with a lower risk of cardiovascular disease (CVD). This study focuses on the effects produced by the consumption of these nutrients, supplemented in milk, in PVD patients. Methods. 30 PVD male patients were given 0.5 L/day of an enriched milk (Puleva Omega 3) containing n-3 PUFAs (333 mg DHA+EPA), oleic acid (5.1 g), folic acid (150 µg) and vitamins B6 (1,5 mg) and E (7,5 mg) (? group). The other 30 PVD male patients were provided with 0.5 L/day of semi-skimmed milk (placebo group). Blood was sampled at times T0, T3, T6, T9 and T12 months. Results. At T12, the ? group increased the walking distance (105±22 vs 394±61 m; p=0.000) and the ABI index (0.46±0.022 vs 0.52±0.027, p=0.01), showing a reduction in total cholesterol (5.58±0.18 vs 5.26±0.14 mmol/L; p=0.013), and a lower LDL-C than the placebo (3.22±0.13 vs 3.43±0.21mmol/L; p=0.027). Serum and RBC folate and vitamin B6 increased (p<0.01) only in the ? group. When total homocysteine was high (>15 µmol/L), reductions were only found in the ω group (17.4±0.7 vs 14.8±0.8 µmol/L; n=8, p=0.032). No changes were found in plasma or LDL oxidation markers. Plasma VCAM-1 and E-selectin increased only in the placebo group (p<0.05). This combination of fats and nutrients in the daily diet, together with healthy lifestyle habits, may be useful in PVD patients for lowering CVD risk and improving the clinical signs.

(2-3) DIETARY FISH OIL AND FATTY ACID COMPOSITION OF PLASMA AND MYOCARDIUM IN PIGS
Dullemeijer C1,2, Brouwer IA1,2, Coronel R3, Katan MB1,2, Zock PL1,2 ,1Wageningen Centre for Food Sciences, Wageningen, The Netherlands, 2Division of Human Nutrition, Wageningen University, The Netherlands, 3Department of Experimental Cardiology, Academic Medical Centre, Amsterdam, The Netherlands

N-3 fatty acids are suggested to have anti-arrhythmic properties potentially through incorporation into the myocardium. We examined whether pigs fed extra dietary n-3 fatty acids have higher amounts of n-3 fatty acids in plasma and myocardial phospholipids than pigs fed a control diet. We performed a randomized, blinded, placebo-controlled trial in male pigs. The experimental group (n=12) received a semi-purified diet containing 4% (w/w) fish oil and the control group (n=11) a semi-purified diet containing 4% (w/w) high oleic sunflower oil for a period of 8 weeks. Fatty acid profiles in phospholipids of plasma (obtained in a fasted state) and myocardium were determined after 8 weeks. We compared the percentages of several n-3 polyunsaturated fatty acids in myocardial and plasma phospholipids between the two groups. Values are means ± SD *Significantly different from oleic oil-fed pigs (p<0.05) Fish oil-fed pigs had higher levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and lower levels of linoleic acid (LA) and arachidonic acid (AA) in both plasma and myocardium compared with the oleic oil-fed pigs (p<0.05). Dietary fish oil, relative to the control diet, results not only in higher amounts of EPA and DHA in phospholipids in pigs in plasma, but also in myocardium. In both plasma and myocardium this increase in EPA and DHA occurs at the expense of LA and AA.

Values are means ± SD

* significantly different from oleic oil-fed pigs (P < 0.05)

(2-4) PLATELET FATTY ACID COMPOSITION AND CORONARY HEART DISEASE RISK IN THE SUDAN: A CASE-CONTROL STUDY
A.Shikieri, R.Armstrong, J.Landman, R.Wilson, M.Elameen, R.Rimeresma, D.Sutherland, The National Ribat University, Khartoum, Sudan, The Chief Scientist Office, Edinburgh, UK, The Nutrition Society, London, UK University of Edinburgh, UK, Dietetics, Nutrition and Biological Sciences, Queen Margaret University College, Edinburgh, UK

Coronary heart disease (CHD) event and mortality rates are lower in Sudan than many developed and developing countries. The objective of the current study was to determine whether a high intake of dietary polyunsaturated fatty acids (PUFA) is responsible for the low CHD event and mortality rates in the Sudan. 61 CHD cases and 63 apparently healthy controls aged 40-65 yrs with blood pressure < 160/95 mmHg, glucose level of < 6.5 mmol/L and cholesterol level of <5.2 mmol/L (healthy subjects) and < 6.5 mmol/L (CHD cases). Dietary pattern was estimated using repeated 24 hr-recall records which were validated and platelet fatty acid composition was determined by gas chromatography. Cases and controls had similar PUFA levels. The platelet PUFA was primilary n-6 (43 ± 1.1%) with only 2 ± 0.1% being n-3 PUFA thus resulting in a higher ratio of n-6:n-3 PUFA. Both cases and controls had high levels of platelet LA AND DGLA. The ratio of platelet PUFA:SFA was > 1 amongst both cases (1.3 ± 0.0%) and controls (1.4 ± 0.0%). Both cases and controls had lower intake of fish (< 35 g/d) and fresh fruit intake. Results were comparable with other countries such as Scotland where cases and controls had lower LA levels and higher n-3 PUFA. The high intake of dietary LA, DGLA AND PUFA:SFA ratio may be responsible for or at least contribute to the low CHD mortality rates in the Sudan.

(2-5) N-3 LONG-CHAIN POLYUNSATURATED FATTY ACIDS IN PATIENTS UNDER CHRONICHEMODIALYSIS AND THEIR PULSE WAVE VELOCITY
Kei Hamazaki1, Hitoshi Inagaki2, Miho Itomura1, Shigeki Sawazaki1, Shin Tomita3, Hitoshi Hirata3, Masahiro Kuroda2, Tomohito Hamazaki1 1: Toyama Med and Pharm Univ, Toyama Japan; 2: Asanagi Hospital, Toyama; 3: Jounan Clinic, Toyama

n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs) are known to prevent cardiovascular disease (CVD). However, it has not yet been reported whether n-3 LCPUFAs are related to arteriosclerosis of patients under chronic hemodialysis (HD). Methods: Pulse wave velocity from the brachium to the ankle (baPWV) was measured as a marker of arteriosclerosis with an ABI-form (COLIN Medical technology Co., Ltd., Komaki, Japan) in 149 chronic HD patients [non-diabetic (non-DM): 51 males/ 44 females, 62±14 y; and DM: 33 m/21 f, 67±9 y]. The fatty acid composition of the total phospholipid fraction from washed RBCs was analyzed by gas chromatography. Analyses were adjusted for age, sex, systolic blood pressure (SBP), pulse, body mass index (BMI), period of HD treatment and smoking status. Results: The mean baPWV was 18.9±5.2 and 23.7±6.3 m/s in non-diabetics and diabetics, respectively. The mean diabetic baPWV was significantly higher than that of non-diabetics after adjustment (p=0.005). Multivariable (p=0.02) in non-DM patients after adjustment but not in DM patients. Discussion: We suggest that n-3 LCPUFAs are also a negative risk factor of CVD in non-DM HD patients. In DM patients the effects of n-3 PUFAs on the vascular system became undetectable probably because DM overwhelmingly affected it. Further studies in a prospective manner are necessary.

(2-6) MODIFIED OMEGA-3 FATTY ACIDS PROFILES IN PATIENTS WITH IDIOPATHETIC ATRIAL FIBRILLATION

EL Mejaber W.1, Gutowski N.1, Miguet J.1, Gleize B.1, Payet M.1, Duran MJ.2 , Sennoune S.2 ,Paganelli F.1, Pieroni, G.1, Maixent JM.1 1 :Université de Poitiers & Marseille, France 2 : Texas Tech University, USA

Background: Recent developments confirm and extend the concept that n-3 (omega 3) fatty acids (FA) are beneficial in the prevention of cardiovascular diseases and sudden cardiac death. Many reports have shown positive correlation between n-3 polyunsaturated FA (PUFA) and coronary artery disease (CAD). The mechanism of prevention by n-3 PUFA involves cardiac antiarrhythmic properties. All the evidences could be applied to atrial fibrillation (AF) but nothing is known in about n-3 PUFA status from patients with idiopathic AF. Objective: To examine FA patterns associated in Mediterranean patients (pts) with AF. Methods: A total of 38 consecutive pts with idiopathic AF were included. Exclusion criteria were diabetes and CAD. The gas chromatography method was used to analyze The erythrocyte membrane FA patterns from pts with idiopathic AF were analyzed by gas chromatography. Pts without coronary stenosis were used as controls (n=12). Results: Pts with AF showed increased percentages of arachidonic acid (AA) [(n-6) serie) (15.9±0.6 vs. 17.2±0.4%, p<0.05) as well as decreased percentages of docosahexaenoic acid (DHA) [(C22 :6 (n-3) (6.6±0.2 vs. 5.6±0.2%, p<0.05)]. The n-3/n-6 PUFA balance expressed by the DHA to AA ratio is also significantly altered from 0.42±0.02 to 0.33±0.02 between control and AF groups. Conclusion: Knowing the metabolic competition between both n-3 and n-6 PUFA families, n-3 FA dietary interventions could also be of benefit for pts with idiopathic AF.

(2-7)THE OMACOR CAROTID ENDARTERECTOMY (OCEAN) TRIAL - STUDY DESIGN
AL Cawood1, FL Napper1, J Williams2, PJ Gallagher3, CP Shearman2, RF Grimble1, PC Calder1, 1Institute of Human Nutrition, University of Southampton; 2Vascular Surgery, Southampton General Hospital; 3Chemical Pathology University of Southampton, UK

It is possible that long chain n-3 polyunsaturated fatty acids (PUFAs) from fish oil contribute to stabilisation of atherosclerotic plaques through an anti-inflammatory mechanism. Recent data from an intervention study suggests that greater proportions of EPA and DHA are present in carotid plaque lipid fractions in patients consuming fish oil and that this is associated with fewer infiltrating macrophages and a plaque morphology indicative of increased stability (Thies et al. (2003) Lancet 361, 477-485). It is important to confirm these findings and to understand more about the mechanisms involved in plaque stabilisation by n-3 PUFAs. Therefore a new intervention study using Omacor in carotid endarterectomy patients is being undertaken. 140 patients awaiting carotid endarterectomy will be recruited into a randomised double-blind, placebo controlled study of Omacor (2 g daily, provided by Pronova Biocare). Patients will consume placebo or Omacor from study entry until surgery. Fasting blood will be collected at study entry and immediately prior to surgery. At surgery the carotid plaque will be collected. Plasma will be analysed for lipid, soluble adhesion molecule and C-reactive protein concentrations. Plaque specimens will be used for determination of fatty acid composition, morphology (AHA and modified AHA gradings), infiltration of macrophages and T lymphocytes (immunohistochemistry), activation state of T lymphocytes (immunohistochemistry), presence of inflammatory cytokines (immunohistochenmisyry; mRNA) and presence of matrix metalloproteinases (mRNA). This research is supported by Pronova Biocare and results will be available from mid-2005.

