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Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei-infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei-infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs.

Babesia divergens was cultivated in RPMI 1640 (25 mM HEPES) supplemented with 10% human serum (RPMI-10% HS) with a high percentage of parasitized erythrocytes (PPE) ([ges ]40%). Standardization of in vitro tests, purification of exoantigens, biochemical studies and the safety of the culture handler motivated the development of a serum-free defined medium. Removal of serum greatly reduced the PPE but, after a period of adaptation, the culture was continuous and the parasite was able to develop a 3% routine PPE. Addition of vitamins or reduced glutathione in basal medium (RPMI) did not improve the PPE. The supplementation of basal medium with lipidic carrier (Albumax I or bovine serum albumin–Cohn's fraction V) promoted the growth of B. divergens with high PPE (>30%) close to those obtained in RPMI–10% HS. Neither protein nor lipid fractions alone were able to restore the growth of B. divergens. Nevertheless, the whole lipid fraction from serum or Albumax I added to delipidated albumin partially restored the growth (7% PPE), indicating that the presentation of specific lipids by a carrier is crucial for the parasite. All the data indicate that Albumax I can replace human serum offering the advantages of safety, standardization for chemosensitivity tests, and exoantigen purification.

BACKGROUND:Plasmodium falciparum parasitization of erythrocytes causes a substantial increase in the levels of intracellular fatty acids, notably oleic acid. How parasites acquire this monounsaturated fatty acid has remained enigmatic. Here, we report on the biochemical and enzymatic characterization of stearoyl-CoA desaturase (SCD) in P. falciparum. METHODOLOGY/PRINCIPAL FINDINGS:Metabolic labeling experiments allowed us to demonstrate the production of oleic acid from stearic acid both in lysates of parasites incubated with [(14)C]-stearoyl-CoA and in parasite-infected erythrocytes labeled with [(14)C]-stearic acid. Optimal SCD activity was detected in schizonts, the stage of maximal membrane synthesis. This activity correlated with a late trophozoite stage-specific induction of PFE0555w transcripts. PFE0555w harbors a typical SCD signature. Similar to mammalian SCDs, this protein was found to be associated with the endoplasmic reticulum, as determined with PFE0555w-GFP tagged transgenic P. falciparum. Importantly, these parasites exhibited increased rates of stearic to oleic acid conversion, providing additional evidence that PFE0555w encodes the plasmodial SCD (PfSCD). These findings prompted us to assess the activity of sterculic acid analogues, known to be specific Delta9-desaturase inhibitors. Methyl sterculate inhibited the synthesis of oleic acid both with parasite lysates and infected erythrocytes, most likely by targeting PfSCD. This compound exhibited significant, rapid and irreversible antimalarial activity against asexual blood stages. This parasiticidal effect was antagonized by oleic acid. CONCLUSION/SIGNIFICANCE:Our study provides evidence that parasite-mediated fatty acid modification is important for blood-stage survival and provides a new strategy to develop a novel antimalarial therapeutic based on the inhibition of PfSCD.

International audience; Rhoptries are secretory organelles involved in the virulence of the human pathogen Toxoplasma gondii. We have used high performance liquid chromatography and capillary gas-liquid chromatography to isolate and quantify lipids from whole Toxoplasma cells and their purified rhoptries. This comparative lipidomic analysis revealed an enrichment of cholesterol, sphingomyelin and, most of all, saturated fatty acids in the rhoptries. These lipids are known, when present in membranes, to be contributing to their rigidity and, interestingly, fluorescence anisotropy measurements confirmed that rhoptry-derived membranes have a lower fluidity than membranes from whole T. gondii cells. Moreover, while rhoptries were initially thought to be highly enriched in cholesterol, we demonstrated it is present in lower proportions and provided additional evidence towards a lack of involvement of rhoptry cholesterol in the process of host cell invasion by the parasite. Indeed, depleting the cholesterol content of the parasites did not prevent the secretion of protein-containing rhoptry-derived vesicles and the parasites could still establish a structure called the moving junction, which is necessary for invasion. Instead, the crucial role for host cholesterol for invasion, which has already been demonstrated (Coppens, I. and Joiner, K. A. (2003), Mol.Biol.Cell 14, 3804-3820), might be explained by the need of a cholesterol-rich region of the host cell we could visualise at the point of contact with the attached parasite, in conditions where parasite motility was blocked.

