Effect of procaine on the loss of erythrocyte phospholipids during blood preservation in ACD-AG-stabilizer]

The phospholipid changes of erythrocytes were investigated following the storage under blood bank conditions up to 42 days (ACD-AG-stabilisator) without and with addition of procaine (25 mM). The phospholipid loss of erythrocytes is described as two-step occurrence. The rapid decrease in the first three days is followed by a further loss up to the end of the storage period. The concomitant increase of phospholipid fractions of supernatant shows that the erythrocyte membranes lose their phospholipids mainly by a passive way. For the further loss of phospholipids the action of a phospholipase A is adopted. The addition of procaine hinders strongly the phospholipid loss of erythrocytes. The clinical importance of these procaine action is shortly discussed.     

Effect of Intralipid-infusion on erythrocyte membrane in rabbits

Through Intralipid infusion in rabbits, the phospholipids derived from Intralipid were incorporated into erythrocytes, although Intralipid is mainly composed of triglycerides. This is supported by the increase in oleic acid and the compensatory decrease in linoleic acid of the phospholipids in the erythrocyte membrane, corresponding to the content of linoleic acid in the phospholipids from Intralipid. The excess phospholipid rendered the membrane more fluid, probably by overwhelming the rigidifying effect of the increased cholesterol content. Furthermore, the shape of erythrocytes was changed from biconcave to spur, dose dependently. The morphological alterations in erythrocyte membranes could not be completely elucidated by the changes in lipid. These results suggested that the alteration in lipid metabolism in Intralipid-infused rabbits caused various effects on the erythrocyte membrane, through the elevation of triglyceride, cholesterol, and phospholipid contents in plasma.     

Schistosoma mansoni: L-lysil residues in agglutination of mouse erythrocytes by acid phospholipids of parasite membranes

The agglutination of mouse erythrocytes caused by acid phospholipids of Schistosoma mansoni membranes is strongly inhibited by l-lysine; other amino acids do not interfere with the reaction. The inhibitory effect observed after treatment of mouse erythrocytes with low concentrations of glutaraldehyde confirms that l-lysil residues are present on the surface of the erythrocytes and that, probably, protonated ?-amino groups are responsible for the susceptibility of these cells to the agglutination by worm membranes in physiological conditions. There is a possible role for lysil-phospholipid interactions in the host-parasite relationships.     

Molecular Factors Responsible for Host Cell Recognition and Invasion in Plasmodium falciparum1

ABSTRACT. In Plasmodium falciparum. the rhoptries involved in the invasion process are a pair of flask-shaped organelles located at the apical tip of invading stages. They, along with the more numerous micronemes and dense granules, constitute the apical complex in Plasmodium and other members of the phylum Apicomplexa. Several proteins of varying molecular weight have been identified in P. falciparum rhoptries. These include the 225-, 140/130/110-, 80/60/40-, RAP-1 80-, AMA-1 80-, QF3 80-, and 55-kDa proteins. Some of these proteins are lost during schizont rupture and release of merozoites. Others such as the 140/130/110-kDa complex are transferred to the erythrocyte membrane during invasion. The ring-infected surface antigen (RESA). a 155-kDa polypeptide located in dense granules also associates with the erythrocyte membrane during invasion. Erythrocyte-binding studies have demonstrated that both the 140/130/110-kDa rhoptry complex and RESA bind to inside-out-vesicles (IOVs) prepared from human erythrocytes. The 140/130/110-kDa complex also binds to erythrocyte membranes prepared by hypotonic lysis. These proteins, however, do not bind to intact human erythrocytes. In a heterologous erythrocyte model, both the 140/130/110-kDa complex and RESA are shown to bind directly to mouse erythrocytes. Other studies have shown that RESA associates with spectrin in the erythrocyte cytoskeleton. We have recently developed a liposome-binding assay to demonstrate the lipophilic binding properties of the P. falciparum rhoptry complex of 140/130/110 kDa. The rhoptry complex binds to liposomes containing neutrally, positively, and negatively charged phospholipids. However, liposomes containing phosphatidylethanolamine compete effectively for rhoptry protein binding to mouse erythrocytes. The rhoptry complex also binds to membrane and inside-out-vesicles prepared from human erythrocytes and erythrocytes from other species. The rhoptry complex associated with the erythrocyte membrane in ring-infected erythrocytes is accessible to cleavage by phospholipase A. Studies are in progress to identify the molecular epitopes on the individual proteins within the complex responsible for lipid interaction in the erythrocyte bilayer and to determine the specificity of the phospholipid interaction using erythrocyte phospholipids.     