(2-8) APPLICATION OF DIRECT FATTY ACID ANALYSIS IN A BLOODROP FROM A FINGERTIP IN A POPULATION GROUP. CORRELATIONS WITH DIETARY HABITS, PHYSIOLOGIC CONDITIONS, LIFE STYLES AND FATTY ACID SUPPLEMENTATIONS
Galli C., Colombo C. and Marangoni F.,Department of Pharmacological Sciences, School of Pharmacy, University of Milan, Italy

Dietary fatty acids (FA), e.g. the intakes of n-3 PUFAs, are important in the maintenance of health. However the assessment of the FA status in large groups is limited by the complexity of conventional methods for sample preparation and analysis. A new approach (Marangoni et al. Anal.Biochem. 2004) for the analysis of FA in a drop of blood collected from a fingertip was applied to screen 100 volunteers (54 M vs 46 F, 22-73 y), and questionnaires for food consumption were also used. The following differences were observed : Gender : higher LA and lower OA in F vs M. Dietary habits : higher AA in high vs low meat consumers, higher levels of n-3 FA (especially DHA) in high vs low fish consumers. Life styles: lower n-3 FA in smokers. Physiologic conditions: lower PUFA in pregnant women. FA intakes: both the consumption of salmon (200 g/week) and the intake of 1capsule /day of n-3 FA for two weeks resulted in distinct elevations of blood EPA and DHA levels. The new method has the following advantages: rapid, simple, less expensive, no requirement of health personnel for sample collection, applicable to population groups, it provides information on the impact of dietary habits, life styles and FA supplementation on blood FA.

(2-9) EFFECT OF AA, EPA AND DHA IN VASCULAR FUNCTION OF SMALL MESENTERIC ARTERIES IN VITRO
1 Golfetto I,2 Kahn I,2 Poston L,1 Crawford MA.1 Institute of Brain Chemistry & Human Nutrition, Metropolitan University,North Campus .2 Fetal Health Research Unit, Division of Gynecology, St Thomas Hospital

Vascular endothelial disorders are now considered to be an important underlying pathology which predisposes to atherosclerosis, thrombosis and hypertension. Beneficial effects have been reported in individuals that consume fish oil (rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA). Arachidonic acid (AA) is an important component of vascular endothelium. Aims: We tested the effect of AA, EPA and DHA on normal rat mesenteric arteries to determine whether the vasodilatatory and vasoconstrictor response is modified by noradrenaline and acetylcholine (ACh) in vitro. Methods: Small mesenteric arteries Sprage Dawley rats (n=10) fed with a standard breeding diet were dissected and mounted on a small vessel wire myograph and incubated with AA, EPA and DHA. Subsequently dose-response curve were performed. Results: Significant relaxation of mesenteric arteries preincubated with AA was obtained between 10-9 to 10-6 molar ACh compared with controls. pEC50 changed significantly (7.32 ± 0.13 treated vs. 6.82 ± 0.05 control. p<0.01). No significant effect was described for EPA and DHA. Conclusion: AA enhances endothelium-dependent vasodilatation to acetylcholine and an increased sensitivity to ACh.

(2-10) EFFECT OF OMEGA-3 SUPPLEMENTATION AND MODERATE INTENSITY EXERCISE ON CARDIOVASCULAR (CV) HEALTH
AM Hill, KJ Murphy, DA Saint, JD Buckley & PRC Howe,Nutritional Physiology Research Group, School of Molecular and Biomedical Science, University of Adelaide and School of Health Science, University of South Australia, Adelaide, Australia

Regular exercise and ω3 consumption are strategies that can improve CV health. Two studies examining the combination of these strategies found contradictory effects on blood lipids (Brilla et al. 1990, Warner et al. 1989). The aim of the present study was to compare effects of dietary ω3 supplementation and regular moderate-intensity exercise, both alone and in combination, on CV risk factors including obesity in individuals with symptoms of the metabolic syndrome. Overweight volunteers (mean BMI=34) with raised plasma TAG (mean 2.3mM), or blood pressure (BP; mean 136/84 mmHg) were assigned to one of four groups; fish oil and exercise (FOX); sunflower oil and exercise (SOX); fish oil (FO) and sunflower oil (SO, control) in a double blinded dietary intervention trial. The FOX and FO groups consumed 6 g/day of HiDHA tuna fish oil (~1.6g DHA), while the SOX and SO groups took 6 g/ day of sunflower oil for 12 weeks. The FOX and SOX groups walked for 45 min, 3 days/wk at 75% of the predicted maximal heart rate for each subject. Blood lipids, BP, arterial stiffness, endothelial function and compliance with diet and exercise regimes were assessed at 0, 6 and 12 weeks. Body composition (% body fat) was assessed by DEXA at 0 and 12 weeks. All 24 of the first intake of subjects (6/group) completed the requirements. Despite increased total energy intake in all groups over 12 weeks, there was a trend toward greater reductions of weight, body fat, BP and small artery stiffness in the FOX group and a significant treatment interaction for both total body fat (P=0.03) and abdominal fat (P=0.01). Evaluation of a second cohort of 36 subjects is now in progress.

(2-11) EFFECTS OF n-3 POLYUNSATURATED FATS ON INDUCIBLE VENTRICULAR TACHYCARDIA IN PATIENTS UNDERGOING CARDIAC ELECTROPHYSIOLOGY TESTING: A PILOT STUDY
RG Metcalf, 1GD Young, *MJ James, LG Cleland . Rheumatology Unit and 1Cardiology Unit, Royal Adelaide Hospital, Adelaide,
SA, Australia 5000. (*Presenting author)

Increased consumption of fish and / or n-3 polyunsaturated fatty acids (PUFA) is associated with a reduced risk of sudden cardiac death, suggesting a cardiac anti-arrhythmic effect. We have undertaken a pilot study to determine whether supplementation with n-3 fatty acids has any effect on inducible, sustained ventricular tachycardia (VT) in patients undergoing cardiac electrophysiology (EP) testing. Fifteen patients with inducible, sustained VT at EP testing were supplemented with 3 g/d fish-oil (900 mg/d n-3 fatty acids) for at least 3 weeks before repeat EP studies. (NB 11/15 patients had inducible VT at enrolment despite antiarrhythmic drug therapy) The inducibility of VT was examined by a standardised protocol which incrementally increased in aggressiveness until a VT was induced or until the end of the protocol was reached, after which the patient was considered ‘non-inducible’. At repeat EP testing after 3 weeks of fish oil supplementation: • 8/15 patients no longer had inducible VT • 5/15 patient required more aggressive stimulation to induce the VT • 1/15 patients was unchanged • 1/15 patients required less aggressive stimulation to induce the VT These fish oil induced differences were significant (Wilcoxon’s Signed Rank Test, p=0.001). Thus, there was a significant shift towards non-inducibility, providing preliminary evidence of an anti-arrhythmic effect of fish oil in humans.
(Berg Lipid Tech AS (BLT) has provided oil for our studies)

(2-12) DO n-3 POLYUNSATURATED FATTY ACIDS PROTECT AGAINST ATHEROSCLEROSIS? FACT OR FICTION?
Gerhard Spiteller, Am Lehrstuhl für Organische Chemie 1 University of Bayreuth, Bayreuth

Consumption of fish is protective against atherosclerosis. Fish tissue distinguishes from mammalian tissue by a high content of n-3 polyunsaturated fatty acids (PUFAs). As a consequence it was concluded that consumption of n-3 PUFAs protects against atherosclerosis. Fish fats contain besides n-3 PUFAs also about 1 % of furan fatty acids (F-acids) which scavenge peroxyl radicals algae and plants. Fish or vegetable derived F-acids are incorporated in human blood plasma and serve there probably to remove peroxy radicals which oxidise low density lipoprotein and thus induce atherosclerosis. On the other hand fish tissue is poor in linoleic acid. Therefore linoleic acid in blood plasma of people living on a fish diet is reduced for 2/3. This is only partly compensated by incorporation of n-3 PUFAs. As aconsequence the total amount of PUFAs is reduced for 1/3. Because n-3 and n-6 PUFAs are equally easy subjected to peroxidation reduction of the total level of PUFAs should diminish the risk of atherogenesis.In conclusion the statement that consumption of n-3 PUFAs protect against atherosclerosis deserve a reinvestigation, especially considering that consumption of plant fats rich in n-3 PUFAs has been found to have not the same effect as fish consumption.

3. Bone Health

(3-1) IS DOCOSAHEXAENOIC ACID MORE EFFECTIVE THAN EICOSAPENTAENOIC ACID ININCREASING CALCIUM BIOAVAILABILITY?
Marlena C Kruger, *Linda M Schollum. Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, Fonterra Research Centre, Palmerston North, New Zealand

Experimental animal and human studies have indicated that essential fatty acids (EFAs) enhance calcium absorption, educe urinary calcium excretion, and increase bone calcium content.In the present study, the effect of differing ratios of EFAs on calcium bioavailability was investigated. The EFAs were rovided by evening primrose oil, fish (18:12 eicosapentaenoic acid: docosahexaenoic acid) and tuna (6:26eicosapentaenoic acid:docosahexaenoic acid) oils. Male weanling rats were fed a semi-synthetic diet containing 5% /w fat for 6 weeks. The fish oil and tuna oil diets contained 4% of test oil and 1% corn oil to prevent n-6 deficiency.After 6 weeks calcium balance, bone mineral density (ex vivo), bone calcium content, bone breaking strength and rinary excretion of type 1 collagen degradation products (RatLaps) were measured.Calcium balance, ex vivo bone mineral density, and bone calcium content were significantly higher in the animals fed una oil as compared with the group fed corn oil (p<0.05). Tuna oil had no effect on the excretion of collagencrosslinks (RatLaps). Fatty acid analyses of the red blood cell membranes of the animals as well as the plasma, revealed that the feeding of tuna oil significantly changed the ratio of DHA (22:6n-3) versus EPA (20:5n-3) in themembranes and plasma. Significant correlations were found between the DHA content of the red cell membranes and the plasma and bone density as well as bone calcium content. It is feasible that DHA may affect bone formation,rather than resorption, since there was no observed effect by DHA on urinary collagen cross-link excretion.This study was funded by New Zealand Milk Ltd.

4. Cell Cycle, Apoptosis and Cancer

(4-1) MODULATION OF LIPID METABOLISM AND OXIDATIVE STATUS IN HEPATIC PRENEOPLASTIC TISSUE AS A DIETARY INTERVENTION TOOL FOR HUMAN LIVER CANCER DEVELOPMENT
S. Abel1, M.C. Kew2, M. De Kock3, C.M.Smuts4, C. de Villiers5 and W.C.A. Gelderblom1,1PROMEC Unit, 2Molecular Hepatology Research Unit, 4Nutritional Intervention Research Unit and 5Diabetes Research Group/ Primate Unit, Medical Research Council, Tygerberg, South Africa. 3Department of Physiology, University of the Western Cape, South Africa

Cell growth is normally a closely regulated process involving a homeostatic balance in the regulation of cell differentiation, proliferation and apoptosis. Tumour growth is the breakdown or dysfunction of this finely controlled balance. The process of carcinogenesis is known to be associated with alterations in lipid metabolism affecting cellular function and growth. In rat hepatocyte nodules, the long-chain polyunsaturated fatty acid (LCPUFA) content was effectively modulated utilising different diets containing sunflower, soybean or fish oil with or without gammalinolenic acid (GLA). The low n6/n3 FA ratio diets significantly increased the n3 LCPUFA content and oxidative status of the nodules. Only the low n6/n3 FA ratio diet containing GLA decreased the number of GSTP+ foci/nodules indicating that a combination of specific dietary fatty acids could affect neoplastic development. Detailed lipid analyses of human hepatocellular carcinoma indicated a reduced level of n6 PUFA in PC and n3 PUFA in PC and PE. The low level of LCPUFA was associated with a low oxidative status in the tumour. Modulation of the FA profiles and oxidative status of neoplastic tissue with dietary LCPUFA could provide an important tool for altering neoplastic development in the liver.

