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%.

Adjuvant properties of non-phospholipid liposomes (Novasomes) in experimental animals for human vaccine antigens.

Non-phospholipid liposomes composed of dioxyethylene cetyl ether, cholesterol and oleic acid were evaluated as adjuvants with human vaccine antigens, tetanus toxoid (TT) and diphtheria toxoid (DT), in mice and rabbits. Antigens encapsulated in or mixed with liposomes elicited antitoxin levels similar to those elicited by antigens given with Freund's adjuvant or adsorbed onto aluminum phosphate. All liposomal antigen preparations, antigen given with Freund's adjuvant or adsorbed onto aluminum phosphate, elicited significantly higher IgG antibodies and antitoxin levels than soluble antigens in mice after a single injection and in rabbits after each of three injections. TT encapsulated in liposomes elicited sustained anti-TT IgG antibody levels in mice after a single injection as compared to TT mixed with liposomes. TT mixed with or encapsulated within liposomes containing monophosphoryl lipid A/squalene or squalene alone, as well as aluminum phosphate adsorbed TT elicited greater primary responses in mice than TT mixed with or encapsulated within plain liposomes. Liposomal TT preparations produced a slightly higher anamnestic response in mice than aluminum phosphate adsorbed TT. Subclass analysis of anti-TT antibodies showed that the majority of the antibodies belong to IgG1 subclass. Liposomal TT preparations, particularly those with encapsulated monophosphoryl lipid A/squalene or squalene alone, consistently elicited higher levels of anti-TT IgG2a and IgG2b than aluminum phosphate adsorbed or soluble TT. None of the preparations elicited IgG3 or IgM antibodies. It appears that non-phospholipid liposomes are as potent adjuvants as the currently employed adjuvant for human vaccines (aluminum phosphate) or a benchmark adjuvant for experimental immunology (Freund's adjuvant), and may be able to modulate the immune response towards the Th1 type.

Encapsulation of hemoglobin in non-phospholipid vesicles.

The efficiency of encapsulating hemoglobin in non-phospholipid liposomes by rapidly mixing hemoglobin with lipids heated above their solid-liquid phase transition temperature was examined. Human hemoglobin was mixed at 55-60 degrees C with a lipid solution containing polyoxyethylene-2 cetyl ether and cholesterol (molar ratio, 3:1) at 60-65 degrees C. Repeated mixing was carried out through a high-shear orifice, followed by rapid cooling and additional mixing. Lipid vesicles were heterogeneous in size, with diameters from approximately 300 nm to 10 microns. The non-encapsulated aqueous phase was removed by centrifugation, and total hemoglobin was determined spectrophotometrically. Encapsulation efficiency was calculated as the percentage of hemoglobin associated with the liposome phase (i.e., encapsulated) as a function of hemoglobin concentration and the aqueous:lipid hydration ratio. Hemoglobin concentrations were varied from 1 to 10 nM (in heme). Aqueous:lipid ratios of 8:1 and 4:1 were tested. Percent encapsulation varied from 13-30%, with the greatest efficiency, i.e., 30%, at a 4:1 hydration ratio of hemoglobin:lipid at 5.6 mM hemoglobin.

Synthesis of a somatostatin-like peptide by Plasmodium falciparum.

The intracellular parasite, Plasmodium falciparum, was found to synthesize a peptide similar to mammalian somatostatin. High performance liquid chromatography of acetic acid extracts of Plasmodium-infected erythrocytes revealed a metabolically labeled peptide that co-eluted with rat somatostatin and that was reactive with antibody against rat somatostatin. Bioassay of partially purified Plasmodium peptide demonstrated somatostatin activity. Acetic acid extracts from non-synchronized infected cultures were shown by radioimmunoassay to contain the equivalent of 150 molecules of somatostatin per parasite. Somatostatin was not detectable in erythrocytes of non-infected cultures.

Membrane potential of erythrocytic stages of Plasmodium chabaudi free of the host cell membrane.

Free parasites were isolated from Plasmodium chabaudi-infected rat erythrocytes by N2-cavitation and purified on Percoll gradients. The membrane potential of the free parasites determined from the transmembrane distribution of the lipophilic cation, tetraphenylphosphonium, was -93 +/- 10 mV for late stage parasites and -90 +/- 3 mV for ring forms. Studies with intact infected erythrocytes demonstrated that the membrane potential of ring forms was much smaller compared to late trophozoites and schizonts and thus the present findings with free parasites suggest that host cell cytoplasmic factors may determine the magnitude of the parasite membrane potential. Both extracellular pH and [Na+] were found to modify the membrane potential of free parasites. Electrogenic protonophores, the H+-ATPase inhibitor dicyclohexylcarbodiimide and orthovanadate collapsed the potential of free parasites. Ouabain (or its membrane permeant derivative, strophanthidin), and oligomycin were without effect. These inhibitor studies suggest that an electrogenic H+-ATPase similar to that found in yeast generates in part the membrane potential of malaria parasites. Using weak acid distribution or a pH sensitive fluorescent dye, it was demonstrated that free parasites maintain an alkaline intracellular pH at extracellular pH greater than 6.5. The pH gradient was partially collapsed by orthovanadate or dicyclohexylcarbodiimide and by substitution of Na+ for K+ in the suspending buffer. The H+-ATPase and K+:H+ exchange may therefore both contribute to regulation of intracellular pH in Plasmodium.