Survival of rabbit and horse erythrocytes in vivo after changing the fatty acyl composition of their phosphatidylcholine

The phospholipid composition and the distribution of phospholipids over the two leaflets of the membrane have been investigated for rabbit and horse erythrocyte membranes. Phosphatidylcholine (PC) comprises 39.4% and 41.3% of the total phospholipid complement of the rabbit and horse erythrocytes, respectively. In both membranes the distribution of this phospholipid is asymmetric: 70% of the PC is present in the outer layer of the rabbit membrane and 60% in that of the horse. The major species of this phospholipid class are the (1-palmitoyl-2-oleoyl)- and the (1-palmitoyl-2-linoleoyl)PC. The disaturated species, (1,2-dipalmitoyl)PC, is present in limited amounts only. Partial replacement of the native PC from intact erythrocytes was accomplished with a purified PC specific transfer protein from bovine liver. Replacement of the native PC species with (1-palmitoyl-2-oleoyl)PC up to 40% of the total PC complement had no effect on the osmotic fragility, the shape and the in vivo survival time of both erythrocyte species. Replacement of the native PC in both rabbit and horse erythrocytes with (1,2-dipalmitoyl)PC up to 20% gave rise to an increased osmotic fragility, a shape change from discocytic to echinocytic and a significant reduction in survival time measured after reinjection of the modified cells. At 30% replacement with (1,2-dipalmitoyl)PC the resulting spheroechinocytes appeared to be cleared from the circulation within 24 h after reinjection. The conclusion can be drawn that the repair mechanisms which may exist in vivo are insufficient to cope with the drastic changes in properties of the erythrocyte membrane which are induced by replacing more than 15% of the native PC by the dipalmitoyl species.     

Schistosoma mansoni: a comparative study of plasma and erythrocyte lipid alterations in the experimentally infected mouse and in selected human patients

Alterations of plasma and erythrocyte lipids associated with hepatosplenic schistosomiasis mansoni were studied in the mouse and in human patients. Qualitative and quantitative differences were observed between the two species which indicated that the experimentally infected mouse should not be used as a model for altered lipid metabolism associated with Schistosoma mansoni infections in man. Also blood lipid values should not be used as prophylactic indicators for experimental therapeutical studies in the infected mouse, although lipid determinations could have clinical value in studies of human patients. In infected mice plasma cholesterol and phospholipid were significantly reduced (40 and 25%, respectively), but proportions of individual plasma phospholipids were unchanged. In contrast, only plasma cholesterol was reduced in human patients with compensated or decompensated hepatosplenic schistosomiasis (16 and 29%, respectively); of the individual phospholipids, lecithin was significantly increased and lysolecithin was decreased. The percentage of plasma total cholesterol was reduced in infected mice and patients suggesting that hypocholesterolemia is due mainly to decreased cholesteryl ester. Lipid changes also occurred in erythrocytes. Those of infected mice had significantly elevated membrane phospholipid content and no changes in cholesterol or in the proportions of the individual phospholipid fractions. In marked contrast, the erythrocytes of two groups of human patients had significantly higher levels of cholesterol without a raised total phospholipid concentration. Moreover, decreased proportions of lysolecithin and increased proportions of lecithin were apparent although only the increased membrane lecithin associated with compensated patients was statistically significant.     

Interaction of the 140/130/110 kDa rhoptry protein complex of Plasmodium falciparum with the erythrocyte membrane and liposomes

During Plasmodium falciparum merozoite invasion into human and mouse erythrocytes, a 110-kDa rhoptry protein is secreted from the organelle into the erythrocyte membrane. In the present study our interest was to examine the interaction of rhoptry proteins of P. falciparum with the erythrocyte membrane. It was observed that the complex of rhoptry proteins of 140/130/110 kDa bind directly to a trypsin sensitive site on intact mouse erythrocytes, and not human, saimiri, or other erythrocytes. However, when erythrocytes were disrupted by hypotonic lysis, rhoptry proteins of 140/130/110 kDa were found to bind to membranes and inside-out vesicles prepared from human, mouse, saimiri, rhesus, rat, and rabbit erythrocytes. A binding site on the cytoplasmic face of the erythrocyte membrane suggests that the rhoptry proteins may be translocated across the lipid bilayer during merozoite invasion. Furthermore, pretreatment of human erythrocytes with a specific peptide derived from MSA-1, the major P. falciparum merozoite surface antigen of MW 190,000-200,000, induced binding of the 140/130/110-kDa complex. The rhoptry proteins bound equally to normal human erythrocytes and erythrocytes treated with neuraminidase, trypsin, and chymotrypsin indicating the binding site was independent of glycophorin and other major surface proteins. The rhoptry protein complex also bound specifically to liposomes prepared from different types of phospholipids. Liposomes containing PE effectively block binding of the rhoptry proteins to mouse cells, suggesting that there are two binding sites on the mouse membrane for the 140/130/110-kDa complex, one protein and a second, possibly lipid in nature. The results of this study suggest that the 140/130/110 kDa protein complex may interact directly with sites in the lipid bilayer of the erythrocyte membrane.     

