Incorporation of free fatty acids can explain alterations in the molecular species composition of phosphatidylcholine and phosphatidylethanolamine in human erythrocytes as induced by Plasmodium falciparum.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species composition of human erythrocytes changes upon intraerythrocytic development of Plasmodium falciparum. Though the activity of the phosphotransferases which catalyze the last step of the Kennedy pathway for the synthesis of PC and PE is dependent of the species on diacylglycerol, it appeared that this cannot, by itself, explain the alterations found in PC and PE molecular species composition. When the incorporation of radiolabeled palmitic and stearic acids in PC and PE was studied, it became clear that differences in the incorporation of fatty acids in those phospholipids might be responsible for the observed alterations in their molecular species composition.     

Phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection

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 the membrane of nonparasitized cells, the erythrocyte membrane of parasitized cells and 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: erythrocytes from healthy monkeys, nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and 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.     

Carbohydrate metabolism of malarial parasites--I. Metabolism of lactate in Plasmodium knowlesi infected monkey erythrocytes

Lactate metabolism by Plasmodium knowlesi infected erythrocytes was examined after careful removal of leucocytes from cell preparations. Infected cells were able to metabolize glucose, pyruvate and lactate. Respiration of infected erythrocytes was maximally stimulated by lactate and to a lesser degree by pyruvate and glucose. Respiration of infected cells was insensitive to stimulation by succinate or glutamate or inhibition by malonate. Mepacrine was found to be a potent respiratory inhibitor. Chromatographic analysis of end products of lactate metabolism showed incorporation of carbon from [2-C14]lactate into phosphoenol pyruvate, 3-phosphoglycerate and malate. Experimental data failed to provide evidence for the presence of a functional citric acid cycle activity in infected cells.     

Plasmodium knowlesi: glycolytic intermediate levels of enzyme activities of infected rhesus monkey erythrocytes

In vitro glycolytic enzyme activities and in vivo glycolytic intermediate concentrations were assayed in Plasmodium knowlesi-infected rhesus monkey erythrocytes and control erythrocytes. The enzyme activities of infected erythrocytes were greater than controls indicating that P. knowlesi had its own glycolytic system and that parasite glycolysis was the source of the increased rate of glucose consumption by infected erythrocytes. The P. knowlesi glycolytic enzymes phosphofructokinase and hexokinase were less sensitive to acid inhibition than uninfected red cells.P. knowlesi-infected monkey erythrocytes and Plasmodium berghei-infected mouse erythrocytes had similar in vivo glycolytic profiles and in vitro enzyme activity increases.     

Changes of membrane phospholipid composition of human erythrocytes in hyperlipidemias. II. Increases in distinct molecular species of phosphatidylethanolamine and phosphatidylcholine containing arachidonic acid.

The molecular species composition of red blood cell diacyl-phosphatidylcholine (PC), diacyl-phosphatidylethanolamine (PE) and alkenylacyl-PE (plasmalogen PE) has been analyzed in normolipidemic and hyperlipidemic donors. In all three phospholipid subclasses the percentages of the species 16:0/20:4 were increased in hyperlipidemic patients. In diacyl-PE, 18:1/20:4 was also elevated. No changes were observed in the other quantitatively important molecular species containing arachidonic acid at sn-2, namely 18:0/20:4. The rise in 16:0/20:4 in diacyl-PC and diacyl-PE of hyperlipidemic donors was accompanied by a fall in molecular species with linoleic acid (18:2) at sn-2 (in particular 18:1/18:2). In alkenylacyl-PE the elevation of 16:0/20:4 was compensated by a decrease in species with docosatetraenoic acid (22:4) at sn-2 in particular by a fall in 16:0/22:4. Among all donors, the percentages of 16:0/20:4 in diacyl-PC and PE were positively associated with plasma total cholesterol levels. The changes in molecular species composition of PC and PE in hyperlipidemia are expected to alter the function of erythrocyte membrane transport proteins and--if present also in other cell types--to affect eicosanoid metabolism.     