Membrane orientation and antigenic peptides of an immunoprotective 74 kDa Plasmodium knowlesi glycoprotein.

Vaccination trials have shown that a purified, 74 kDa glycoprotein, GP74, isolated from the host cell membrane of Plasmodium knowlesi-infected rhesus erythrocytes, can provide protective immunity against P. knowlesi malaria. We have extended this work by a tryptic peptide analysis of the disposition of GP74 in the host cell membrane. Of the 18 peptides characterized by high-performance liquid chromatography only four were accessible to lactoperoxidase-catalyzed radioiodination of non-leaky, schizont-infected host cells from the extracellular space. Metabolic labeling with radioactive glucosamine indicates that two of the surface exposed peptides are glycopeptides, and one of these, peptide 12 appears to carry a dominant antigenic site, according to its reactivity with immunoglobulin from sera of monkeys protected against P. knowlesi malaria.

Receptors for the malarial parasite.

During the erythrocytic cycle of Plasmodium, the parasite develops within an enclosed space, the parasitophorous vacuole, formed by endocytosis of an invasive stage, the merozoite. Among the erythrocyte membrane proteins possibly acting as a receptor for the attachment of P. falciparum merozoites to human erythrocytes is glycophorin A. Isolated glycophorin inhibits merozoite entry in a competitive manner, perhaps via association with a 155 kDa surface protein. Another protein that competitively inhibits merozoite invasion, is band 3, the erythrocyte anion transport protein. The protein bearing Duffy blood group antigens may act to modulate invasion, but does not behave as a receptor.

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.

Extracellular development of Plasmodium knowlesi erythrocytic stages in an artificial intracellular medium.

The development of erythrocytic stages of Plasmodium knowlesi separated from their host cells has been determined in terms of the capacity of the isolated organisms to carry out the synthesis and secretion of proteins. P. knowlesi trophozoites and schizonts were released from host cells by nitrogen decompression and cultivated in a medium consisting of 20 mM Na+; 120 mM K+; 1 mM Mg2+; no Ca2+; 100 mM Cl-; 20 mM HCO3-; 5 mM Hepes [pH 6.73], glucose, vitamins, amino acids and 10% fetal calf serum. The yield was about 97% intact parasites, judging by their ability to maintain a membrane potential, and these parasites had more than 80% the capacity of infected cells for nuclear replication and macromolecule biosynthesis. Pulse and pulse-chase labeling studies with [35S]methionine show that parasite-synthesized proteins with Mr 160 000, 140 000, 100 000 and 58 000 are exported from the parasite in soluble form. Proteins with Mr 140 000, 100 000, 58 000-60 000, 40 000 were recovered in a particulate fraction isolated from the parasite culture fluid. An Mr 62 000 protein synthesized in large amounts by isolated parasites during the last 2h of the developmental cycle, could not be detected in infected erythrocytes, and a minor early Mr 74 000 protein becomes prominent in free parasites but not infected cells toward the end of the developmental cycle. Parasite-synthesized proteins with Mr 230 000, 160 000, 140 000, 62 000, 58 000 and 45 000 were labeled by incubation with radioactive N-acetylglucosamine during short term incubation in vitro. About 80% of label incorporation occurred via N-glycosylation supported by dolichol derived from the blood, and about 20% via glycolytic intermediates.

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.

Resonance Raman spectra of beta-carotene in native and modified low-density lipoprotein.

Low-density lipoproteins isolated between density 1.02 and 1.063 g/cm3 from normal fasting human plasma, show strong resonance Raman spectra due to the presence of beta-carotene. Three intense bands, at 1010, 1160 and 1530 cm-1, are assigned to the stretching vibrations of -C-CH3, = C-C = and -C = C- bonds, respectively, of beta-carotene. High-resolution spectra of the 1500-1600 cm-1 region reveal multiple features, suggesting the coexistence of several structural populations of beta-carotene. The modifications of lipoproteins with pH and temperature (30 degrees-42 degrees) change the resonance Raman spectra of beta-carotene. The specific binding of LDL at pH 7.0 by fibroblast cells is suppressed. Our experiments thus suggest that physical and chemical perturbations of plasma lipoproteins modify the lipid-protein interactions and thereby alter the configurational distribution of beta-carotene molecules within these particles.

