Preparation and characterization of F-protein vesicles isolated from Sendai virus by means of octyl glucoside.

We have demonstrated that Triton X-100 is always present in F-protein vesicles at concentrations that can provoke cell lysis. In order to avoid any misinterpretation of the fusogenic capacity of this protein, we solubilized the Sendai virus using octyl glucoside, which can be totally removed from the F protein preparation in less than 16 h by dialysis in the presence of absorbent beads. F-glycoprotein preparations preserved their ability to lyse erythrocytes in the presence of lectins and to induce cell-vesicle fusion as demonstrated by ESR studies. These vesicles were characterized by electron microscopy and SDS-polyacrylamide gel electrophoresis. Lipid analysis of these preparations by thin-layer chromatography indicated that they had the same proportion of lipids as virus envelopes, with slight variations in the sphingomyelin content and the cholesterol/phospholipid molar ratio. F-protein vesicles of different sizes can be obtained by adding exogenous lipids before detergent removal. The hemolytic activity of the vesicles was retained over a large range of lipid concentrations. We conclude that F-protein vesicles prepared with octyl glucoside are convenient tools for studying the fusogenic mechanism of this protein and improving the fusion process between liposomes and cells.

Extemporaneous preparation of large unilamellar liposomes.

Direct contact between lipids solubilized by octyl glucoside and Amberlite XAD-2 beads yielded large liposomes (240 nm diameter) with no residual detergent molecules, in less than 10 min. This extemporaneous preparation of liposomes was prepared with a detergent/bead ratio no higher than 0.12 (mumol/mg) and a phosphatidylcholine/phosphatidylserine/cholesterol molar ratio of 1:1:1. The liposomes were mainly unilamellar, as deduced from thin section and freeze-fracture electron micrographs and from measurement of calcein incorporation into the vesicles. The relatively large internal volume of these vesicles (8.9 l/mol lipid) accounts for the high percentage of entrapped material observed. The percentage increased with lipid concentration, but could not be increased above 20% corresponding to 20 mM total lipids.

Non-phospholipid fusogenic liposomes.

We have demonstrated the capacity of non-phospholipid liposomes composed primarily of dioxyethylene acyl ethers and cholesterol to fuse with membranes composed primarily of phospholipid. Phase-contrast microscopy, freeze-fracture electron microscopy and a macromolecular probe indicate that these non-phospholipid liposomes can fuse with the plasma membranes of erythrocytes and fibroblasts. Furthermore, fluorescence probe experiments have demonstrated fusion between phosphatidylcholine liposomes and non-phospholipid liposomes. Mixing of internal contents was shown by a terbium/dipicolinate assay. Mixing of membrane lipid components was demonstrated by measuring (i) fluorescence resonance energy transfer between N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine, after phosphatidylcholine liposomes were mixed with non-phospholipid liposomes, and (ii) reduced concentration quenching of rhodaminephosphatidylethanolamine and octadecylrhodamine incorporated into phosphatidylcholine liposomes after mixing with the non-phospholipid liposomes. The degree of apparent fusion reported by the different probe techniques ranged from 25% to 64%.

Anitroxide-sterol derivative potently modifies cholesterol biosynthesis by normal and neoplastic guinea pig lymphocytes.

Leukemic guinea pig lymphocytes (L2C) synthesise cholesterol in vitro at a forty-fold greater rate than normal cells. Equilibration (18 h) with lecithin or lecithin-cholesterol liposomes, respectively, enhances or suppresses sterol manufacture by normal lymphocytes but does not influence sterol production by L2C cells. In contrast, greater than 5-10(9) molecules/cell of a nitroxide-derivative of androstane, (17 beta-hydroxy-4',4'-dimethylspiro [5 alpha-androstan-3,2'-oxazolidin]-3'-yloxyl), commonly used as a membrane spin-probe, drastically inhibit sterol roduction by both normal and leukemic cells (maximum within 2 H). At less than 5-10(9) molecules/cell, this sterol stimulates cholesterol synthesis. 25-Hydroxycholesterol at low concentrations also stimulates sterol manufacture, whereas high concentrations are also inhibitory in both cell types.

Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes. Their use as parasite-specific markers.

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.

Modifications of LDL-receptor-mediated endocytosis rates in CEM lymphoblastic cells grown in lipoprotein-depleted fetal calf serum.

The efficiency of supplying cholesterol by the LDL endocytic pathway of lymphoblastic T CEM cells was compared when incubated in the presence of either fetal calf serum (FCS) or lipoprotein-depleted fetal calf serum (LDFCS). In the presence of FCS, there were 8600 +/- 2000 LDL receptors/cell with a Kd of (2.2 +/- 0.8).10(-8) M and a receptor cycling time of about 7 min; about 90% of the internalized LDL was degraded. LDL degradation produced 98% of total cellular cholesterol and only 2% came from endogenous synthesis. The absence of LDL in the culture medium of lymphoblastic CEM cells deeply modified certain metabolic and structural characteristics of the cells. Their cholesterol content decreased; the total number of LDL receptors increased 6-fold, whereas their affinity for the ligand decreased by the same factor (Kd = (1.2 +/- 0.2).10(-7) M); the receptor cycling time increased 3-fold. Finally, LDL degraded by cholesterol-depleted CEM cells amounted to about 40% of that degraded by untreated CEM cells.

