Abstract. Several intraerythrocytic growth cycles of Plasmodium falciparum could be achieved in vitro using a serum free medium supplemented only with a human high density lipoprotein (HDL) fraction (d = 1.063-1.210). The parasitemia obtained was similar to that in standard culture medium containing human serum. The parasite development was incomplete with the low density lipoprotein (LDL) fraction and did not occur with the VLDL fraction. The lipid traffic from HDL to the infected erythrocytes was demonstrated by pulse labeling experiments using HDL loaded with either fluorescent NBD-phosphatidylcholine (NBD-PC) or radioactive [aH]palmitoyl-PC. At 37°C, the lipid probes rapidly accumulated in the infected cells. After incubation in HDL medium containing labeled PC, a subsequent incubation in medium with either an excess of native HDL or 20% human serum induced the disappearance of the label from the erythrocyte plasma membrane but not from the intraerythrocytic parasite. Internalization of lipids did not occur at 4°C. The mechanism involved a unidirectional flux of lipids but no endocytosis. The absence of labeling of P. falciparum, with HDL previously [125I]iodinated on their apolipoproteins or with antibodies against the apolipoproteins AI and All by immunofluorescence and immunoblotting, confirmed that no endocytosis of the HDL was involved. A possible pathway of lipid transport could be a membrane flux since fluorescence videomicroscopy showed numerous organdies labeled with NBD-PC moving between the erythrocyte and the parasitophorous membranes. TLC analysis showed that a partial conversion of the PC to phosphatidylethanolamine was observed in P. falciparum-infected red cells after pulse with [3H]palmitoyl-PC-HDL. The intensity of the lipid traffic was stage dependent with a maximum at the trophozoite and young schizont stages (38 t~ h of the erythrocyte life cycle). We conclude that the HDL fraction appears to be a major lipid source for Plasmodium growth.URING its intraerythrocytic phase, the human malarial parasite Plasmodium falciparum reproduces at a rapid rate, completing its development from the infective merozoite to the mature schizont (16-20 nuclei) in <48 h. After the invasion step, the parasite is isolated from the erythroeyte cytoplasm by a parasitophorous vacuole membrane and undergoes a sequential development through a ring form, trophozoite, schizont, and finally the differentiation of 10-20 merozoites. The new merozoites are released into the bloodstream by erythrocyte bursting.Throughout the schizogonic phase, the parasite sequential development that takes place, corresponds to considerable nuclear and cytoplasmic transformations, including a fivefold increase of total phospholipid content and a decrease in the cholesterol-phospholipid ratio of the infected cell (Holz, 1977;Sherman, 1979;Vial et al., 1982a). Studies of the fatty acid composition have shown that the ability of P/asmodium to perform saturation or desaturation reactions of aliphatic chains, as well as chain lengthening and s...

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors lovastatin and simvastatin inhibit the in vitro intraerythrocytic development of Plasmodium falciparum and Babesia divergens, with concentrations inhibiting parasite growth by 50% in the ranges of 10 to 20 and 5 to 10 ug -ml-', respectively. For P.falciparum, the 50% inhibitory concentrations were in the same range whatever the chloroquine susceptibility of the strains tested (strain F32/Tanzania [chloroquine susceptible] or FcB.1/Columbia [resistant]). The stage-dependent susceptibility of P. falciparum to simvastatin was studied by subjecting synchronized cultures to 6-h pulses of drug throughout the 48-h erythrocytic life cycle. The most important inhibitory effects were observed between the 12th and 30th hours of the cycle, corresponding to the trophozoite stage. This period precedes the S phase and the nuclear divisions. Parasites in the newly formed ring stage (time zero to the 6th hour of the cycle) and the schizont stage (30th to 48th hour of the cycle) were weakly or not susceptible to simvastatin pulses.The Apicomplexa hemoparasites Plasmodium falciparum and Babesia divergens, the causative agents of human malaria and bovine babesiosis, respectively, still constitute major health and economic problems in most developing countries (12,24). The (8,21,22) and depend on their import from the host's plasma. With the use of a serum-free medium, we have recently found a unidirectional transfer of phospholipids from human high-density lipoproteins to the intraerythrocytic P. falciparum by ducts and vesicles moving from the erythrocyte to the parasitophorous vacuole membranes (6). A similar phospholipid transfer was observed in B. divergensinfected erythrocytes (RBC), but no ducts or vesicles were observed (21). High-density lipoproteins support the in vitro growth of B. divergens and P. falciparum in the absence of other major serum components and appeared to be a lipid source for the parasites (6,7,21 by P. falciparum-infected RBC, we have shown evidence for an isoprenoid metabolisnm until, at least, the farnesyl PPi step (16).This pathway, with numerous end products, is essential for various cellular functions such as mitochondrial electron transport, tRNA synthesis, control of cell growth, protein glycosylation, and intracellular targeting (4). In both prokaryotic and eukaryotic cells, the isoprenoid pathway is highly regulated through feedback regulations at the level of two sequential enzymes involved in the synthesis of mevalonate: 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMGCoA reductase (4). HMG-CoA reductase inhibitors are available as hypocholesterolemic agents in humans. The goal of our study was to establish whether the HMG-CoA reductase inhibitors lovastatin and simvastatin are able to inhibit the in vitro development of P. falciparum and B. divergens. MATERIALS AND METHODSLovastatin and simvastatin, both in the lactone form, were kindly provided by J. C. Mazi&re (Hopital St-Antoine, Paris, France) and by C. Fous...