(4-2) DIFFERENCES IN EXPRESSION BETWEEN GLIOMAS AND MENINGIOMAS OF MOLECULES INVOLVED IN TUMOUR INVASION AND PERITUMOURAL OEDEMA
Panagopoulos AT1, Aguiar PHP2, Colquhoun A3: 1Hospital Santa Casa de Misericórdia, 2Hospital das Clínicas, 3Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brasil

Two of the most common forms of primary brain tumours are the gliomas, including the highly malignant glioblastoma multiforme, and the benign meningiomas (bMNG). Both have a high rate of reincidence after surgical resection but with differing behaviour, bMNGs do not invade the brain parenchyma while gliomas are extremely invasive. The aim of the present study was to compare the protein expression of metalloprotease-2 (MMP2), metalloprotease-9 (MMP9), tenascin C (TNC), cyclooxygenase-2 (COX2) and brain fatty acid binding protein (bFABP) between glioma and intracranial meningioma samples. Tumour tissue was collected from patients undergoing surgery for the removal of gliomas or bMNGs and sections of 5mm were used for H&E staining or immunohistochemical reactions. Gliomas and bMNGs were found to express both MMP2 and MMP9. bMNGs expressed more MMP9 than MMP2 while gliomas expressed the two in similar quantities. TNC, COX2 and bFABP were present in both gliomas and bMNGs, although the degree of expression varied considerably among the subtypes of bMNGs studied and less so among the gliomas. The strong staining for COX2 seen in some bMNG’s may be related to peritumoural oedema. The role that bFABP plays in the biology of bMNG’s and gliomas remains to be determined and is of particular interest for future studies. The study was approved by the Ethical Committee of the Biomedical Sciences Institute of the University of São Paulo (420/2003). Research funded by FAPESP 02/13121-6.

(4-3) 2-HYDROXY-9-CIS-OCTADECENOIC ACID MEDIATES GROWTH INHIBITION AND CASPASE-8-DEPENDENT APOPTOSIS IN THE JURKAT T-LYMPHOBLASTIC CELL LINE
A. Gutiérrez1, J. Martínez2, J. Casas2, C. Baamonde2, V. Llado2, R. Villavicencio1, A. Galmes1, J. Besalduch1, PV. Escribá2, Hematology Department, Hospital Son Dureta1; Laboratory of Molecular and Cellular Biomedicine2, UIB, Mallorca, Spain

Several epidemiological studies have suggested an association between dietary fat and the incidence of cancer. Free fatty acids can be selectively cytotoxic to tumor cells or inhibit cell and animal models of carcinogenesis. It has been shown that upon their incorporation into the plasma membrane, several monounsaturated fatty acids like oleic acid (OA) induce a perturbation of lipid domains, altering the membrane physical/chemical properties as well as lipid-protein interactions. We studied the cytotoxic effect of 2-hydroxy-9-cis-octadecenoic acid (2-OHOA), a novel synthetic OA derivative, in the Jurkat cell line. The effects of 2-OHOA (25-75 µM) and OA (50-200 µM) on cell proliferation were studied with an automated cell counter. Apoptosis was analyzed by DNA content (flow cytometry) and caspase-3 and PARP fragmentation was assessed by western blot. We observed that 2-OHOA but not OA proliferation by 50% (IC50, 48 h) was 50 µM. In addition, 2-OHOA but not OA induced apoptosis in Jurkat cells in a time- and dose-dependent manner. Caspase 8 and 9 inhibitors were able to block 2-OHOA-mediated apoptosis by 80% and 30%, respectively. These results indicate that that 2-OHOA but not OA mediates a dose- and timedependent caspase-8-dependent apoptosis and inhibition of Jurkat cells proliferation. Theses effects of 2-OHOA are most likely due to modulation of the Jurkat cell membrane.

(4-4) 2-HYDROXY-9-CIS-OCTADECENOIC ACID INHIBITS A549 CELL LINE PROLIFERATION THROUGH G1 CELL CYCLE ARREST
Martínez J1, Gutierrez A2, Casas J1, Baamonde C1, Lladó V1, Villavicencio R2, Besalduch J2, Pablo V. Escribá1,Laboratory of Molecular and Cellular Biomedicine, Department of Biology, UIB1, Hospital Son Dureta2, Palma de Mallorca, Spain

Many studies have shown that Mediterranean diet has a protective effect against some types of cancer. The Mediteranean diet, rich in olive oil, leads to a high consumption of its major component, the natural monounsaturated fatty acid (MUFA) oleic acid (OA). In addition, MUFAs regulate the proliferation of some cell lines in vitro and in vivo. For this reason we studied the role of 2-hydroxy-9-cis-octadecenoic acid (2-OHOA), a novel synthetic OA derivative, in the regulation of the cell cycle in lung adenocacinoma (A549) cells. The effects of 2-OHOA and OA were investigated at the level of cell proliferation, DNA content and expression regulation, measured by western blot and RT-PCR. DNA content analysis demostrated that inhibition of A549 cell growth ocurrs via G1 arrest being 2-OHOA more potent inhibitor than OA. In addition, a significant reduction in populations of cells in S, G(2) and M phases was observed. 2-OHOA elicited dose- and time-dependent inhibitions of proliferation of A549 cells (IC50, 48 Hours, 100 µM) and a marked down-regulation of cell cycle-related proteins like Cyclin D3, A, B, E, Cdk2, tigthly associated to aberrant regulations in many types of cancer cells. 2-OHOA markedly reduced phosphorilation of retinoblastoma protein and decreased E2F1 expression at the transcriptional level. These results could explain in part the protective effects of Mediterranean diets against cancer and the anti-tumor action of the synthetic fatty acid, 2OHOA.

(4-5) POSITIVE ASSOCIATION BETWEEN DNA STRAND BREAKS IN PERIPHERAL BLOOD MONONUCLEAR CELLS AND POLYUNSATURATED FATTY ACIDS IN RED BLOOD CELLS
1AY Thorlaksdottir, 2Skuladottir GV, 3Tryggvadottir L, 3Stefansdottir S, 3Hafsteinsdottir H, 3Ogmundsdottir HO, 3Eyfjord JE, 1Jonsson JJ, 1Hardardottir I.1Department of Biochemistry and Molecular Biology and 2Department of Physiology, School of Medicine, University of Iceland, 3The Icelandic Cancer Society

DNA modification is believed to be an important step in carcinogenesis. Increased consumption of omega-6 polyunsaturated fatty acids (PUFA) increases several markers of DNA damage in humans. However, less is known about the effects of omega-3 PUFA on DNA damage. Endogenous DNA strand breaks and DNA damage induced ex vivo by H2O2 in peripheral blood mononuclear cells (PBMC) from healthy women (n=99) were evaluated with comet assay. Fatty acid composition of red blood cells (RBC) was analyzed by gas chromatography. Association between variables was evaluated with Pearson´s correlation coefficient and multiple regression. A positive correlation was found between endogenous DNA strand breaks in PBMC and total PUFA and omega-3 PUFA in RBC. In a multivariate analysis total PUFA still showed a significant independent contribution. Among individual PUFA there was a positive correlation between endogenous DNA strand breaks and linoleic acid (LA) and DHA. In a multivariate analysis both LA and DHA showed significant independent contributions. Statistically significant association was not seen between DNA omega-6 PUFA contribute to the positive association seen between DNA strand breaks in PBMC and PUFA in RBC from healthy women.

(4-6)REPRESSION OF CYCLIN D3 EXPRESSION AFTER TREATMENT WITH 2- HYDROXYOLEIC ACID IN HUMAN LUNG CANCER A549 CELLS
Casas J, Martinez J, Gutierrez A, Lladó V, Baamonde C and Pablo V. Escribá,Laboratory of Molecular and Cellular Biomedicine, Department of Biology, University of the Balearic Islands, Palma de Mallorca, Spain

Anthracyclines exert in part their antitumor effects through modulation of the membrane structural properties. We have designed and anti-cancer drug , 2-hydroxioleic acid (2-OHOA), that also alters the membrane structure. Many anticancer drugs trigger DNA damage and cause cell cycle arrest or cell death. In order to indentify the molecular bases of its cytostatic effect, we examined gene expression profiles in human lung cancer A549 cells before and after 2-OHOA treatments , using cDNA arrays. In this manner, we identified several up- or down-regulated genes in cells undergoing G1 arrest following 2-OHOA treatment. Among them, reduction in cyclin D3, which plays a crucial role in the G1/S transition, expression was markely reduced (decreases of 80% with respect to the control untreated cells) . The reduction in cyclin D3 expression was confirmed by real time RT-PCR (decreases of about 70%) and Western Blot (decreases of about 70% with respect to the control untreated cells) . These results suggest that the novel anti-tumor drug 2-OHOA provokes a down-regulation of cell cycle associated proteins, such as cyclin D3, leading to a cell cycle arrest in A549.

(4-7) THE ANTICARCINOGENIC EFFECT OF TRANS-11 18:1 IS DEPENDENT ON ITS CONVERSION TO CIS-9, TRANS-11 CLA BY DELTA-9 DESATURASE
Lock, A.L., Corl, B.A., Barbano, D.M., Ip, C.*, and Bauman, D.E.,Cornell University, Ithaca, USA and *Roswell Park Cancer Institute, Buffalo, USA

Dairy products are the principal source of cis-9, trans-11 conjugated linoleic acid (CLA) in human diets and this fatty acid is a potent anticarcinogen in a number of animal tumor models. We have previously shown that dietary VA caused a dose-dependent increase in the accumulation of CLA in the mammary fat pad which was accompanied by a parallel decreased risk of mammary tumorigenesis. The objective of the present study was to determine whether treatment with sterculic oil (SO), a potent inhibitor of Δ9-desaturase, would reverse the cancer-preventative effect of VA by inhibiting its conversion to cis-9, trans-11 CLA. Female Sprague-Dawley rats were injected with a single dose of carcinogen (methylnitrosourea), and fed one of 4 diets: 1) low VA, 2) low VA+SO, 3) high VA, and 4) high VA+SO for 6 wk. Total premalignant lesions were 83, 80, 43 and 68 for treatment 1, 2, 3 and 4, respectively (P<0.05). CLA concentrations (g/100g fatty acids) in the mammary fat pad were 2.13, 2.14, 4.75 and 2.98 (P<0.001), while the VA concentrations were 0.54, 0.74, 4.89 and 8.20 (P<0.001). The feeding of VA increased mammary tissue level of CLA effects of VA. We conclude that the anticarcinogenic effects of VA are mediated through its conversion to cis-9, trans- 11 CLA via Δ9-desaturase, and when this conversion is blocked by SO, the biological response to VA is reduced.