Synthesis of molecular species of phosphatidylcholine and phosphatidylethanolamine by germinating soya bean

The composition and metabolism of phosphatidylcholines and phosphatidylethanomines of germinating soya bean Glycine max have been examined. Both phospholipids have a very similar fatty acid composition and distribution, with saturated acids located at the 1- position. The fatty acid composition and relative amounts of individual molecular species of the two phospholipids were also very similar. The relative amounts of the species were in the order tetraenoic pentaenoic trienoic = dienoic = monoenoic. In contrast, the labelling of the molecular species from choline Me[¹⁴C] or ethanolamine [2-¹⁴C] showed considerable differences. Phosphatidylethanolamine-[¹⁴C] showed 58% label in trienoic, 17% in tetraenoic, 18% in pentaneoic and 5% in dienoic species 48 hr after germination. The equivalent figures for phosphatidylcholine-[¹⁴C] were 37, 34, 13 and 15% respectively. An increase in labelling of the more unsaturated species was seen with time.     

Lipid compartmentalization in erythrocytes parasitized by Plasmodium spp

Although reasonably well protected from the host immune system by the erythrocyte membrane, the intraerythrocytic malaria parasite has to make that membrane compatible with its own requirements for development and multiplication. The development of Plasmodium spp brings about major changes in the lipid composition of the host cell membrane, as well as in its physical properties. The parasite itself has a lipid composition that differs from that of the host cell and an intense lipid trafficking seems to occur between intracellular parasite and host cell membrane. Here, Ana Paula Sim?es, Ben Roelofsen and Jos Op den Kamp discuss how, despite serious methodological limitations and the existence of some conflicting results, an overall picture of lipid compartmentalization within the parasitized erythrocyte is perceived.     

Individual molecular species of phosphatidylcholine and phosphatidylethanolamine in myelin turn over at different rates.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of the myelin membrane exhibit heterogeneity with respect to metabolic turnover rate (Miller, S. L., Benjamins, J. A., and Morell, P. (1977) J. Biol. Chem. 252, 4025-4037). To test the hypothesis that this is due to differential turnover of individual molecular species (which differ in acyl chain composition), we have examined the relative turnover of individual molecular species of myelin PC and PE. Phospholipids were labeled by injection of [2-3H]glycerol into the brains of young rats. Myelin was isolated at 1, 15, and 30 days post-injection, lipids were extracted, and phospholipid classes were separated by thin-layer chromatography. The PC and PE fractions were hydrolyzed with phospholipase C, and the resulting diacylglycerols were dinitrobenzoylated and fractionated by reverse-phase high performance liquid chromatography. The distribution of radioactivity among individual molecular species was determined. The labeled molecular species of myelin PC were 16:0-16:0, 16:0-18:0, 16:0-18:1, and 18:0-18:1, with most of the label present in 16:0-18:1 and 18:0-18:1. Changes in distribution of label with time after injection indicated that 16:0-18:1 turned over more rapidly than 18:0-18:1. The labeled molecular species of myelin PE were 18:0-20:4, 18:1-18:1, 16:0-18:1, 18:0-18:2, and 18:0-18:1. As with myelin PC, 16:0-18:1 (and 18:1-18:1) turned over more rapidly than 18:0-18:1. The relative turnover of individual molecular species of PC in the microsomal fraction from forebrain was also examined. The molecular species profile was different from myelin PC, but again, 16:0-18:1 turned over more rapidly than the other molecular species. Thus, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Comparison of our results with previous studies of turnover of molecular classes of phospholipids indicates that in addition to polar head group composition (Miller et al., 1977), fatty acid composition is very important in determining the metabolic fate of a phospholipid.     

CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes

Erythrocytes infected with P. falciparum express knob-like adhesion structures that allow the infected cells to cling to the postcapilliary endothelium of characteristic host organs. At present, the mechanism of cytoadherence is not fully understood. While parasitized erythrocytes have been shown to specifically bind to the platelet/matrix molecule thrombospondin, adherence to suitable target cells can also be blocked by monoclonal antibody OKM5, which recognizes a surface molecule expressed by hematopoietic cells and endothelium. In apparent reconciliation of these findings, it has been reported that the OKM5 antigen (CD36) is a receptor for thrombospondin. Here we report that expression of a CD36 cDNA clone in COS cells supports cytoadherence of parasitized erythrocytes but does not support increased binding of purified human thrombospondin.     

Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolamine and phosphatidylcholine

Dolichol promotes the leakage of membranes in liposomes composed of phosphatidylethanolamine and phosphatidylcholine but not liposomes composed only of phosphatidylcholine. The membrane leakage was assayed by measuring the entrapment of TEMPOcholine, a cationic spin probe, in liposomes using ESR methods. The percent of membrane leakage induced by dolichol was found to be linearly proportional to the concentrations of dolichol. It is proposed that dolichol enhances the formation of non-bilayer configurations in liposomes containing phosphatidylethanolamine, thereby membrane leakage.     