Toxoplasma gondii is a unicellular parasite characterized by unique extracellular and intracellular membrane compartments. The lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control, and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. The occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE). Immunolabeling characterization supports the notion that the DGLE polar head is similar to that of DGDG. Mass spectrometry analyses indicated that dihexosyl lipids having various hydrophobic moieties (ceramide, diacylglycerol, and acylalkylglycerol) might react with anti-DGDG, but we cannot exclude the possibility that more complex dihexosyl-terminated lipids might also be immunolabeled. DGLE localization was analyzed by immunofluorescence and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE was scattered in pellicle membrane domains in extracellular tachyzoites and was relocalized to the anterior tip of the cell upon invasion in an actin-dependent manner, providing insights on a possible role in pathogenetic processes. DGLE was detected in other Apicomplexa (i.e., Neospora, Plasmodium, Babesia, and Cryptosporidium).

International audience; Using high performance liquid chromatograpy and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.

We propose a serial assay of both protein kinase C activity and the related [ 3 H]phorbol 12,13-dibutyrate binding, each carried out in 96-multiwell dishes, started and stopped row by row using a multipipet. Protein kinase C activity is observed through the transfer of the γ-phosphoryl group of radioactive ATP onto histone H1 type III-S. Enzymatic reactions are started by adding enzyme extracts and stopped by adding trichloroacetic acid. Acidic precipitates of each row are simultaneously collected on glass fiber paper using a cell harvester. The addition of bovine serum albumin and cold ATP at the end of the reaction and the addition of trichloroacetic acid in the washing fluid lead to a high recovery of protein kinase C activity and reproducible results. Measurement of [ 3 H]phorbol 12,13-dibutyrate binding to protein kinase C was carried out in a mixed micellar solution as described elsewhere (Y. Hannun and R. M. Bell (1987) in Methods in Enzymology, Vol. 141, pp. 287–293) . The quaternary complex formed from protein kinase C, phosphatidylserine, calcium, and [ 3 H]phorbol 12,13-dibutyrate was then bound to a beaded anionic exchanger which was automatically separated from the free phorbol 12,13-dibutyrate by microfiltration using a cell harvester. The binding reaction was highly calcium- and phosphatidylserine-dependent and calcium had to be added to washing fluid for optimal recovery. Determination of protein kinase C activity and phorbol 12,13-dibutyrate binding gave results similar to those of other published methods and the signal/noise ratio was greatly increased. Using a semi-automated cell harvester, the system is partially automated and provides accurate and reproducible results.

The transbilayer distribution of glycerophospholipids in the plasma membrane of Plasmodium knowlesi infected erythrocytes was studied by using lysine-116-ε-N-palmitoyl amidinated pancreatic phospholipase A2. As a consequence of its superior membrane penetrating capacities, this modified enzyme rapidly degrades its substrates in the outer membrane leaflet of intact erythrocytes, a property that makes the enzyme an excellent tool to study the malaria parasitized red cell. The modified phospholipase A2 caused a nonlytic hydrolysis of up to 12–15% of the phosphatidylethanolamine and none of the phosphatidylserine in the red cell membrane, irrespective of whether the cells harboured trophozoite and schizont stages of parasites or no parasites at all. The absence of phosphatidylserine at the exterior surface of Plasmodium infected erythrocytes was confirmed by applying the prothrombinase assay on Plasmodium falciparum infected human erythrocytes. Consequently, the results from these and previous studies indicate that the plasma membrane of Plasmodium infected erythrocytes exhibit a normal transbilayer phospholipid asymmetry.Key words: malaria, plasma membrane, phospholipid asymmetry, erythrocyte, prothrombinase assay.

L2C B lymphocytes have a constant high DNA synthesis due to their continuous proliferative state. The addition of polymyxin B (PmB), a rather selective inhibitor of protein kinase C, stopped (3H)thymidine incorporation with an IC50 of 10 microM when added 18 h before measuring DNA synthesis. Interestingly, PmB inhibition of DNA synthesis was suppressed when 4 nM 12-O-tetradecanoylphorbol-13-acetate was added along with PmB, indicating that PmB may act through inhibition of protein kinase C. In the node and spleen lymphocytes of normal guinea pigs, protein kinase C activity was entirely cytosolic and was eluted at 0.12 M NaCl when adsorbed on DEAE-cellulose. In L2C leukemic lymphocytes, total protein kinase C activity was of the same order of magnitude, but 20% of it was associated with the membrane fraction. The lipid-dependent activity, eluted at 0.12 M NaCl from cytosolic and membrane fractions, was suppressed by staurosporine with an IC50 of 10-40 nM and by polymyxin B with an IC50 of 2-6 microM. Phosphoinositide metabolism was studied in the transformed cells. Incorporation of 32Pi into polyphosphoinositides was considerable, whereas much more time was required for a tiny incorporation of inositol. We detected no release of radioactive inositol triphosphate. Taken together, these results suggest that protein kinase C function is indispensible for triggering L2C leukemic lymphocyte proliferation. The causes of this permanent activation merit further investigation.