In plasma, Ph4-OH is mainly transported by high-density lipoproteins (HDL). This high affinity for HDL may explain the selective accumulation of lipophilic pheophorbide derivatives in the intracellular parasites. Photosensitization with pheophorbide derivatives may be a promising approach to inactivation of transfusion-transmissible parasites and viruses in blood bank units.

A two-fold increase in the amount of phospholipids was observed in Babesia divergens infected human red blood cells. In vitro incubation with [32P]-phosphorus and [3H]-glycerol demonstrated that B divergens has the ability to synthesize the phospholipid backbone. On the other hand, the low incorporation of [14C]acetate indicated the absence of a de novo fatty acid synthesis and suggested the necessity of an exogenous lipid source for the parasite. Several intra-erythrocytic growth cycles of B divergens could be achieved in vitro, using a serum-free medium supplemented only with fractions of human high density lipoproteins (HDL). At an HDL concentration of 0.5 mg/ml (protein concentration) and with a 1% starting parasitaemia, parasite growth was similar to that observed under standard culture conditions with 10% human serum, at least for the first 24 h, a time equivalent to three parasite erythrocytic life-cycles. Lipid transfer from HDL to the intra-erythrocytic parasites was demonstrated by uptake and exchange of fluorescent NBD-phosphatidylcholine (NBD-PC) loaded HDL at different temperatures. Kinetic experiments with [3H]-oleyl-PC-loaded HDL demonstrated a unidirectional transfer of lipids from radiolabelled HDL to the parasite; partial conversion of PC to phosphatidylethanolamine (PE) was also observed. In the semi-defined medium, the HDL fraction appeared to be the major source of lipids for the growth of B divergens in human erythrocytes.

No abstract

139In Bacillus subtilis, the fatty acid moiety of the phospholipids was affected differently during growth in the presence of 1.1 M-methanol or 0.7 M-ethanol, though at these concentrations methanol and ethanol had the same effects on growth rate and completely inhibited sporulation. Synthesis of phosphatidylglycerol was also strongly inhibited and the amount of total cell phospholipids was reduced by 50 % by both alcohols. The composition of fatty acids, especially the relative concentration of 12-methyltetradecanoic acid, was modified only by ethanol; in bacteria grown in the presence of methanol, changes in fatty acid composition were negligible. In non-sporulating mutants, synthesis of phosphatidylglycerol was much less affected than in the wild-type and synthesis of phosphatidylethanolamine was increased. In these strains, fatty acid composition was also modified by ethanol but unaffected by methanol.

The growth rate of Bacillus subtilis is lowered but the final cell yield is unchanged when certain concentrations of ethanol are present in the culture medium. At the concentration allowing growth at half-maximal rate, practically no spores are formed. Blockage of spore formation generally occurs at stage 0-I. Sensitivity to ethanol of the capacity to form spores is limited, in a nonsynchronized culture, to a period of at most 45 min around t1. Postexponential events such as excretion of certain enzymes and modification of ribonucleic acid polymerase are altered or suppressed in the presence of ethanol, possibly as the results of a physical change upon the cell membrane. In effect, ethanol is turning wild-type cells into phenocopies of spoO mutants.

No abstract