(4-8) INDUCTION OF APOPTOSIS IN PROSTATE CANCER CELLS BY A NOVEL POLYUNSATURATED FATTY ACID INVOLVES PROTEIN SYNTHESIS
Schmidt, L1, Hii, CS2, Murray, AW1, Easton, CJ3 and Ferrante, A2,1Flinders University of South Australia, 2Women's & Children's Hospital, Adelaide, South Australia, 3Australian National University, Canberra

As prostate cancer has been linked to dietary fatty acid intake, our group hypothesised that fatty acids may form the basis for potential therapeutic agents to treat prostate cancer. A range of novel fatty acids were synthesised with an oxa group inserted at the β position of the fatty acid. One of these, β-oxa 23:4n-6, was shown to kill DU145 prostate cancer cells in a dose- and time-dependent manner. The death showed apoptotic characteristics including PARP cleavage and DNA laddering. Death was partially inhibited by the protein synthesis inhibitor cycloheximide and the translation inhibitor actinomycin D. This implied that de-novo protein synthesis was involved in β-oxa 23:4n-6 induced death. We then studied the effect of β-oxa 23:4n-6 on the transcription factor NFκB. DU145 cells treated with β-oxa 23:4n-6 showed increased levels of NFκB in the nucleus compared to controls and translocation of NFκB preceded cell death. Therefore, protein synthesis is involved in β-oxa 23:4n-6 induced death in DU145 prostate cancer cells.

5. Eicosanoids

(5-1) EICOSANOID SYNTHESIS BY ALVEOLAR MACROPHAGES OF PROGENY ISSTIMULATED BY MATERNAL DIETARY VITAMIN E LEVEL
Charlotte Lauridsen & Søren Krogh Jensen, Danish Institute of Agricultural Sciences, Research Centre Foulum, Tjele, Denmark

The influence of vitamin E (all-rac-?-tocopheryl acetate) level and fat sources in sow diets was investigated on the eicosanoid synthesis, vitamin E concentration and fatty acid profile in alveolar macrophages of the progeny. One weekprior to parturition sows were randomly allotted to one of four diets, which were added 8 % of either fish oil or sunflower oil, with or without addition of 500 ppm vitamin E. Milk samples were obtained from the sows throughout the sucklingperiod. After the end of the suckling period (at 28 days of age) and until day 49 of age, piglets were fed the same grower diet. At day 28, 35, and 49 of age piglets were killed and alveolar macrophages were harvested. Concentrationof vitamin E and fatty acids in milk and immune cells, and synthesis of eicosanoids (prostaglandin E2, leukotriene B4, and tromboxane B2) by immune cells, was determined. The concentration of fatty acids and vitamin E in the milk, andin the immune cells of the progeny reflected the dietary treatments of the sows. The synthesis of all eicosanoids was enhanced with the maternal addition of vitamin E, but was not statistically influenced by the dietary fat or the age of thepiglets.

6. Fatty Acid Synthesis and Metabolism

(6-1) HALF-LIVES OF DOCOSAHEXAENOIC ACID IN RAT BRAIN PHOSPHOLIPIDS ARE PROLONGED BY 15 WEEKS OF DEPRIVATION OF N-3 POLYUNSATURATED FATTY ACIDS
James C. DeMar, Kiazong Ma, Jane Bell, and Stanley I. Rapoport. Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, Bethesda, MD , USA

Docosahexaenoic acid (DHA), an n-3 PUFA, is obtained from the diet or synthesized in vivo from a-linolenic acid. Depletion of brain DHA by dietary n-3 PUFA deprivation can lead to abnormal neurological development. We determined if the loss half-life of DHA is prolonged in rats deprived of n-3 PUFA for 15 weeks. Methods: Male rat pups (21-day old) were fed a diet deficient in n-3 PUFA or an n-3 PUFA adequate diet containing alinolenic acid. After 15 weeks, [3H]-DHA was injected into the lateral cerebral ventricle, and rats were killed at fixed times over a period of 60 days. Total lipids in brain and plasma were extracted. TLC was used to isolate brain phospholipid classes. HPLC, scintillation counting, and GC were used to determine DHA radioactivity and concentrations. Half-lives of [3H]-DHA in brain phospholipids were calculated from slopes of plots of log 10 DHA radioactivity vs. time post-injection. Results: Compared with the adequate diet, 15 weeks of n-3 PUFA deprivation reduced plasma DHA by 89% and brain DHA by 37%; the DHA concentrations were stable thereafter. In n-3 PUFA adequate rats, [3H]-DHA half-lives equalled 33 days in brain total phospholipids and 23, 32, 24, and 58 days in PC, PE, PI, and PS classes, respectively. In n-3 PUFA deprived rats, these half-lives were prolonged 2-fold or more. Conclusions: Mechanisms exist in the adult rat brain to minimize DHA metabolic loss during reduced n-3 PUFA availability for only 15 weeks.

(6-2) ACTIVATION OF THE BODY WALL MUSCLE OF THE C. ELEGANS FAT-3 MUTANT BY DHA
S. Luke Hillyard1, Raymond C. Valentine2, J. Bruce German1,1Food Science and Technology Department, University of California, Davis, USA 2Professor Emeritus, University of California, Davis, USA

Research on the role of DHA/EPA in muscle action is the most advanced in the nematode C. elegans of all model organisms. Pivotal experiments on the role of EPA in C. elegans have recently been published by Watts et al (Genetics, 163, 581-589 (February 2003) using body bends per minute measured in a buffer solution as an assay for body wall muscle (BWM) activation. They reported that maximum muscle activity of the fat-3 mutant (which is lacking a functional delta 6 desaturase and therefore EPA) is restored by EPA to wild type levels. We have confirmed this finding and found that DHA levels of 200 µg of DHA spread on the surface of 100 mm plates restores BWM activity to wild type levels from approximately 86 body bends/minute for fat-3 mutants without DHA compared to 135 body bends/ minute for fat-3 mutants with DHA. Both the free fatty acid as well as the triglyceride forms of DHA were found to be active. The restoration to wild type muscle activity is dose and time dependent over a range of 40 µg to 800 µg per 100 mm plate, with a time period of 16-18 hours is required for activation. A linear response in growth rate was also effective in rescuing the phenotype of the fat-3 mutant.

(6-3) BIOHYDRATION AND BIOHYDROGENATION OF LONG-CHAIN POLYUNSATURATED FATTY ACIDS BY A BACTERIUM FROM THE OVINE RUMEN
K.N. Joblin and H. Hussein, Rumen Biotechnology, Grasslands Research Centre, AgResearch, Palmerston North, New Zealand

A key step in the conversion of dietary polyunsaturated fatty acids (PUFAs) into fats found in meat and dairy products is their transformation by microbes in the rumen. However, these rumen processes are poorly understood. During a study on ruminal biohydrogenation, we isolated a bacterium which biohydrogenated linoleic acid and linolenic acid to trans-vaccenic acid. Further investigation showed that linoleic, linolenic and oleic acids, but not trans-vaccenic acid, were converted by biohydration into hydroxy-acids. The products, 10-hydroxy-12-octadecenoic acid and 13-hydroxy-9- octadecenoic acid, appeared in cultures simultaneously with the trans-vaccenic acid. Linoleic acid and linolenic acid had an inhibitory effect on bacterial growth and biohydrogenation and biohydration occurred after logarithmic growth ceased. The isolate was a Gram-positive coccus which utilised a range of carbohydrates. The 16S rRNA gene was isolated and sequenced. Alignment against database sequences showed that the isolate is closely related (99.7%) to the type strain of Enterococcus avium. To the best of our knowledge, this is the first report of biohydrogenation by an Enterococcus sp. The properties of E. avium OV77 were different to those of Fusocillus babrahamensis, the only previous ruminal bacterium with the capacity to both biohydrate and biohydrogenate PUFAs and which has been lost from culture collections.

(6-4) DETERMINATION OF THE KINETICS OF N-6 FATTY ACID METABOLISM IN TERM INFANTS
Robert J. Pawlosky2,Yuhong Lin2, Adolfo R Llanos1,3,Patricia Mena1, Ricardo Uauy 1 Norman Salem Jr.2,1Clinical Nutrition, INTA Universidad de Chile, Santiago, Chile, 2LMBB, NIAAA, Rockville, MD, USA and 3NEORED Neonatal Network, Santiago, Chile

The objective of this study was to determine the quantitative contributions of linoleic acid, 18:2n6, toward the synthesis of desaturated and elongated n-6 fatty acids in the plasma of ten full term infants (mean weight: 3130 g; range: 2450- 4150 g) at birth. Infants were given an oral dose of 100 mg/kg 13C-18:2n-6 ethyl ester on day 2 of life and fed with expressed breast milk that had been collected and/or a commercial infant formula that did not include long-chain polyunsaturated fatty acids. The plasma was sampled at 0, 4, 8, 24, 48, 96, and 168 h after dosing and the concentrations of unlabeled and labeled fatty acids were determined by capillary GC and GC-MS. A multicompartment model was used to fit the plasma concentration-time curves for 13C-18:2n-6, 13C-18:3n6, 13C-20:2n6, 13C- 20:3n6 and 13C-20:4n6 (arachidonic acid) for determination of the in vivo rate constant coefficients. Based on an analysis of the ratios of these coefficients for the ten infants, it was observed that approximately 4.4% of the labeled fatty acid that exited the 18:2n6 compartment was desaturated to yield 18:3n6 (mean synthetic rate: 25.5 ug/hr) and also about 1.5% of the label exiting the 18:2n6 compartment appeared within the 20:3n6 compartment (mean synthetic rate: 9.1 ug/hr). A much higher percentage of the label (47%) that exited the plasma 20:3n6 compartment was further desaturated to form 20:4n6 (mean synthetic rate: 35.5 ug/hr). The dietary n-6 fatty acid intake data was incorporated into the model and the daily synthetic and utilization rates for the n-6 fatty acids were determined. In this group of infants, the mean 18:2n6 plasma concentration was 115.4 +/- 15 ug/mL. The plasma 18:2n6 concentrations for each of the subjects appeared to approach equilibrium with an estimated daily intake of 2.3 +/- 0.8 g/day and utilization of 2.09 g/day.

(6-5) DEVELOPMENTAL CHANGES IN DHA SYNTHESIS IN RAINDOW TROUT
Michael V. Bell and James R. Dick,Institute of Aquaculture, University of Stirling, Scotland, UK

Freshwater fish can synthesise 22:6n-3 from dietary 18:3n-3 but the pathway is repressed in the presence of dietary C20 and C22 polyunsaturated fatty acids. The rate of synthesis of 22:6n-3 from 18:3n-3 was measured in rainbow trout at the whole animal level from first feeding to circa 8 g weight using a deuterated tracer. In fully induced first-feeding rainbow trout of ca. 0.2 g weight the rate of synthesis was 5.4 µg D5-22:6n-3/g fish/mg D5-18:3n-3 eaten/7 days. Synthetic capacity rose rapidly to a peak of ca. 50 µg D5-22:6n-3/g fish/mg D5-18:3n-3 eaten/7 days at around 1 g weight, and then declined almost exponentially to 3.5 µg D5-22:6n-3/g fish/mg D5-18:3n-3 eaten/7 days in 8 g fish. Quantitative analysis showed that the fish were unable to maintain their body concentration of 22:6n-3 during this period of growth even though the pathway was fully induced.