Chain length dependent modification of lipid organization by low levels of 25-hydroxycholesterol and 25-hydroxycholecalciferol. A laser Raman study.

We have used Raman spectroscopy to investigate the thermal transitions of multibilayered liposomes composed of lecithins, i.e., dilauroyllecithin, dimyristoyllecithin, dipalmitoyllecithin, distearoyllecithin, or egg lecithin, plus 5-cholesten-3,25-diol (25-hydroxycholesterol), 25-hydroxycholecalciferol, and vitamin D3. We recorded the CH-stretching (2800-3000-cm-1) regions of the Raman spectra at various temperatures and employed plots of temperature vs. the intensity of the 2880- or 2930-cm-1 bands relative to that of the 2850-cm-1 feature, i.e., the ratios I2880/I2850 and I2930/I2850, to estimate thermal transitions. These plots show multiple discontinuities, each of which may be ascribed to a state change of a separate phase with distinctive proportions of lecithins and cholesterol derivatives. Low concentrations of 25-hydroxycholesterol and 25-OH-D3 (greater than or equal to 0.2 mol%) abolish the pretransition and split the main transitions of dilauroyllecithin (4 degrees C) and dimyristoyllecithin (23 degrees C) into two. The midpoint of the new small transition centers at about 3-4 degrees C lower than those of the respective main transitions of dilauroyllecithin and dimyristoyllecithin. A further increase in the molar ratio of 25-hydroxycholesterol and 25-hydroxycholecalciferol decreases the amplitudes of the new and the main transitions (dilauroyl- and dimyristoyllecithin). The transitions of dipalmitoyllecithin and distearoyllecithin at 2 mol % concentrations of either sterol remain unaffected. There was no splitting in the main transition of either dipalmitoyllecithin or distearoyllecithin in the presence of these sterols. The perturbing effect of the 25-hydroxysterols follows the order dilauroyllecithin greater than dimyristoyllecithin greater than dipalmitoyllecithin greater than distearoyllecithin.(ABSTRACT TRUNCATED AT 250 WORDS)

A protein anomaly in erythrocyte membranes of patients with Duchenne muscular dystrophy.

Raman spectroscopic comparisons of erythrocyte membranes from 20 patients with Duchenne muscular dystrophy and 8 age-matched controls indicate a prominent and consistent protein anomaly in the patient samples. This was apparent in the following: (a) CH-stretching signals from control membranes reveal a thermotropic transition at 15.6 degrees C, attributable to a protein/lipid phase that is lacking in dystrophic membranes. (b) CH-stretching signals from control membranes also show a protein transition at 39 degrees C [pH 7.4] that is shifted to 45 degrees in dystrophic membranes. (c) A reduction in pH to 5.7 shifts this transition from 39 degrees C to 7 degrees C in normal membranes and from 45 degrees C to 24 degrees C in dystrophic membranes. (d) The Amide I/Amide III regions indicate a significant proportion of beta-structured peptide in dystrophic but not normal membranes. (e) Analysis of tyrosine signals indicates greater polar exposure of tyrosine hydroxyl groups in dystrophic vs normal membranes. All of the differences between dystrophic and normal membranes are highly significant (P less than 0.001).

Transport of ions in erythrocytes infected by plasmodia.

Throughout its erythrocytic cycle the plasmodial parasite modifies the plasma membrane of its host cell. Some changes derive from parasite metabolism. Intraerythrocytic forms use glucose at more than 10-fold normal red cell rates. The H+ accompanying the lactate end-product is exported into the host cell cytoplasm by an electrogenic proton pump in the parasite membrane. This maintains a pH greater than 7.0 in the parasite cytoplasm, but lowers erythrocyte cytoplasmic pH from approximately 7.2 to 6.5. Ca2+ transport across parasite membranes is coupled to the proton pump, possibly a Ca2+/H+ antiporter. The Ca2+, Mg2+-ATPase and Na+,K+-ATPase activities of erythrocyte membranes from schizont-infected erythrocytes have been studied. Under optimal assay conditions (pH = 7.0; [ATP] = 1 mM; +/- calmodulin) membranes from infected cells showed a 30% reduction in Ca2+,Mg2+-ATPase activity but no difference from normal in Na+,K+-ATPase activity. The calmodulin levels of infected cells were depressed by about 30%. The [ATP] in the cytoplasm of infected erythrocytes was only 0.2 mM (as against 1.3 mM in normals) and at this ATP concentration the activities of both ATPases are only 30% of normal. Shifting the pH from 7.0 to 6.5 decreases Na+,K+-ATPase activity by an additional 50% but is without effect on the Ca2+,Mg2+-ATPase. The results provide a partial explanation for the increased Ca2+ permeability and altered Na+/K+ content of plasmodia-infected erythrocytes.