Choline kinase (EC 2.7.1.32) was investigated in plasmodium falciparum-infected erythrocytes. Disrupted infected erythrocytes had a choline kinase activity of 1.9 +/- 0.2 nmol phosphorylcholine/10(7) infected cells per h, whereas the activity in normal uninfected erythrocytes was less than 6 pmol/10(7) cells per h. A broad alkaline optimal pH (7.9-9.2) was observed. The Km values for choline and ATP were 79 +/- 20 microM, and 1.3 +/- 0.3 mM, respectively. ATP concentrations higher than 12 mM inhibited choline kinase. Maximal activity was registered with a Mg2+ concentration of 10 mM, whereas its replacement by Mn2+, or other divalent cations, involved a decrease in choline kinase activity of at least 75%. Inhibition by products of the reaction, such as phosphorylcholine and ADP was investigated. In plasmodium knowlesi-infected erythrocytes, choline kinase had similar properties, but with a much higher specific activity of 16.4 +/- 2.1 nmol/10(7) infected cells per h. Subcellular fractionation of P. knowlesi-infected erythrocyte suspensions revealed that choline kinase was located exclusively in the cytosol of the parasite. We show that this enzyme is a useful index of parasite cytosolic content leakage, when infected erythrocytes are fractionated by saponin lysis or nitrogen decompression.

International audience; The intraerythrocytic malaria parasite depends on the surrounding medium for a supply of phospholipid precursors. Efficient inhibition (IC50 7–90 μM) of Plasmodium falciparum growth in vitro was achieved using modified fatty acids. The fatty acid analogues most effective in suppressing P. falciparum growth in culture were also the most active inhibitors of acyl-CoA synthetase from the monkey parasite P. knowlesi.

Human alveolar macrophages (AMs) from allergic asthmatics (AAs) showed continuous Li + -sensitive production of IP 1 , indicating that the cells were continuously activated. Furthermore, whereas the accumulation of IP 1 , IP 2 and IP 3 rapidly increased by as much as 125—175% in chemotactic-factor-stimulated AMs from healthy subjects, stimulation of cells from AAs increased these inositol phosphates only slightly. This moderate production could be due to a permanent state of activation leading to a depleted pool of polyphosphoinositides, corroborating the greater capacity of these cells to generate superoxide anion after stimulation by a chemoattractant. The activation state could be due to the action of priming agents, which are known to be released into the inflammatory sites.

Alteration of uninfected erythrocytes from Plasmodium (the malaria parasite)-infected blood remained an open question. In this study we compared the in vivo fatty acid compositions of control and uninfected monkey erythrocytes. A large (40%) increase in the linoleic acid level was observed, which was recovered mostly in neutral lipids. An in vitro system was developed to study medium-mediated alterations of cultured erythrocytes by Plasmodium falciparum. The increase in the linoleate level was reproduced in vitro and was also localized in the neutral lipid fraction, especially in triacylglycerols. Studies using proteolytic digestion and heat denaturation showed that a heat-labile serum protein is indispensable for the increase in the linoleate level of red cells treated with the supernatant of P. falciparum cultures. Both the function and the mechanism of this modification of uninfected erythrocytes still remain unknown.

The effect of the antimalarial drug chloroquine (CQ) on the phospholipid metabolism in Plasmodium knowlesi-infected simian erythrocytes has been studied by incubating cells with different labeled precursors and various concentrations of CQ. The drug induced considerable modifications of this metabolism but at the same time decreased nucleic acid and protein synthesis as well as the output of 14CO2 from radioactive glucose. Phosphatidylcholine biosynthesis was severely reduced. However, under these conditions, CQ had the early effect of markedly increasing phosphatidylinositol labeling from radioactive inositol, fatty acids, 1-(14C)palmitoyl-lysophosphatidylcholine, but not from glycerol. Synthesis of phosphatidylserine from (14C)serine and of phosphatidylethanolamine from labeled glycerol, ethanolamine, and serine was increased, especially at high CQ concentrations when the whole metabolism of the parasite was severely reduced. These effects reflect a deep differential effect of CQ on the intense phospholipid metabolism of the Plasmodium-infected erythrocytes, which might involve a redirecting of phospholipid metabolism similar to that induced by other cationic amphiphilic drugs, and a compensatory synthesis resulting from the severe blockage of phosphatidylcholine synthesis.