(6-6) LA AND ALA MAY INHIBIT DHA INCORPORATION INTO PLASMA PHOSPHOLIPIDS
Robert A Gibson,Child Health Research Institute, Flinders Medical Centre, Flinders University and the University of Adelaide, Adelaide, Australia

We examined the effect of altering the linoleic acid (LA, 18:2n-6) to alpha-linolenic acid (ALA, 18:3n-3) ratio in the dietary fats of 3 day old piglets fed formula for 3 weeks. The LA:ALA ratios of the experimental formulas were 0.5:1, 1:1, 2:1, 4:1 and 10:1. The level of LA was held constant at 13% of total fats while the level of ALA varied from 1.3% (10:1 group) to 26.8% (0.5:1 group). Incorporation of the n-3 long chain PUFA EPA and 22:5n-3 into erythrocytes, plasma, liver and brain tissues was linearly related to dietary ALA. Conversely, incorporation of DHA into all tissues was related to dietary ALA in a curvilinear manner with the maximum incorporation of DHA appearing to be between the LA:ALA ratios of 4:1 and 2:1. Feeding LA:ALA ratios of 10:1 and 0.5:1 resulted in lower and similar levels of DHA in tissues despite the very different levels of dietary ALA (1.3% vs 26.8% of total fats respectively). Plasma DHA levels were inversely correlated with plasma LA (r2=0.64) and plasma ALA (r2=0.54) indicating that high PUFA intakes may inhibit incorporation of DHA. This may explain the apparent curvilinear effect of dietary ALA on synthesis of DHA. Past stable isotope experiments may not taken this into account.

(6-7) GROUP TYPE SEPARATION OF NATURAL FATS AND OILS BY COMPREHENSIVE TWO DIMENSIONAL GAS CHROMATOGRAPHY (GC X GC)
Luigi Mondello and Giovanni Dugo, Dipartimento Farmaco- Chimico, Facoltà di Farmacia, Università di Messina, Italy

Comprehensive gas chromatography (GC x GC) is an adequate methodology for the separation and identification of entire complex samples. It is based on the coupling of two capillary columns that each give a different but substantial contribution to the unprecedented resolving power of this technique. The 2D space chromatograms that derive from GC x GC analysis have a great potentiality for identification. This is due to the fact that the contour plot positions in the 2D plane, pinpointed by two retention time coordinates, give characteristic patterns for specific families of compounds that can be mathematically translated. This investigation concerned the application of this principle to FAMEs that were grouped on an equal double bond number basis. The ester samples were derived from various lipids and all underwent bidimensional analysis on two sets of columns. Peak attribution was supported by MS spectra, LRI and information reported in the literature.

(6-8) COMPARISON OF PLASMA DYNAMICS AND TISSUE INCORPORATION OF 14C-DHA AND 3H OLEIC ACID IN MICE
Alla Polozova and Norman Salem, Jr.,Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, USA

It is known that certain tissues such as brain, retinas, testes, and heart, specifically incorporate and retain essential omega-3 fatty acids, and in particular docosahexaenoic acid (DHA). The mechanisms responsible for selective uptake of DHA by these organs are unknown. We conducted a study aimed at the direct comparison of plasma dynamics and uptake by the organs of DHA and non-essential oleic acid. 14C-DHA and 3H-oleic acid were complexed to albumin and co-injected via the tail vein into 8-12 weeks old C57Bl/6J mice. Blood, brain, liver, heart, muscle, brown and white adipose tissues were harvested at 0.5, 1.5, 3, 5, 19 and 24 hours after injection. The distribution of radioactivity in plasma fractions was analyzed by HPLC coupled to a flow radioisotope detector. Total activity levels of both isotopes in blood, plasma and all collected tissues were determined. We found that at 30 min after injection, more than 40% of the 14C-DHA, but less than 20% of the injected 3H-oleic acid activities were associated with liver. Heart uptake of 14CDHA was 3-4 times higher compared to 3H-oleic acid label. Brain incorporation of 14C-DHA slowly rose from 0.5% at 30 minutes to 1% at 24 hours, but it remained at the 1.5% level for 3H oleic acid at all time points. Uptake of both labels by the other tissues was not significantly different. Total 14C activity in plasma was less than 1% of injected dose after 30 minutes, and later it leveled off at 0.5%. Less than 30% of this activity was associated with non-esterified fatty acids. A major portion of the 14C-DHA activity was found in VLDL and LDL fractions at 0.5 and 1.5 hours after injection, and later it redistributed into the HDL fraction. In contrast to that, only 10% of 3H activity originally associated with oleic acid was found in the VLDL fraction at 30 minutes after injection. All 3H activity in plasma at later time points was in fractions of low molecular weight metabolites, which are likely to contribute to the 3H activity, found in brain. No 14C activity was detected in the low molecular weight fractions. We believe that these finding demonstrate that liver plays an important role in the initial selectivity and targeting of DHA to other organs. It is likely that DHA is specifically taken up by liver, esterified, loaded into lipoproteins, and then delivered to brain, heart and other target tissues.

(6-9) LCPUFA LEVELS IN LA- AND ALA- SUPPLEMENTED HEPG2 CELLS ARE INDEPENDENT OF Δ6 DESATURASE MRNA ABUNDANCE
R Portolesi1, BC Powell2 RA Gibson1, 2, 1Department of Paediatrics and Child Health, Flinders Medical Centre, Flinders University, South Australia, 2Child Health Research Institute, Women’s and Children’s Hospital, North Adelaide, South Australia

Δ6 desaturase (D6D) is the rate-limiting enzyme in the conversion of linoleic acid (LA) and αlinolenic acid (ALA) to long chain polyunsaturated fatty acids (LCPUFAs). The HepG2 cell line was used to examine the effect of LA, ALA, arachidonic acid (AA) and eicosapentaenoic acid (EPA) on D6D mRNA and fatty acid levels in membrane phospholipids. Individual fatty acids, bound to BSA, were added to serum free media. After 48h, cells were analysed for fatty acids and D6D mRNA expression. The incorporation of LA and ALA into cell phospholipids was concentrationdependent, as was the incorporation of endogenously synthesised AA and EPA. After supplementation with 20µg/ml LA, the level of AA in cell phospholipids increased from 2.9±0.29% to 10.6±1.7%. Following ALA supplementation, the level of membrane EPA increased from 0.15±0.04% to 7.1±1.5% and that of DHA increased from 2.6±0.13% to 4.4±0.5%, total fatty acids. Oleic acid, LA, ALA, AA and EPA suppressed D6D mRNA abundance (39±6.6%, 40±2.2%, 31±5.2%, 55±4.8%, 52±5.0%, respectively) relative to control cells in serum-free media. Although the relative abundance of D6D mRNA was significantly reduced in cells supplemented with LA and ALA, the level of conversion products (AA, EPA and DHA) were elevated above those observed in control cells. Our results suggest that a 50% decrease in abundance of D6D mRNA does not influence the conversion or incorporation of LCPUFAs into cell phospholipids in HepG2 cells.

(6-10) GROWTH HORMONE (GH) STATUS IN RATS AFFECTS THE FATTY ACID (FA) PROFILES OF PLASMA AND TISSUE PHOSPHOLIPIDS (PL)
BG Gabrielsson1, V Palsdottir2, DF Carmignac3, T Elfverson2, ICAF Robinson3, B Strandvik2. Depts of Internal Medicine1 and Paediatrics2, Sahlgrenska Academy, Gothenburg, Sweden, and the National Institute for Medical Research3, London, UK

We have previously shown that the composition of essential FA in the maternal diet affects endocrine parameters in the pups, as leptin, IGF1 and growth. However, little is known whether endocrine factors could influence the FA composition of tissue PL. We therefore investigated FA composition of PL in plasma, liver, white adipose tissue (WAT) and skeletal muscle (SkM) in GH-deficient dw/dw rats compared with normal AS rats, all on the same diet. Blood, liver, WAT and SkM were collected from normal and dwarf female rats (12 weeks, n=6/ group). After extraction of total lipids, the lipid metabolomics were analysed with HPLC and PL FA were analysed by capillary GLC. The PL FA profiles in all three tissues and plasma differed; the ratio of total n-6/n-3 FA was lowest in SkM (2.5) followed by liver (4.0), plasma (7.9) and WAT (17.0). The dwarves consistently showed higher ratios (p<0.05) compared with controls in all tissues. The highest activities of delta9-desaturases (18:1w9/18:0) and (24:1w9/24:0) were detected in WAT and SkM, respectively. Dwarf rats had lower delta9-(24:1w9)-activity in SkM (p<0.05) while there was no difference of delta9-(18:1w9)-activity in WAT. The delta6-desaturase activity was highest in the liver and lower in dwarf rats (p<0.05), whereas the delta5-desaturase activity was similar in liver and SkM, and higher in the dwarves (p<0.05). Distinct differences were found in FA pattern of tissues, indicating lower delta9- and delta6-desaturase and higher delta5-desaturase activities in the dwarves suggesting enhanced insulin sensitivity.

(6-11)POSITRON EMISSION TOMOGRAPHY IMAGING OF INCORPORATION OF DOCOSAHEXAENOIC ACID FROM PLASMA INTO BRAIN AND HEART IN HUMANS
John C. Umhau1, Giuseppe Esposito2, Stanley Rapoport2, William Eckelman3, Peter Herscovitch3, Richard E. Carson3, Joseph Hibbeln1, Michael Channing3, B.K. Vuong3, Norman Salem, Jr1. 1NIAAA, NIH; 2NIA, NIH; 3PET Dept., NIH Clinical Center, Bethesda, MD, USA

Docosahexaenoic acid (DHA) is selectively concentrated in brain and is also important for cardiac function. Here we report the first images of uptake of DHA into the human brain and heart. Based on an established model of DHA incorporation and turnover, positron emission tomography (PET) with [1-11C] labelled DHA was used to evaluate incorporation of DHA. In addition, regional cerebral blood flow was measured using [15O]water, allowing us to relate regional neuronal activity to DHA incorporation. In healthy adults, we injected 30 mCi of [1-11C]DHA intravenously followed by dynamic PET scans and arterial radioactivity measurements. We visualized robust uptake into the heart and liver and determined DHA incorporation into brain and heart normalized to integrated plasma radioactivity due to DHA. These techniques may permit us to estimate an index of daily brain incorporation of DHA in different dietary states and thereby refine our understanding of the daily dietary requirement for DHA. The PET measurements can be repeated in the same subject to explore differences in DHA uptake and turnover subsequent to dietary changes such as fish oil supplementation or alcohol abstinence. This methodology may also be use to explore functional aspects of DHA in the brain such as stimulated turnover due to neuronal activation.