Simian virus 40 (SV40)-specific isoelectric point-4.7--94,000-Mr membrane glycoprotein: major peptide homology exhibited with the nuclear and membrane-associated 94,000-Mr SV40 T-antigen in hamsters.

Tryptic peptide maps of electrophoretically purified 94,000-molecular weight (relative) (Mr) nuclear and membrane-associated simian virus 40 (SV40) T-antigens, TN and TM, respectively, were compared to those of the SV40-specific isoelectric point (pI)- 4.7--94,000-Mr plasma membrane component reactive with anti-T-sera from Syrian golden hamsters. Bidimensional thin-layer electrophoresis and chromatography of TN labeled with 125I revealed about 27 tryptic peptides. A similar number of peptides was identified for TM and the pI-4.7--94,000-Mr component. A peptide homology between TN and TM or TN and the pI-4.7-94,000-Mr protein exists and indicates that the previously described pI-4.7--94,000-Mr membrane component represents TM. Only 4 of 27 peptides were labeled when TM was subjected to lactoperoxidase-catalyzed radioiodination from the outer surface of the plasma membrane. One of these TM peptides was metabolically labeled with [14C]glucosamine. The data indicate that TM is partially exposed on the cell surface and represents a glycosylated form of TN. Closely associated with TM is a pI-4.5--55,000-Mr membrane component. This component does not exhibit significant peptide homology with the 94,000-Mr SV40 protein and, therefore, appears to be coded for by the host cell genome.

Calcium transport of Plasmodium chabaudi-infected erythrocytes.

The calcium content and transport processes of Plasmodium chabaudi-infected rat erythrocytes were analyzed by atomic absorption spectroscopy and 45Ca2+ flux measurements. Infected erythrocytes, after fractionation on metrizamide gradients according to stage of parasite development, exhibited progressively increasing levels of Ca2+ with schizont and gametocytes containing 10- to 20-fold greater calcium levels than normal cells (0.54 +/- 0.25 nmol/10(8) cells). 45Ca2+ flux experiments showed both increased influx and decreased efflux in infected erythrocytes. Tris/NH4Cl lysis of normal erythrocytes preloaded with 45Ca2+ with the Ca2+ ionophore A23187 released less than 90% of cell calcium after incubation in ethyleneglycol bis(aminoethylether) N,N'-tetraacetic acid containing buffer, whereas lysis of the infected erythrocyte membrane resulted in release of 10-20% cell Ca2+, with the remaining portion associated with the isolated parasite fraction. This information together with the effects of various metabolic inhibitors indicates the presence of a parasite Ca2+ compartment in P. chabaudi-infected erythrocytes. Dicyclohexylcarbodiimide (DCCD) an inhibitor of proton ATPases of chloroplasts, bacteria, yeast, and mitochondria, and the proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), inhibited Ca2+ influx and stimulated efflux from infected cells. These results combined with evidence for a DCCD- and CCCP-sensitive membrane potential in P. chabaudi-infected cells (Mikkelsen et al., accompanying manuscript) suggest that Ca2+ transport of intraerythrocytic parasites is coupled to a proton-motive force across the Plasmodia plasma membrane.

Membrane potential of Plasmodium-infected erythrocytes.

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.

Immunogenic antigens common to Plasmodium knowlesi and Plasmodium falciparum are expressed on the surface of infected erythrocytes.

Sera of Gambian individuals and rhesus monkeys immune against infections with Plasmodium falciparum and plasmodium knowlesi, respectively, were reacted with triton X-100-solubilized membranes of infected erythrocytes. Indirect immune precipitation with Staphylococcus aureus, Cowan strain A, followed by dodecylsulfate-polyacrylamide gel electrophoresis, were used to identify interspecies plasmodial antigens that were immunogenic in vivo. Both types of sera specifically precipitated Plasmodium-specific antigens with Mrs of 125,000, 90,000, and 65,000 to 50,000 from membranes of P. knowlesi-infected erythrocytes that had been labeled with 125I using the lactoperoxidase-catalyzed radioiodination or metabolically with 14C-amino acids. In addition, P. falciparum inhibited the precipitation of P. knowlesi antigens by the Gambian immune sera. Our results indicate, that during erythrocytic schizogony, interspecies Plasmodium antigens are exposed on the surfaces of infected erythrocytes.