Structural abnormality in LDL from diabetic patients as revealed by resonance Raman spectroscopy.

Resonance Raman spectra of low-density lipoprotein (LDL) isolated from the plasma of diabetic patients (age range 13-77 yr, mean age 37 yr) and age- and sex-matched control subjects were recorded in the 1000- to 1600-cm-1 region as a function of temperature (0-50 degrees C). Both nondiabetic and diabetic LDL yield spectra characterized by two major bands near 1160 and 1530 cm-1 due to the carotenoid component of lipoproteins. The relative intensity of 1530- and 1160-cm-1 bands, assigned to -C = C- and = C-C = stretchings, respectively, i.e., I1530-I1160 ratio, was plotted against temperature. For nondiabetic control subjects, the plots showed an inflection in the temperature range of 30-39 degrees C, which corresponded to the thermal transition of LDL. This transition was abolished in the LDL of diabetic patients (P less than 0.001), suggesting an altered lipid structure. The transition (30-39 degrees C) was also abolished in the in vitro glycosylated nondiabetic LDL. Lipid analysis did not show any appreciable change between nondiabetic control subjects and diabetic patients. The change in the thermal transition properties of diabetic LDL has been attributed to the organizational change in the LDL protein.

Comparison of the internalization efficiency of LDL and transferrin receptors on L2C guinea pig lymphocytes.

We demonstrate that L2C lymphocytes have about 10-times more receptors for transferrin (Tf) than healthy lymphocytes, as has been shown in the case of LDL receptors. The dissociation constant is the same in the two cell types (about 4 X 10(-7) M). In contrast to LDL, Tf enters L2C lymphocytes with very rapid kinetics. It is shown by cross-reaction that each receptor is internalized independently of the other.

The influence of coupling transferrin to liposomes or minibeads on its uptake and fate in leukemic L2C cells.

Coupling transferrin to liposomes or minibeads did not affect its uptake by L2C lymphocytes via the Tf specific receptors. The uptake kinetics of Tf conjugated with particles about 50 nm in diameter was as rapid as in the case of native Tf, and the receptors were recycled with a similar turnover time (about 15 min). Contrary to the generally accepted scheme, we found some Tf degradation provoked by cellular uptake. The degradation represented about 10% of the amount of ligand taken up by the cells. It occurred when transferrin was coupled to liposomes, but not when coupled to minibeads.

Effects of benzyl alcohol on transferrin and low density lipoprotein receptor mediated endocytosis in leukemic guinea pig B lymphocytes.

We demonstrated that benzyl alcohol, a neutral local anesthetic drug, inhibits the uptake and degradation of lowdensity lipoprotein and endocytosis of transferrin receptors of guinea pig leukemic B lymphocytes (L2C). This inhibition is very rapid, concentration dependant and reversible by simple washing. Membrane fluidity of the living cells is also modified.

Inhibition of the in vitro growth of Plasmodium falciparum by D vitamins and vitamin D-3 derivatives.

D vitamins are effective inhibitors of the in vitro intraerythrocytic growth of Plasmodium falciparum. Disappearance of the parasitemia was observed after 48 h contact between infected cells and 5 x 10(-6) M 1 alpha-hydroxycholecalciferol, 5 x 10(-5) M 25-hydroxycholecalciferol (25-OH-D-3), 1 alpha, 25-dihydroxycholecalciferol or 2.5 x 10(-4) vitamin D-2 and D-3. A 48 h pretreatment of healthy erythrocytes with 5 x 10(-5) M 25-OH-D-3 did not change their susceptibility to invasion by the parasite and their ability to support the growth of P. falciparum. Ionomycin, a calcium ionophore, and EGTA prevented parasite development at concentrations greater than 2 x 10(-7) M and 4 x 10(-4) M, respectively, but did not antagonize the inhibitory activity of 25-OH-D-3. Addition of 25-OH-D-3 for 12 or 24 h duration to synchronized cultures, showed that the drug had a schizonticidal action, but was without effect when parasites were in the ring form.

A reevaluation of the status of cholesterol in erythrocytes infected by Plasmodium knowlesi and P. falciparum.

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.

Differential effects of chloroquine on the phospholipid metabolism of Plasmodium-infected erythrocytes.

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.

Phospholipid metabolism as a new target for malaria chemotherapy. Mechanism of action of D-2-amino-1-butanol.