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.

Using a recently described technique of electron spin resonance spectroscopy, we determined the rate of transbilayer mobility (flip) of the four major simian-erythrocyte phospholipids in the erythrocyte membrane after infection by Plasmodium knowlesi. The development of the malarial parasite induces a very large increase in the flip rate of these phospholipids (mainly during the ring stage and at the beginning of trophozoite maturation). The half flip time fell from 2 and 3 hr in the case of choline phospholipids in healthy erythrocytes to less than 15 min in erythrocytes infected with the last stage of the parasite.

CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in Plasmodium knowlesi-infected erythrocytes obtained from Macaca fascicularis monkeys. Disrupted infected erythrocytes possess a cholinephosphotransferase activity (1.3 +/- 0.2 nmol phosphatidylcholine/10(7) infected cells per h) 1.5-times higher than the ethanolaminephosphotransferase activity. Optimal activities of both enzymes were observed in the presence of 12 mM MnCl2, which was about 3-times as effective as 40 mM MgCl2 as a cofactor. The two activities had similar dependences on pH and thermal inactivation. Their Arrhenius plots show an identical break at 17 degrees C and the corresponding activation energies below and above the critical temperature were similar for the two activities. Sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, beta-D-octylglucoside and lysophosphatidylcholine strongly inhibited the two activities above their critical micellar concentration, but the first three detergents stimulated the activities at lower concentrations. Saponin (0.004-0.5%) either did not affect the two activities or else increased them. Cholinephosphotransferase and ethanolaminephosphotransferase activities had apparent Km values for the CDP ester of 23.4 and 18.6 microM, respectively. CDPcholine and CDPethanolamine competitively inhibited the ethanolaminephosphotransferase and cholinephosphotransferase activities, respectively. The high selectivity of these activities for individual molecular species of diradylglycerol suggests that substrate specificity is responsible for the various molecular species of Plasmodium-infected erythrocyte phospholipids. However, cholinephosphotransferase and ethanolaminephosphotransferase had different dependences on 1,2-dilauroylglycerol and 1-oleylglycerol, which were substrates for cholinephosphotransferase but not for ethanolaminephosphotransferase under our conditions. These data provide the first characterization of an enzyme involved in the intense lipid metabolism in Plasmodium-infected erythrocytes, and the presence of cholinephosphotransferase demonstrates a biosynthesis of phosphatidylcholine by the Kennedy pathway after infection. Our data suggest that cholinephosphotransferase and ethanolaminephosphotransferase activities could be catalyzed by the same enzyme. Furthermore, since host erythrocytes are devoid of these enzymatic activities, cholinephosphotransferase is a parasite-specific membrane-associated enzyme which can be used as a probe or marker.

International audience; A new method of simultaneous quantitation of total fatty acids, plasmalogens, and cholesterol using thin-layer chromatography with flame-ionization detection (TLC-FID) is presented. This Method only requires methanolysis of the crude lipid extracts, an operation usually performed for fatty acid analysis by gas-liquid chromatography (GLC), and both analyses can thus be carried out in one step. Because of its resolution power and sensitivity, the TLC-FID technique can be used to analyze microsamples routinely assayed in capillary GLC (less than 15 μg of total fatty acids). The data obtained by capillary GLC and those provided by TLC-FID after careful individual calibration of the rods are almost identical. Reproducibility of the TLC-FID measurements is lower for fatty acid methyl esters and dimethylacetals, but equivalent for cholesterol quantitation, if the samples are analyzed twice on chromarods.

International audience; The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gel beads. In this way, the phospholipid content and composition of (i) the membrane of nonparasitized cells, (ii) the erythrocyte membrane of parasitized cells and (iii) the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phospholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phospholipids in: (i) erythrocytes from healthy monkeys, (ii) nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and (iii) erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.

The cholesterol synthesis of rhesus monkey erythrocytes parasitized by Plasmodium knowlesi and human erythrocytes infected by P. falciparum, as measured by incorporation of [1-14C]acetate and 3H2O, was almost undetectable, concordant with very low levels of measurable 3-hydroxy-3-methyl glutaryl-CoA reductase activity. In addition, both types of infected cells exchanged cholesterol with the plasma at the same rate as uninfected cells. The data do not exclude the possibility of cholesterol transfer from uninfected to infected cells.