Selective changes to phosphatidylcholine and phosphatidylethanolamine molecular species in the developing fetal guinea pig liver and plasma

The molecular species composition of membrane phospholipids influences the activities of integral proteins and cell signalling pathways. We determined the effect of increasing gestational age on fetal guinea pig liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and plasma PC molecular species composition. The livers were collected from fetuses (n = 5/time point) at 5 day intervals between 40 and 65 days of gestation, and at term (68 days). Hepatic PC and PE molecular species composition was determined by electrospray ionisation mass spectrometry. An increasing gestational age was accompanied by selective changes in individual molecular species. The proportion of the sn-1 18:0 species increased relative to the sn-1 16:0 species in liver PC, but not PE, with an increasing gestational age. 1-O-alkyl-2-acyl PC species concentrations decreased significantly between 40 and 45 days of gestation (40%), and 65 and 68 days (54%). Total 1-O-alkenyl-2-acyl PE species concentration increased between days 60 and 65, due to a rise in 1-O-16:0 alkyl/20:4 content, and then decreased until term. Between day 40 and term, PC and PE sn-2 18:2n-6 species concentrations increased 3-fold. PC16:0/18:2 increased gradually throughout gestation, while PC18:0/18:2 content only increased after day 65. The overall increase in PE18:2n-6 content was due to PE18:0/18:2 alone. The composition of plasma PC essentially reflected hepatic PC. Overall, these data suggest differential regulation of hepatic PC and PE molecular species composition during development which is essentially independent of the maternal fatty acid supply.     

The apparent transfer of fatty acid from phosphatidylcholine to phosphatidylethanolamine in human erythrocytes

In previous studies an apparent transfer of (14)C-labeled fatty acid from phosphatidylcholine to phosphatidylethanolamine was observed in prelabeled human erythrocytes reincubated in fresh serum. These data could have been explained by direct fatty acid transfer from phosphatidylcholine to phosphatidylethanolamine or by an apparent transfer simulated by either demethylation of labeled phosphatidylcholine to phosphatidylethanolamine or base-exchange of phosphatidylcholine with ethanolamine. To explore these possibilities, RBC containing phosphatidylcholine doubly labeled with palmitic acid-9,10-(3)H and with choline-1,2-(14)C were prepared. Upon reincubation in fresh serum, incorporation of (3)H (fatty acid) into phosphatidylethanolamine was observed without incorporation of (14)C (choline). In similar experiments in which RBC labeled with (3)H-labeled fatty acid alone were used, (14)C-ethanolamine added to the incubation was not incorporated into the isolated phosphatidylethanolamine which again showed incorporation of the fatty acid-(3)H. The data indicate that direct transfer of fatty acid from phosphatidylcholine to phosphatidylethanolamine can occur in human erythrocytes incubated in fresh serum.     

Febrile temperature leads to significant stiffening of Plasmodium falciparum parasitized erythrocytes

Parasitic infection with Plasmodium falciparum is responsible for the most severe form of human malaria in which patients suffer from periodic fever. It is well established that during intra-erythrocytic maturation of the parasite in the red blood cell (RBC), the RBC becomes significantly more cytoadhesive and less deformable; these and other biochemical factors together with human host factors such as compromised immune status are important contributors to the disease pathology. There is currently substantial interest in understanding the loss of RBC deformability due to P. falciparum infection, but few results are available concerning effects of febrile conditions or parasitization on RBC membrane rheology. Here, for the first time, we report rheology of the single, isolated RBC with and without P. falciparum merozoite invasion, spanning a range from room temperature to febrile conditions (41 degrees C), over all the stages of parasite maturation. As expected, stiffness increased with parasite maturation. Surprisingly, however, stiffness increased acutely with temperature on a scale of minutes, particularly in late trophozoite and schizont stages. This acute stiffening in late falciparum stages may contribute to fever-dependent pathological consequences in the microcirculation.     