A number of choline and ethanolamine analogs were evaluated as inhibitors of P. falciparum growth in vitro. 1-Aziridineethanol, DL-2-amino-1,3-propranediol and D- or L-2-amino-1-butanol were the most efficient inhibitors of parasite multiplication, with an IC50 of 50-80 microM, whereas numerous other analogs were less active. The effect of D-2-amino-1-butanol on various metabolisms of P. knowlesi-infected simian erythrocytes was studied by incubating these cells with different labeled precursors of phospholipids, nucleic acids, proteins, and with radioactive glucose. In the presence of radioactive glycerol, oleate or lysophosphatidylcholine, the appearance of radioactivity in an unnatural phospholipid indicated that 2-aminobutanol was incorporated into a new PL which accounted for up to 30-40% of the total biosynthesized lipids. This new phospholipid accumulated primarily at the expense of PE biosynthesis and decreased the decarboxylation of phosphatidylserine. These effects were not accompanied, over a large range of concentrations, by any parallel change in nucleic or protein synthesis, nor in glucose metabolism. These data demonstrate that the incorporation of analogs, instead of the natural polar head groups, into cellular phospholipids, and/or modification of phospholipid composition have a deleterious impact on the growth of Plasmodium. It follows that PL metabolism is a crucial process for Plasmodium growth and may constitute a potentially fruitful chemotherapeutic approach to malaria.

Raman studies of structural rearrangements induced in human plasma lipoprotein carotenoids by malondialdehyde.

Raman and resonance Raman spectra of plasma lipoproteins +/- malondialdehyde were studied at concentrations which block the normal receptor-mediated uptake by cells. The strong resonance Raman bands at about 1010, 1162 and 1530 cm-1, due to the presence of carotenoids in the lipoproteins, are envisaged as structural probes. High resolution resonance Raman spectra of the 1500-1600 cm-1 region reveal multiple features suggesting the coexistence of several structural populations of beta-carotene whose precise assignment is complex. When plasma lipoproteins are reacted with malondialdehyde, a complex change occurs in the resonance Raman banding of beta-carotene in the 1500-1600 cm-1 region. Malonaldehyde (MDA) also modifies the acoustical region (70-200 cm-1 of low density lipoprotein (LDL) lipids. We suggest that malondialdehyde association with plasma lipoproteins alters the lipid structure via apoprotein or apoprotein/lipid associations.

Reevaluation, using marker enzymes, of the ability of saponin and ammonium chloride to free Plasmodium from infected erythrocytes.

Saponin and ammonium chloride lysis have been applied for some time to the separation of erythrocyte membranes from malarial-infected erythrocytes, allowing easy isolation of the parasites. We present a reevaluation of the use of saponin and ammonium chloride as tools for isolating Plasmodium (knowlesi or falciparum) parasites. Acetylcholine esterase (EC 3.1.1.7) was used as an erythrocyte membrane marker and CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) as a parasite membrane marker to monitor fractionation by these agents. Both saponin and ammonium chloride produced hemolysis of uninfected and infected erythrocytes, but failed to separate host erythrocyte membrane from the parasite, regardless of its stage. Thus, saponin and ammonium chloride can be used to isolate whole infected erythrocytes, depleted of hemoglobin, by selective disruption of uninfected cells.

Phospholipid biosynthesis by Plasmodium knowlesi-infected erythrocytes: the incorporation of phospohlipid precursors and the identification of previously undetected metabolic pathways.

Metabolic pathways leading to phospholipid biosynthesis in Plasmodium-infected simian erythrocytes were tested and quantified by incubating leucocyte-free erythrocytes in the presence of labelled precursors. Plasma fatty acids and lysophospholipids both served as sources of the fatty acids required for cellular phospholipid biosynthesis. However, the entry of free fatty acids and lysophospholipids appeared to be controlled by a competitive mechanism. A powerful deacylase-acylase system was detected, the nature and specificity of which remain to be defined. Glycerol-3-phosphate incorporation into cellular lipids accounted for most of the new phospholipid molecules formed in parasitized cells, and into cellular lipids accounted for most of the new phospholipid molecules formed in parasitized cells, and this compound, rather than the lysophospholipids, appeared to be the natural acceptor of the acyl groups. By incorporation of nitrogenous bases into cellular phospholipids, we identified significant pathways not previously detected in Plasmodium-infected erythrocytes: the formation of phosphatidylethanolamine by phosphatidylserine decarboxylation, and the formation of phosphatidylcholine by the methylation of phosphatidylethanolamine. These results, associated with the absence of lipid synthesis in host cells, mean that the enzymes controlling these two pathways could serve as enzymatic markers of parasites.

Encapsulation of drugs into large unilamellar liposomes prepared by an extemporaneous method.

The method we developed for extemporaneous preparation of large unilamellar liposomes (Philippot et al. 1984) was applied to the encapsulation of 12 different antibiotic, anti-asthmatic and anti-inflammatory drugs. The behaviour of these drugs, during encapsulation, assigns them to one of the three classes: hydrophilic, hydrophobic or both. Alone, the first type of compound gave an appreciable encapsulation. However, the entrapment yield depended on the respective charges of the liposome and the drug. The amphiphile molecules tested are permeant and thus did not stay inside the liposomes. Only one of the hydrophobic drugs analysed associated with the liposome membrane lipids with a good yield.