Membrane phospholipid asymmetry as a factor in erythrocyte-endothelial cell interactions

The tendency of human erythrocytes to adhere to vascular endothelial cells was assessed as a function of the transbilayer distribution of the phospholipids of the erythrocyte membrane, using erythrocyte ghosts in which transbilayer lipid arrangement was manipulated by varying the conditions under which the ghosts were prepared. By two different assays, ghosts with symmetric lipid bilayers adhered strongly to monolayers of cultured endothelial cells, whereas ghosts with normal asymmetric membranes, like normal erythrocytes, did not. These results provide direct evidence that changes in phospholipid asymmetry can alter the tendency of erythrocytes to adhere to endothelial cells, and therefore imply that transbilayer phospholipid arrangement may influence the behavior of erythrocytes in the circulatory system and may contribute to the formation of microvascular occlusions.     

An early childhood erythrocyte phospholipid distribution as further indication of persistence of neonatal erythrocyte characteristics in Diamond-Blackfan anemia

Comparisons of erythrocyte and plasma phospholipids made among adults, newborns, and a female patient with Diamond-Blackfan anaemia (DBA) revealed some indications for the continual existence of a neonatal phospholipid distribution in DBA. The relative percentage of phospholipids in erythrocytes and plasma were similar in newborns and in the female patient. The other peculiarities characteristic of newborns, such as deviations in the absolute phospholipid content, typical fatty acid patterns of phospholipids, could not be identified in DBA.     

Comparative analysis of phospholipid composition in blood erythrocytes of various species of mouse-like rodents

Comparative analysis of quantitative phospholipid composition of blood erythrocytes was performed on various species of mouse-like rodents. In erythrocyte phospholipids of common voles there was revealed a relatively low sphingomyelin content, not characteristic of mammals. A hypothesis is put forwards that the unique lipid composition in common vole erythrocytes might be one of adaptation mechanisms for survival of the animals under conditions of pathogenic stress.     

Effect of oxidative stress on membrane phospholipid and protein organization in human erythrocytes

Membrane phospholipid and protein organization was studied in intact human erythrocytes exposed to phenylhydrazine, an oxidative agent inducer. The evaluation of the membrane phospholipid and protein organization was carried out in terms of asymmetric distribution across the membrane bilayer for the phospholipids, and in terms of accessibility of cleavable sites present on the outer membrane surface for the proteins. Treatment of phenylhydrazine-exposed erythrocytes either with bee venom phospholipase A2 or with trinitrobenzenesulfonic acid indicated that phosphatidylserine (PS), which is the only phospholipid not formally present on the outer leaflet of the membrane, was translocated to the outer surface of the cell membrane. The extent of this phenomenon was directly proportional to the concentration of the oxidant having a peak value at 0.1 mM. Phosphatidylcholine and phosphatidylethanolamine conserved their original distribution across the erythrocyte membrane throughout the study. The oxidant, at a dose which did not induce any modification of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis cytoskeleton membrane protein pattern, did not provoke any alteration of the membrane protein surface architecture, although the translocation of PS to the membrane outer leaflet in intact erythrocytes was present.     

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.     