(6-12) OXIDATION OF U-13C-ALPHA-LINOLENIC ACID IS NOT CHANGED BY THE AMOUNTS OR RATIO OF ALPHA-LINOLENIC AND LINOLEIC ACID IN THE DIET
Petra L.L. Goyens and Ronald P. Mensink, Maastricht University, Department of Human Biology, Maastricht, The Netherlands

Background: Conversion of alpha-linolenic acid (ALA, C18:3n-3) is influenced by the ALA and linoleic acid (LA, C18:3n-3) content of the diet. It is not known however if beta-oxidation of ALA is also influenced by dietary ALA and LA. Methods: Twenty-nine subjects consumed a control diet for four weeks (7 En% LA, 0.4 En% ALA, ALA-to-LA ratio of 1:19). For the next six weeks, nine subjects continued on the control diet, ten subjects consumed a low-LA diet (3 En% LA, 0.4 En% ALA) and ten subject followed a high-ALA diet (7 En% LA, 1.1 En% ALA). The low-LA and high-ALA diet had an identical ALA-to-LA ratio of 1:7. Ten days prior to the end of the run-in and intervention period, 30 mg U-13CALA was administered orally. Breath was sampled and CO2-production measured at baseline and during regular intervals up to 9 hours after tracer intake. Results: During the run-in period, the total percentage of tracer dose recovered equaled 19.2 ± 2.4% (mean ± SD) in the control group, 17.8 ± 5.4% in the low-LA group and 20.7 ± 2.1% in the high-ALA group. Tracer recovery following intervention decreased slightly but insignificantly with -3.5 ± 2.6%, -2.5 ± 3.9% and -3.3 ± 3.3% on respectively the control, low-LA and high-ALA diet. Conclusion: Short-term oxidation of ALA is not influenced by a change in the absolute amounts of ALA and LA nor by a change in the ALA to LA ratio of the diet.

(6-13) Δ9-DESATURASE EXPRESSION REDUCES D6-DESATURASE ACTIVITY
Guillou H, D’Andrea S, Rioux V, Barnouin R, Pedrono F, Bouriel M, Catheline D, Legrand P,Laboratoire de Biochimie ENSAR-INRA, Agrocampus, Rennes cedex, FRANCE

In animals, Δ6-desaturase catalyses the rate-limiting step for long-chain polyunsaturated fatty acid (PUFA) biosynthesis of n-6 and n-3 series. Unlike Stearoyl-CoA Desaturase 1 (SCD1), the main hepatic Δ9-desaturase, ratΔ6-desaturase has never been purified and its structure remains poorly characterised. SCD1 interacts with cytochrome b5 and uses NADH cytochrome b5 reductase as primary electron donor. Through an immunoprecipitation study, performed with recombinant proteins, we showed that Δ6-desaturase, like SCD1, binds the cytochrome b5 and the NADH cytochrome b5 reductase in vitro. We questioned whether both desaturases may use and compete for the same electron transport proteins in the endoplasmic reticulum. Then, we investigated whether SCD1 co-expression interferes with Δ6-desaturase activity in cultured cells. We showed that an increase in SCD1 expression reduces the Δ6-desaturase activity in COS-7 cells. A dose-dependent effect of SCD1 expression reaching a 50% inhibition was obtained for Δ6-desaturation of both linoleic acid (C18:2n-6) and alinolenic acid (C18:3n-3). These data support a competition between Δ9-desaturase and Δ6-desaturase activities. This work provides a biochemical evidence for an indirect mechanism modulating Δ6-desaturase activity. It may contribute to elucidate how 6-desaturase activity decreases in vivo when its expression remains unchanged.

(6-14) FATTY ACID ALTERATIONS WITHIN PHOSPHOLIPID SPECIES AND NEUTRAL LIPIDS OF THE PANCREAS IN CFTR-/- KNOCKOUT MICE. EFFECT OF DHA TREATMENT
Mario Ollero1,2, Steven D. Freedman1, Munir M. Zaman1, Paola G Blanco1, Charlotte Andersson1, Yana Urman1, Michael Laposata3,1 Beth Israel Deaconess Medical Center – Harvard Med S, Boston, MA, USA 2 Faculté de Médecine Necker, INSERM U467, Paris, France, 3 Massachusetts General Hospital - Harvard Med School, Boston, MA, USA

For several decades, an association has been made between alterations in fatty acid metabolism and the presence of cystic fibrosis (CF). In general, reported observations include decreased 18:2 n-6 and 22:6 n-3, and increased 20:4 n- 6. We hypothesized that the fatty acid defects found in cystic fibrosis are specific for a particular lipid compartment within the cell, and are the result of a relevant pathogenic mechanism. We attempted to analyze specific lipid compartments for the fatty acid changes found previously at the organ/tissue level, using pancreatic homogenates from cftr-/- mice. Fatty acid methyl esters were analyzed in total extracts, lipid classes obtained by aminopropyl column chromatography, and in phospholipid classes separated by HPLC. We observed that: 1) 18:2 n-6 decreases were present in phospholipids but not in neutral lipids; 2) there is an increase in the 20:3 n-6 metabolite of 18:2 n-6 more consistently than an increase in 20:4 n-6; 3) there was a consistent increase in all phospholipids and in neutral lipids in 22:5 n-6, the terminal fatty acid in the n-6 pathway; and 4) that the 22:5 n-6/22:6 n-3 ratio was significantly elevated in all phospholipid species analyzed in cftr-/- mice. These fatty acid defects were reversed by oral treatment of mice with 40 mg/day of 22:6 n-3 (DHA). The observed metabolic alterations suggest that enhanced flux through the n-6 pathway from 18:2 n-6 to 22:5 n-6, the terminal fatty acid in the n-6 pathway, results in competition in the n-3 pathway at the level of 22:5 n-3 and thereby reduces the synthesis of 22:6 n-3.

(6-15) DIET AND GENDER INTERACTIONS IN THE METABOLISM OF N-3 FATTY ACIDS IN HUMAN SUBJECTS
Robert J. Pawlosky, Joseph R. Hibbeln, Yuhong Lin and Norman Salem Jr. LMBB, NIAAA, NIH, Rockville, MD, USA

A compartmental model was used to determine the coefficients of the in vivo kinetic rate constants from the concentration-time curves of d5-18:3n3, d5-20:5n3, d5-22:5n3, and d5-22:6n3 in the plasma of healthy women (n=5) and men (n=5) subsisting on three different diets (self-select, beef-based, fish-based). For 3 wks, subjects reported their food intake from their normal diets and then were placed on a beef-based diet followed by a fish-based diet for a period of 3 wks each. At the beginning of the 3rd, 6th and 9th wks subjects consumed 1 g of d5-18:3n-3 ethyl ester, blood was drawn over 168 h and the plasma was analyzed for both the labeled and endogenous fatty acids. The coefficients of the kinetic constants of n-3 fatty acid metabolism and the percent utilization of the substrates were determined from an analysis of the isotopic data. Results. Across all diets, less than 0.5% of the plasma d5-18:3n-3 was utilized for synthesis of long-chain PUFA in both men and women. Gender exerted a profound influence on the magnitude of a rate constant coefficient involved in the biosynthesis of 22:6n-3 from 22:5n-3. During the period when subjects subsisted on a beef-based diet, the rate constant coefficient for the conversion of 22:5n-3 to 22:6n-3 was much greater in women (k = 0.041 +/- 0.007) compared with men (k = 0.012 +/- 0.004) (p = .001). The larger rate constant coefficient in women led to a nearly 3-fold greater amount of 22:5n-3 utilized for 22:6n-3 synthesis compared with men. While subjects subsisted on the and this was much less for women than when they had been subsisting on the beef-diet. Neither diet nor gender appeared to exert strong influences on the kinetics of other steps of n-3 fatty acid metabolism. These processes in women may involve a system of feed-back control mechanisms responsive to the plasma concentration of 22:6n-3 which was modified during each of the two experimental dietary periods as well as hormonal influence.

(6-16) EICOSAPENTAENOIC ACID AND 3,10-DITHIA STEARIC ACID INHIBIT THE METABOLISATION OF TRANS-VACCENIC ACID BY Δ9-DESATURASE
Renaville B. (1, 2), Mignolet E. (2), Sergent T. (1), Schneider Y.-J.(1), Larondelle Y. (2) Unité de biochimie cellulaire (2) Unité de biochimie de la nutrition Institut des sciences de la vie, UCL, Louvain-la-Neuve, Belgique

Trans-vaccenic acid (C18:1 trans-11, TVA), the main trans isomer of dairy products, is suspected to have positive effects on health after partial metabolisation in rumenic acid (C18:2 cis-9, trans-11, RA), a conjugated linoleic acid The intestinal metabolisation of TVA by the D9-desaturase was investigated using Caco-2 cells as an in vitro model of the human intestinal barrier. Caco-2 cells desaturate TVA into RA, RA was detected in the cellular lipids only when cells were cultured with TVA. The Δ9-desaturase activity was modulated by eicosapentaenoic acid (EPA) and 3,10 dithia stearic acid (DSA) in acute (300 µM EPA or DSA up to 3h30) and acute after chronic treatment (30 µM EPA or DSA for 6 days followed by 300 µM EPA or DSA up to 3h30). EPA decreases Δ9-desaturase activity only after chronic treatment, suggesting that modulation occurs at the transcriptomic level. By contrast, DSA incubation decreases the ?9-desaturase activity upon acute treatment, which suggests that DSA has an effect on the activity of the enzyme. In conclusion, it appears that EPA and DSA both inhibit the Δ9-desaturase activity, but by different ways. We acknowledge Prof. Spydevold for its kindly supply of DSA. Renaville B. has a fellowship of the belgian FRIA.

(6-17) STATINS DIFFERENTIALLY MODULATE THE Δ9, Δ6 AND Δ5 FATTY ACID DESATURASES IN MONOCYTIC AND HEPATOMA CELL LINES
Risé P., Ghezzi S., Priori I. and Galli C.,University of Milan, Department of Pharmacological Sciences, Milan, Italy

Statins, widely used drugs in the treatment of hypercholesterolemia, also potently increase the production of long chain polyunsaturated fatty acid (PUFA). They increase e.g. arachidonic acid levels in lipid pools in vitro and in vivo, by enhancing the conversion of linoleic acid, through enhanced activities of the Δ5 and to a lesser extent of the Δ6 desaturase (refs). Aims of our study were: 1) to evaluate the effect of statins on the Δ9 desaturase in cells incubated with [14C] stearic acid; 2) to compare their effects on the conversion of [14C] α-linoleic of the n-6 and of [14C] α-linolenic acid of the n-3 FA, to their long chain derivatives using, in THP-1 and in HepG2 cells. Results. Simvastatin (5µM ) did not affect Δ9 desaturase activity evaluated as conversion of [14C] stearic acid to oleic acid, by THP-1 cells, with no difference in the 18:1/18:0 ratios (1.095 vs 0.945, p=0.089) in control vs simvastatin treated cells. As for the n-6 series (?), THP-1 cells, which actively convert (about 70%) the precursor [14C] α-linolenic acid to its products; after treatment with simvastatin also in this case Δ5 and Δ6 desaturases were affected. THP-1 were more efficient than HepG2 cells to convert any type of fatty acid substrate and the effects of simvastatin were less pronounced in HepG2 cells, reflecting their different biochemical roles.