SUMMARYThe biosynthesis of phospholipids is extensive inPlasmodium knowlesi-infected simian erythrocytes due to the synthesis of membranes by this single-cell eukaryote in a host erythrocyte devoid of any pathway for lipid biosynthesis. In the present paper, we show that the incorporation of [3H]glycerol, which reflectsde novobiosynthesis, is better studied at 300 μM−1 mM than at the trace doses, since this non-physiological precursor does not modify the amount of phosphatidylcholine biosynthesis from [3H]choline. Time-course incorporation of radioactive glycerol, oleate, lysophosphatidylcholine, choline, and inositol in RPMI 1640 medium containing nutrients for lipid synthesis showed that the optimum incubation time for phospholipid studies is 60–90 min, after which radioactive incorporation slows considerably. On the other hand, studies with [14C]serine revealed that incubation for 2–3 h is necessary for isotopic labelling of phosphatidylcholine via phosphatidylserine decarboxylation and phosphatidylethanolamineN-methylation. Incorporation of the various fatty acids into individual lipids was related to the molecular species composing each of them. Studies with [14C palmitoyl] lysophosphatidylcholine showed a very fast intracellular release of radioactive fatty acids, which indicates a potent lysophospholipase activity. Taken together, these data define the indispensable conditions for an experimental system suitable for further studies.

International audience; Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlsi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate ( + 23%), oleate ( + 29%) and linoleate ( + 89%), but contained less stearate (−27%) and arachidonate ( −40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI)) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (−60%), but their level is increased in PI.

Mature mammalian erythrocytes lack the enzymatic machinery for synthesizing lipids and there is no net synthesis of major phospholipids (PL). The turnover can be attributed to two main processes: the exchange of PL between plasma lipoproteins and membrane, or the acylation of lyso-phosphatidylcholine, originating from the surrounding plasma and possibly from membrane (1). The synthesis of phosphatidylcholine (PC) by N-methylation of phosphatidylethanolamine (PE) is of minor importance in supplying the cellular PC requirements, but could play a role in transmitting signals acting via cell surface receptors (2).

Separation of fatty acid methyl esters and dimethyl acetals from complex biological samples has been achieved by gas—liquid chromatography on a capillary column coated with free fatty acid phase. Response-correcting factors were determined, showing rather large variations with fatty acid length. Polyunsaturated fatty acid methyl esters were shown to have lower responses than saturated species, whereas dimethyl acetals and equivalent methyl esters were found to give similar responses. Total fatty acid and aldehyde compositions of human and simian erythrocytes were determined and compared, showing a somewhat higher level of linoleate and arachidonate, and a lower level of plasmalogens in simian erythrocytes.

Plasmodium knowlesi-infected erythrocytes efficiently incorporated choline and metabolize it into phosphatidylcholine via the de novo Kennedy pathway. No formation of either betaine or acetylcholine was detected. At physiological concentrations of external choline, isotopic equilibrium between intracellular choline and phosphocholine was reached in less than 1 h, whereas labeled phosphatidylcholine accumulated constantly, until at least 210 min. During this time, intracellular CDP-choline remained quite low compared to phosphocholine, which suggests that choline-phosphate cytidylyltransferase (EC 2.7.7.15) is the rate-limiting step of the Kennedy pathway. However, this activity was probably not saturated in situ by phosphocholine, since the external choline concentration, up to 100 microM, can regulate phosphatidylcholine biosynthesis via the level of intracellular phosphocholine. This was corroborated by the respective velocities and affinity characteristics of the three enzymatic steps involved in the Kennedy pathway. These results, together with the localization of both choline metabolites and enzyme activities, provide a precise scheme of the dynamics of de novo phosphatidylcholine biosynthesis. Concerning the alternative pathway for phosphatidylcholine biosynthesis via the methylation of phosphatidylethanolamine, we show that an increase in de novo phosphatidylcholine biosynthesis could instigate a concomitant decrease in the steps of phosphatidylethanolamine methylation, indicating that the parasite is able to modulate its phosphatidylcholine biosyntheses.

In Plasmodium falciparum-infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 +/- 1.4 nmol phosphoethanolamine/10(7) infected cells per h) was higher than choline kinase specific activity (1.9 +/- 0.2 nmol phosphocholine/10(7) infected cells per h). The Km of choline kinase for choline was 79 +/- 20 microM, and ethanolamine was a weak competitive inhibitor of the reaction (Ki = 92 mM). Ethanolamine kinase had a Km for ethanolamine of 188 +/- 19 microM, and choline was a competitive inhibitor of ethanolamine kinase with a very high Ki of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D-2-amino-1-butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50 degrees C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism.