Endogenous phospholipids of swine liver: effect of fat deprivation on molecular species of phosphatidylcholine and phosphatidylethanolamine

Three 1-yr-old swine and two 2.5-wk-old swine were fed a fat-free diet for 1 month and 5 months, respectively. The hepatic phosphatidylcholine and phosphatidylethanolamine were fractionated by silver ion thin-layer chromatography. A distinctive feature of the chromatographic procedure was the development of the chromatograms at low temperatures: -10 degrees C for phosphatidylcholine and 4 degrees C for phosphatidylethanolamine. The chromatographic fractions were hydrolyzed with phospholipase A(2), and the fatty acids were characterized. Significant concentrations of odd-chain saturated and unsaturated fatty acids were found in the swine deprived of fat for 5 months. The major molecular species of phosphatidylcholine in both groups contained monoenoic fatty acids: 16:0/18:1(n - 9), 18:0/18:1(n - 9), and 18:1(n - 9)/18:1(n - 9). Their concentrations changed only slightly with the diet. The molecular species of phosphatidylethanolamine were more sensitive to dietary changes. In the swine deprived of fat for 1 month, about 50% of the molecular species of phosphatidylethanolamine contained tetraenoic fatty acids: 16:0/20:4(n - 6), 18:0/20:4(n - 6), and 18:1(n - 9)/20:4(n - 6). The phosphatidylethanolamine of animals deprived of fat for 5 months contained only 3% molecular species with tetraenoic acids, 18:0/20:4(n - 6), but 36% molecular species with trienoic acids: 18:0/20:3(n - 9), 18:1(n - 9)/20:3(n - 9), 18:0/19:3(n - 8), 16:0/20:3(n - 9), and 17:0/20:3(n - 9). Doubly unsaturated species, such as 18:1(n - 9)/18:1(n - 9), 18:1(n - 9)/20:3(n - 9), and 18:1(n - 9)/20:4(n - 6), were found in both groups of swine, although their total concentrations were higher in the group deprived of fat for a longer period.     

A large-scale purification of phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylethanolamine, and phosphatidylcholine by high performance liquid chromatography: a partial resolution of molecular species

Egg yolk phospholipids, on a 10 g scale, were resolved by high-performance liquid chromatography on an 8-m silica column with elution by a stepwise chloroformmethanol gradient into homogenous phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and lysophosphatidylethanolamine fractions. Within these fractions, partial resolution on the basis of fatty acyl side chain composition was achieved.     

Limitations of microscopy to differentiate Plasmodium species in a region co-endemic for Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi

Background

The question whether Plasmodium falciparum infection affects the fitness of mosquito vectors remains open. A hurdle for resolving this question is the lack of appropriate control, non-infected mosquitoes that can be compared to the infected ones. It was shown recently that heating P. falciparum gametocyte-infected blood before feeding by malaria vectors inhibits the infection. Therefore, the same source of gametocyte-infected blood could be divided in two parts, one heated, serving as the control, the other unheated, allowing the comparison of infected and uninfected mosquitoes which fed on exactly the same blood otherwise. However, before using this method for characterizing the cost of infection to mosquitoes, it is necessary to establish whether feeding on previously heated blood affects the survival and fecundity of mosquito females.

Methods

Anopheles gambiae M molecular form females were exposed to heated versus non-heated, parasite-free human blood to mimic blood meal on non-infectious versus infectious gametocyte-containing blood. Life history traits of mosquito females fed on blood that was heat-treated or not were then compared.

Results

The results reveal that heat treatment of the blood did not affect the survival and fecundity of mosquito females. Consistently, blood heat treatment did not affect the quantity of blood ingested.

Conclusions

The study indicates that heat inactivation of gametocyte-infected blood will only inhibit mosquito infection and that this method is suitable for quantifying the fitness cost incurred by mosquitoes upon infection by P. falciparum.     

Molecular species of phosphatidylcholine and phosphatidylethanolamine in subcellular membranes from rat liver.

Four subfractions of phosphatidycholine and phosphyatidylethanolamine according to the degree of unsaturation of their fatty acids have been separated from lipid extracts of microsomes, and inner and outer mitochondrial membranes. The predominant species found in the three membranes contained one saturated and one unsaturated fatty acid. In microsomes completely saturated species of both phosphatidylcholine and phosphatideylethanolamine were practically nonexistent. In outer mitochondrial membranes species with two unsaturated fatty acids were absent. In the inner mitochondrial membranes, however, disaturated species and those with two unsaturated fatty acids were found.