(6-18) REGULATION OF DESATURASE EXPRESSION IN HL60 CELLS
K. Thorstensen, M. Kvitland, JE Slagsvold, M. Mack, KS. Bjerve, Dept. Medical Biochemistry, St. Olavs Hospital, Trondheim, Norway

The expression of mRNA for the Δ5-, Δ6- and Δ9-desaturases in HL60 cells was determined by quantitative RT-PCR. During 72 h of culture with 10% foetal bovine serum (FBS) the expression of Δ5- and Δ6-desaturases was upregulated 6-fold, whereas the expression of the ?9-desaturase approximately doubled. Addition of fatty acids (FA) to the culture medium suppressed the upregulation of all desaturases. There was no statistically significant difference in the effect of n-3, n-6, n-9 or saturated FA on the upregulation, although the n-9 and the saturated FA appeared to have slightly less effect than the n-3 and n-6 FA. In cells cultured with limiting amounts of FBS the desaturase expression increased with decreasing concentrations of FBS. Under standard culture conditions (10% FBS) no significant depletion of specific or total FA from the medium occurred. Measurement of cellular FA revealed a 30% fall in total FA over 72 h. When the lipids were separated into neutral lipids, free fatty acids and phospholipids some differences became apparent. The FA content of neutral lipids decreased by 80% from 625 to 130 pmol FA/106 cells, whereas the FA content of the phospholipid fraction decreased by 20% during 72 h of culture. In both fractions the largest decrease was seen in the n-3 and n-6 FA. In fact, the n-3 and n-6 FA practically disappeared from the neutral lipid fraction, whereas the content of these FA in the phospholipid fraction decreased by approximately 40%. These results indicate that when the cells are starved of fatty acids the content of n-3 and n-6 FA decreases leading to an upregulation of the desaturases, particularly the Δ5- and Δ6-desaturases.

(6-19) THIOLACTOMYCIN AND DERIVATIVES AS POTENTIAL THERAPEUTICS FOR MALARIA
Urch, J.E.a, Jones, S.M.b, Gilbert, I.H.b, Berry, C.a and Harwood, J.La.,a Cardiff School of Biosciences, Cardiff University, Wales, UK b Welsh School of Pharmacy, Cardiff University, Wales, UK

Malaria is a parasitic disease that kills over 1 million people every year. The majority of these deaths are caused by the organism, Plasmodium falciparum. Resistance to current anti-malarials is increasing and new drugs are essential to combat this disease. Plasmodium contains a vestigial chloroplast called the apicoplast, thought to be the location of fatty acid synthesis. P. falciparum contains a Type II fatty acid synthase (FAS) consisting of dissociable enzymes common to bacteria and plants. This differs from the multifunctional protein of Type I FAS within the human host, and represents a new target for the development of anti-malarial agents. Thiolactomycin is a specific inhibitor of β-ketoacyl-ACP synthase (KAS) enzymes that catalyse the condensation steps of Type II FAS. We have isolated two KAS enzymes from P. falciparum by recombinant expression of their genes in E. coli. The KAS III enzyme, which catalyses the initial condensation between acetyl-CoA and malonyl-ACP, has been studied in some detail. We have synthesised novel derivatives of thiolactomycin to assay against the parasite. The inhibitory activity of these derivatives was assessed in vitro against purified KAS III and compared with their activity against P. falciparum cultures. The results will be discussed in terms of their structure/activity relationships and our knowledge of the active site of KAS III proteins.

(6-20) VITAMIN A DEFICIENCY ENHANCES LIVER DOCOSAHEXAEXANOIC AND DOCOSAPENTAENOIC ACIDS IN RATS FED α-LINOLENIC ACID ADEQUATE DIET
1D. Zhou, 2R Reifen 1K. Ghebremeskel, and 1M.A Crawford. Institute of Brain Chemistry and Human Nutrition, London Metropolitan,University UK; School of Nutritional Sciences, The Hebrew University of Jerusalem, Israel

Peroxisome proliferator-activated receptor (PPARα) regulates the expression of Δ-5 and –6 desaturases and acyl-CoA oxidase, enzymes vital for the synthesis of docosahexaenoic (DHA) and docosapentaenoic (22:5?6, DPA) acids. The activation of PPAR requires the retinoid X receptor (RXR) 9-cis retinoic acid. We tested the hypothesis that vitamin A deficiency impairs the synthesis of DHA and DPA. Male WISTAR/HsdBrlHan rats (n=24) were randomly fed a 9% cotton or soybean oil diet with (VAS) or without (VAD) vitamin A. The VAD soybean diet fed rats had higher levels of DHA and DPA (P<0.005) in liver choline (CPG) and ethanolamine (EPG) phosphoglycerides compared to the VAS soybean diet group. In contrast, the VAD cotton group had higher DPA (P<0.0001) and lower DHA (P<0.0001) than the corresponding rats fed VAS cottonseed diet in liver CPG and EPG. The data indicate that the synthesis of DHA is reduced and DPA increased by VAD in rats fed ω3 fatty acid deficient diet. The enhanced synthesis of DPA and DHA in the rats fed VAD soybean oil diet suggests that there may be another potent ligand for RXR that is activated when 9- cis retinoic acid, a metabolite of vitamin A, is limiting.

(6-21) ARACHIDONATE AND DOCOSAHEXAENOATE LEVELS IN HEART OF ADULT RATS ARE AGE & GENDER DEPENDENT
1D. Bitsanis, 1K. Ghebremeskel, 2IY. Khan, 2PD. Taylor, 2L. Poston and 1M.A. Crawford. 1IBCHN, London Metropolitan University;
2Maternal and Fetal Research Unit, Division of Reproduction, Endocrinology and Development, King’s College, London

Increasing age is associated with greater risk of cardiovascular disease and there is a gender difference on the age of onset. We have investigated the effect of gender and age on the heart fatty acid composition of adult rats. After weaning, male and female Sprague-Dawley pups were fed on a 5.3% fat diet for 80 or 180 days. The mean levels of arachidonate (AA) in heart choline (CPG; p<0.0001, p<0.005) and ethanolamine (EPG; p<0.005, p<0.0001) phosphoglycerides of the older male and female rats, respectively, were higher than that of the corresponding younger rats. In contrast, the levels of docosahexaenoate (DHA) in CPG (p<0.01, p<0.005) and EPG (p<0.005, p<0.0001) were lower in the older male and female rats. The females had higher DHA in CPG (p<0.001, p<0.0001) and EPG (p<0.0001, p<0.005) at 80 and 180 days than their male counterparts. These findings are consistent with previous reports, which have suggested that the synthesis of the cardio-protective DHA is specifically influenced by age and gender.

(6-22) THE CONVERSION OF DIETARY POLY-UNSATURATED FATTY ACIDS TO ITS LONG CHAIN METABOLITES
Nahed Hussein1, Paul A. Wilkinson1, Clair Leach1, G.C. Burdge2, S.A. Wootton2, Bruce A. Griffin1 and D. Joe Millward1,1Centre for Nutrition & Food Safety, School of Biomedical & Life Sciences, University of Surrey, Guildford, and 2Institute of Human Nutrition, Southampton General Hospital, Southampton, UK

Growing evidence suggests that dietary n-3 polyunsaturated fatty acids (eicosapentaenoic acid; EPA and docosahexaenoic acid ; DHA) reduce the risk of coronary heart disease (CHD) and stroke. α-LNA is the natural precursor of EPA and DHA and is an abundant and accessible source of dietary n-3 PUFA that can be further elongated and unsaturated in vivo. The overall aim of the project is to examine the conversion of α-LNA to its long chain metabolite, most importantly DHA. A human dietary intervention study, was conducted to study the influence of a low n-6 to n-3 diet compared with a standard high n-6 diet on α-LNA conversion to its long-chain metabolites. The human dietary intervention trial was conducted over a 12-week parallel study, to compare a diet enriched in α-LNA (n=21) with a high linoleic acid ‘control’ diet (n=17), in addition to a positive control group; the fish-oil diet (n=19), testing the effect of an increased intake of α-LNA, the intervention also aimed to reduce the ratio of n-6 to n-3 PUFA to 1:1 or less in order to minimize competition between α-LNA and the more abundant linoleic acid (n-6) and, in theory, increase the conversion of α-LNA to LC n-3 PUFA. Subjects participated in the current study were expressing an atherogenic lipoprotein phenotype (ALP), which may represent the most common source of lipid-mediated coronary heart disease. The results from the dietary intervention indicate that, a dietary α-LNA (flaxseed oil) can result in potentially beneficial fatty acid alterations, 3-fold increase in α-LNA (p <0.001)) and 2.5 fold increase in EPA (p<0.001) at week-12, decrease in n-6:n-3 ratio (p =0.001), unchanged level of DHA. In contrast, the fish oil diet increased both EPA and DHA. Dietary α-LNA had 7% of the efficacy of preformed EPA from fish oil to increase membrane EPA levels.

(6-23) DEPOSITION OF POLY-UNSATURATED FATTY ACIDS IN MINK TISSUES IS AFFECTED BY THEIR DIETARY INTAKE
Søren Krogh Jensen & Charlotte Lauridsen, Department of Animal Nutrition and Physiology,Research Centre Foulum, Danish Institute of Agrisultural Sciences

Mink diets are characterised by a high content of poly-unsaturated fatty acids originating from marine lipids. Mink are therefore an excellent animal for studying the effect of very high intakes of poly-unsaturated fatty acids on the deposition of individual fatty acids in the tissues, as well as the effect on different lipid metabolism products. In the present study regression analyses was performed in order to show the effect of feeding increasing levels of herring and mackerel oil on the relative deposition of individual fatty acids in mink tissues. In liver the distribution of the individual fatty acids, were highly correlated with the distribution in the diets. However, the deposition in kidney fat was characterised by an increased relative deposition of the long chain saturated and monounsaturated fatty acids, but a decreased relative deposition of the long chain poly-unsaturated fatty acids. This indicates a relative higher oxidation rate of these fatty acids. Linoleic acid made an exception as the deposition of this fatty acid was highly positively correlated to the dietary intake. Thus, the decrease in (n-6)/(n-3) ratio in kidney fat followed the decreasing intake of linoleic acid more than an increased intake on poly-unsaturated (n-3) fatty acids.

7. Cell Signalling, Transcription Factors, Gene Expression

(7-1) 2-OHOA TREATMENT MODULATES ADENYLYL CYCLASE SIGNALLING PATHWAYS IN A-549 CELLS
J. Martínez, A. Gutierrez, J. Casas, V. Lladó, R. Alemany, P.V. Escribá, C. Baamonde. Laboratory of Molecular and Cellular Biomedicine, Dept. of Biology, Univ. of the Balearic Islands, Palma de Mallorca, Spain

The synthetic monounsaturated fatty acid, 2-hydroxy-9-cis-octadecenoic acid (2OHOA) alters the membrane structure, modifies the PKC signalling pathway and the levels of the antiproliferative proteins, leading to a cell cycle arrest in A549 cells. However, the molecular bases of its anti-tumor action remain largely unknown. Here, we studied the effect of 2OHOA in a crucial signalling pathway involved in cell proliferation and differentiation, the adenylyl cyclase(AC)- cAMP-PKA cascade. AC signalling was investigated by determining cAMP formation after stimulation with NaF (10 mM), cholera toxin (15 unit.ml-1), and forskolin (100 µM) in A549 cells treated 24h with 2OHOA (100µM). cAMP accumulation upon AC stimulation was decreased by 2OHOA treatmen, indicating that the AC activity is downregulated by this fatty acid. Changes in the expression of PKA subunits and caveolin-1 were measured by western blot analyses. 2OHOA-treated A549 cells showed decreased levels of PKARIα (43.13%; t= 8.57; p<0.0001) and PKACα (35.57%; t=4.57; p< 0.001) subunits and increases of PKARIIα (93.9%; t=12.28; p<0.0001) and caveolin-1 (82%; t= certain membrane proteins. Numerous components of the cAMP-based signalling cascade have been localized to caveolae and shown to be regulated by caveolin proteins. The effect of 2OHOA on A549 cells proliferation could be related to modulation of the expression of caveolin-1 and subsequent regulation of the AC-cAMP-PKA cascade leading cell-cycle arrest.