Using electron microscopy, in the human breast cancer cell line T47D, the synthetic progestin R5020, and 5 alpha-dihydrotestosterone were shown to increase significantly the number of lipid droplets per cell section compared to control cells or estradiol- and dexamethasone-treated cells. Lipid accumulation, as measured by Oil Red O dying and by [2-14C]acetate incorporation, was observed at concentrations as low as 10 pM R5020 and 1 nM 5 alpha-dihydrotestosterone, and was always more abundant after progestin treatment. The progestin antagonist RU486 inhibited, in a dose-dependent manner, lipid accumulation initiated by the two hormones, whereas the androgen antagonist flutamide inhibited only the effect initiated by 5 alpha-dihydrotestosterone. Cytoplasmic lipid droplets accumulation was not observed in the BT20 breast cancer cell line, which contains neither progesterone nor androgen receptors. These results indicate that progestins and androgens increase lipid accumulation by interacting with their own receptor. Chromatographic analysis of [2-14C]acetate labeled lipids showed that R5020 and 5 alpha-dihydrotestosterone enhanced the accumulation of cellular triglycerides at least in part by increasing their synthesis and decreased the quantity of lipids released into the medium. To conclude, we have shown that progestins and androgens, via their own receptor, can induce the same triglyceride accumulation in T47D cells. This effect follows fatty acid synthetase induction and precedes cell growth inhibition, two responses also triggered by progestin and androgen in these cells.

Metabolic pathways leading to lipid biosynthesis in four different developmental stages of Schistosoma mansoni were explored and quantified by incubation in the presence of labeled precursors in a chemically defined medium. At the schistosomulum stage and in male, female, or paired worms, glycerol and oleate incorporation into neutral lipids, mainly in the form of triacylglycerols, was greater than into phospholipids, whereas in 11- and 15-day-old worms, synthesis mainly led to phospholipids. Incorporation into phospholipids was recovered largely in phosphatidylcholine, and distribution into other phospholipids depended on the developmental stage. Incorporation of choline and ethanolamine into their respective phospholipids represented up to 15% of the parasitic phospholipid content. The formation of phosphatidylcholine by phosphatidylethanolamine methylation occurred mainly in the immature parasitic stages. Inositol incorporation was also measurable, whereas [14C]serine incorporation was low or undetectable. Addition of 1-palmitoyl-2-[14C]oleyl phosphatidylcholine revealed a very high uptake of this phospholipid by the immature stages but further metabolism was not detectable. In contrast, adult S. mansoni were completely unable to take up or adsorb this exogenous phospholipid. The most striking aspect of this study was the relatively high metabolic activity in 11-day-old worms and the lower but sustained activity on day 15 and at the schistosomulum stage. By comparison, biosynthetic activity in adult S. mansoni, on which research studies have been focused until now, was very low. We also discuss the participation of lipid metabolism in the constant renewal of the membrane complex which is essential to parasitism by S. mansoni.

International audience; In its blood stages the malaria parasite, Plasmodium, displays very high lipid metabolism. We present evidence for an abundant long-chain acyl-CoA synthetase (EC 6.2.1.3) activity in Plasmodium knowlesi-infected simian erythrocytes. The activity was found to be 20-fold higher in the schizont-infected (the last parasite stage) than in control erythrocytes. The cosubstrate requirements of the enzyme were similar to those previously reported for acyl-CoA synthetases from other sources. Among the separated reaction products of oleyl-CoA synthetase, only PPi and oleyl-CoA were inhibitory, with Ki over 350 μM. The fatty acid specificity of the parasite acyl-CoA synthetase activity was fairly marked and depended on the unsaturation state of the substrate. The tested fatty acids displayed similar Vmax, whereas their Km ranged from 11 (palmitate) to 59 μM (arachidonate). Finally, experiments involving heat inactivation and separation on hydroxyapatite excluded the presence of a specific arachidonyl-CoA synthetase identical to those present in other cells. On the other hand, fatty acid competition experiments evidenced the existence of at least two distinct enzymatic sites for fatty acid activation in P. knowlesi-intected simian erythrocytes: one is specific for saturated fatty acids and the other for polyunsaturated species, whereas oleate could be activated at both sites.