(7-2) PHOSPHOLIPASE A1 DISTRIBUTION CHANGES DRAMATICALLY THROUGHOUT TRYPANOSOMA CRUZI LIFE CYCLE
Belaunzarán M. L. , Wainszelbaum, M., Lammel, E. M., Gimenez, G., Gentili, H., Florin-Christensen, J. and Isola, E.L.D.,Department of Microbiology, School of Medicine, University of Buenos Aires, Argentina

We here report that Phospholipase A1 specific activity in amastigotes and trypomastigotes is 15-10fold higher respectively than in epimastigotes and resides mainly on its membrane fraction. Furthermore, only these infective stages secrete the activity to the medium. In agreement with these observations, we demonstrate that, during metacyclogenesis, the secreted activity increases, reaching a peak at aproximately day 8 of the differentiation process, at which time, the proportion trypomastigotes to epimastigotes is maximal. We suggest that membrane associated and secreted Phospholipase A1, are new markers for differentiation of T. cruzi. Our findings may have important biological consequences. When [14C-1]-oleic acid labeled Vero cells are incubated for short periods (15min) with amastigotes, their lipids changes dramatically, with the appearance of free fatty acids, diacylglycerol and lysophosphatidylcholine. This indicates strong effects on membrane lipid composition in target host cells. Since diacylglycerol is increased, we investigated the possible activation of PKC in the host cells. We found that, indeed, this enzyme is clearly stimulated under the experimental conditions employed. In conclusion, the results presented here, support the view that Phospholipase A1 from the infective stages, play a key role in the interaction with the host cells, possibly implicating PKC cascade activation. Supported by UBA and CONICET.

(7-3)REGULATION OF GENE EXPRESSION BY OLEIC ACID USING SUBTRACTIVE HYBRIDIZATION STRATEGY
Lima, TM1, Henrique Bengtson, M2; de Oliveira Rodrigues, L2; Verlengia, R1, Pompéia, C1, Curi, R1 1Department of Physiology and Biophysics, Institute of Biomedical Sciences, 2 Department of Biochemistry, Chemistry Institute, University of São Paulo, Brazil

Recent studies have shown that diets rich in oleic acid have beneficial effects on several disease, such as rheumatoid arthritis and cardiovascular disorders. However the specific effects of oleic acid on the immune system have not been elucidated. In this study the effect of oleic acid on gene expression of Jurkat cells (human T lymphocyte) was examined using subtractive hybridization strategy. Cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, 20 mM Hepes, 10 U/mL ampicilin and 10 µg/mL streptomycin. Oleic acid (50µM) was added to the medium and cells were incubated for 24 hours. This concentration of oleic acid was previously tested and was not toxic for Jurkat cells. Messenger RNA was extracted from control (ethanol 0,05%) and treated cells and cDNA was then prepared. Subtractive hybridization strategy was performed as described by Rebrikov et al. (Nucl. Ac. Res. 2000). This technique allows us to identify genes with higher or lower expression after cell treatment with oleic acid as compared to control cells. Changes in gene expression were validated through northern blot analysis. Oleic acid up regulated gene expression of: translation elongation factor alpha 1, heat shock protein 60, ATP synthase 8 and down regulated: gp96 (human tumor rejection antigen gp96) and subtilisin-like protein (PACE4). The beneficial effects of diets rich in oleic acid are related to regulation on gene expression of lymphocytes. Oleic acid altered the expression of genes related to defense and repair, energy generation, protein synthesis and degradation, indicating a role of this fatty acid in keeping the performance of cell machinery. Financial support: FAPESP.

(7-4) SUB-CLONING AND BACTERIAL EXPRESSION OF HEPATOCYTE-NUCLEAR FACTOR 4α (HNF4α) IN ORDER TO IDENTIFY ACTIVATING LIGANDS
Maria Papachristodoulou1, Nick J. Plant2, Alfred E. Thumser1,1 Nutrition Group, 2 Molecular Toxicology Group, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, UK

HNF4α is a liver-enriched transcription factor that regulates a variety of different genes involved in metabolism, liver differentiation etc. Both fatty-acyl CoA thioesters (Hertz et al, 1998) and Long-chain fatty acids (Dhe-Pagamon et al, 2002) have been identified as potential endogenous ligands, but these findings are controversial. The aim of this study is therefore to identify ligands using a fluorescence displacement assay and the first objective has been to subclone the complete HNF4α cDNA into a protein expression vector. The HNF4α cDNA has been subcloned from pcDNA3 vector into pTriEx using the EcoRI restriction sites and one clone positively identified by DNA sequencing. The pTriEx-HNF4α vector was transformed into 3 different protein-expressing bacterial strains namely BL21DE3, BL21(codon plus)DE3 RP and Rosetta gami. After investigating the effect of IPTG concentration, induction time and incubation temperature on optimal soluble protein expression in all 3 different bacterial strains, best protein expression levels were obtained with BL21DE3. We have now established conditions for satisfactory protein expression and are currently purifying the HNF4α protein in order to develop a fluorescence displacement assay to compare the fatty acids and fatty acyl-coA thioesters binding characteristics of HNF4α.

(7-5) A PUFA HYPOTENSIVE DIET EFFICIENTLY REDUCES LIPID DISORDERS LINKED TO SPONTANEOUS HYPERTENSION (SHR RAT) BY SUPPRESSION OF SCD-1 GENE EXPRESSION

J. Bellenger, S. Bellenger , J.P. Poisson and M. Narce*, UPRES Lipides et Nutrition, IFR 92, Université de Bourgogne, Dijon, France

SCD-1 transcripts indicates a regulation at a transcriptional level. Therefore, such a PUFA hypotensive diet efficientlyWe previously evidenced that oleic acid (18:1n-9, OA) was accumulated in Spontaneously Hypertensive Rats (SHR) liver, concomitant to the pathogenesis of hypertension and that a dietary combination of n-6/n-3 polyunsaturated fatty acids (PUFA) composed of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and gamma-linolenic acid (GLA), (EPA/DHA/GLA) had hypotensive effects. As lipogenesis can be regulated by PUFA, the aim of this work was to evaluate the possible effects of this hypotensive dietary mixture on the 18:1n-9/18:0 ratio in SHR liver and on the expression of stearoyl CoA desaturase-1 (SCD-1), a key enzyme of OA synthesis. The results show that this regimen decreased relative amounts of monounsaturated fatty acids in plasma and hepatocyte lipid fractions, and decreased the delta-9 desaturation index. Suppression of about 50% of hepatocyte reduces lipid disorders linked to spontaneous hypertension. PUFA amounts tended to converge to those of the normotensive control WKY. This is the first time that SCD-1 has been shown to be regulated in such pathophysiological conditions by a dietary n-3/n-6 combination. However, FAS and SREBP-1 remained unchanged. If such a dietary combination is relevant to laboratory studies, how relevant these observations are to humans remains to be determined.

(7-6) LIPID THERAPY: A NEW APPROACH IN MOLECULAR MEDICINE
P. V. Escribá, R. Alemany, O. Vögler, C. Baamonde, F. Barceló, J. Prades, T. Nagy, G. Borchert, J. Martínez, S. Terés, J. Casas, A. Gutierrez, J. Besalduch and C. Egea. Mol. Cell. Biomedicine,IUNICS-Dept. of Biology, Univ. Balearic Islands, Palma de Mallorca, Spain

Currently, most drugs used for therapy in humans are targeted to proteins. In addition, the trend for drug development is mainly focused on the design of molecules that interact and control the activity of target proteins. However, cells are composed of other molecules that may also become targets for the development of new therapies. We have studied the molecular bases of structural biology applied to cell membrane lipids. From our studies we have developed two important new concepts. First, we have demonstrated that the cell membrane lipid structure regulates the localization of pivotal signaling proteins, such as G proteins and protein kinase C (PKC). In fact, the regulation of cell signaling by the membrane lipid structure is a novel mechanism whose molecular bases are currently under study. Second, if the membrane lipid structure regulation modulates cell signaling through parameters that can be rationalized through structural biology, this strategy can be used with clinical purposes. As a matter of fact, we have designed lipid molecules that interact with membranes modulating its structure and signaling protein localization and function. These molecules have proven to be effective for the treatment of important pathologies (cardiovascular, tumoral and obesity). Their oral administration and lack of side-effects add interest to their potential use in humans. Therefore, this novel potential applications.

(7-7) ROSIGLITAZONE TOGETHER WITH NOREPINEPHRINE AND DEXAMETHASONE INDUCE Elovl3 EXPRESSION IN CULTURES OF BROWN ADIPOCYTES
Johanna Jörgensen, Andreas Jakobsson and Anders Jacobsson. The Wenner-Gren Institute, Stockholm University, Sweden

Elovl3 is a member of a highly conserved gene family coding for microsomal proteins involved in the elongation of very long chain fatty acids and sphingolipids. Elovl3 mRNA is expressed in skin, liver and brown adipose tissue. The expression of Elovl3 mRNA is highly elevated in brown adipose tissue when exposing mice to cold. Earlier studies have shown that Elovl3 expression can be induced in cultures of brown adipocytes by the simultaneous addition of norepinephrine (NE) and dexamethasone (Dex). In this study we have investigated the effects of the PPAR-gamma agonist rosiglitazone on the expression of Elovl3 mRNA in cultures of brown adipocytes. We found that the levels of Elovl3 mRNA were increased when the cells were treated with rosiglitazone together with NE and Dex. Treatment with only rosiglitazone did not induce the expression of Elovl3 to the same extent, indicating a requirement of rosiglitazone, NE, and Dex for a maximal expression of the Elovl3 gene.

(7-8) EXPRESSION OF CARDIAC AND SKELETAL MUSCLE SPECIFIC GENES IN P19CL6 CARDIAC CELL LINE
Khodadadi, I., Griffin, B.A., Thumser, A. E.,Centre for Nutrition & Food Safety, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, UK

The cell line P19CL6, a derivative of P19 embryonal carcinoma cells, is used widely as an in vitro model of cardiovascular cells. This cell line has been shown to differentiate into beating cardiomyocytes on exposure to dimethyl sulphoxide (DMSO). The aim of this study was to characterise the P19CL6 cell line with respect to the expression of cardiac (αMHC and βMHC) and skeletal muscle-specific genes (MyoD and Myogenin) under different culture conditions. Cells in adherent culture conditions were plated into media with and without 1% DMSO, whereas cells in non-adherent conditions, aggregates of P19CL6, were exposed to DMSO and re-plated in tissue culture flasks. Cells were then harvested and total RNA extracted. Gene expression was measured relative to GAPDH using RTPCR followed by Nested-PCR. All ge