四种蕈菌凝集素的筛选及活性检测

以长刺猴、白平菇、毛尖蘑、滑菇四种蕈菌为材料,经硫酸铵沉淀、透析,得到蛋白质提取液,用不同类型的红细胞检测凝集活性(人的A型、B型、AB型、O型血,兔血、鸡血、蛤蟆血)。结果表明,长刺猴、白平菇、滑菇3种蕈菌的提取物中均含有凝集素,桦树蘑对兔红细胞凝集性最强。凝集活性可分别被一种或多种类型糖所抑制。同时它们均表现出较好的热稳定性及pH耐受性,金属离子对凝集素的影响也相当大。     

Role of phospholipids in the DDT-induced efflux of potassium in human erythrocytes

Incubation of human erythrocytes for 1–2 h at 37°C in a suspension of dipalmitoylphosphatidylcholine (DPPC) liposomes results in a phospholipid enrichment of erythrocyte membranes by 45–55% and a depletion of cholesterol by 19–24%. The enrichment by DPPC was time and concentration dependent. By contrast, dioleoylphosphatidylcholine (DOPC) liposomes were less effective in enriching the membranes with phospholipid and in depleting the membranes of cholesterol. Concomitantly, the DDT-induced efflux of K⁺ was reduced in the case of DPPC-enriched erythrocytes but enhanced in DOPC-enriched erythrocytes. These results suggest that DDT partitions more readily into the unsaturated than the saturated phospholipids of the erythrocyte membrane. It is concluded that the extent to which DDT affects the flux of K⁺ across the membrane is dependent on the fluidity of the lipid phase. We also report here a rapid method for cholesterol depletion of red blood cells in comparison to previously reported methods.     

Bilirubin induces loss of membrane lipids and exposure of phosphatidylserine in human erythrocytes

Unconjugated bilirubin increasingly binds to erythrocytes as the bilirubin-to-albumin molar ratio exceeds unity, leading to toxic manifestations that can culminate in cell lysis. Our previous studies showed that bilirubin induces the release of lipids from erythrocyte membranes. In the present work, those studies were extended in order to characterize the alterations of membrane lipid composition and evaluate whether bilirubin leads to a loss of phospholipid asymmetry. To this end, human erythrocytes were incubated with several bilirubin-to-albumin molar ratios (0.5 to 5), and cholesterol as well as the total and the individual classes of phospholipids were determined. To detect erythrocytes with phosphatidylserine at the outer surface, the number of annexin V-positive cells was determined following incubation with bilirubin, fixing its molar ratio to albumin at 3. The results demonstrate profound changes in erythrocyte membrane composition, including modified cholesterol and phospholipid content. The release of membrane cholesterol, as well as of total and individual classes of phospholipids at molar ratios ≥1, indicates that damage of erythrocytes may occur in severely ill jaundiced neonates. The loss of the inner-located phospholipids, phosphatidylethanolamine and phosphatidylserine, points to a redistribution of phospholipids in the membrane bilayer. This was confirmed by the exposure of phosphatidylserine at the outer cell surface. In conclusion, this study demonstrates that bilirubin induces loss of membrane lipids and externalization of phosphatidylserine in human erythrocytes. These features may facilitate hemolysis and erythrophagocytosis, thus contributing to enhanced bilirubin production and anemia during severe neonatal hyperbilirubinemia. This revised version was published online in July 2006 with corrections to the Cover Date.     

Steroid-derived phospholipid scramblases induce exposure of phosphatidylserine on the surface of red blood cells

A series of methyl 7alpha,12alpha-bis(phenylurea) cholate derivatives with different cationic substituents at the 3alpha-position were prepared and evaluated for an ability to increase the level of endogenous phosphatidylserine (PS) on the surface of red blood cells (erythrocytes). Some of the compounds induced large fractions of erythrocytes to expose sufficient PS to become stained by the protein annexin V-FITC. In addition, the compounds were found to bind PS in homogeneous solution, and to promote the translocation of fluorescent NBD-labeled phospholipids across vesicle membranes, which supports the hypothesis that cholate-induced exposure of endogenous PS on the erythrocyte surface is due to the ability of the cationic cholates to promote anionic phospholipid flip-flop.     

Identification of a Plasmodium falciparum Phospholipid Transfer Protein

Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.     

The cholesterol content of the erythrocyte membrane is an important determinant of phosphatidylserine exposure

Maintenance of the asymmetric distribution of phospholipids across the plasma membrane is a prerequisite for the survival of erythrocytes. Various stimuli have been shown to induce scrambling of phospholipids and thereby exposure of phosphatidylserine (PS). In two types of patients, both with aberrant plasma cholesterol levels, we observed an aberrant PS exposure in erythrocytes upon stimulation. We investigated the effect of high and low levels of cholesterol on the ATP-dependent flippase, which maintains phospholipid asymmetry, and the ATP-independent scrambling activity, which breaks down phospholipid asymmetry. We analyzed erythrocytes of a patient with spur cell anemia, characterized by elevated plasma cholesterol, and the erythrocytes of Tangier disease patients with very low levels of plasma cholesterol. In normal erythrocytes, loaded with cholesterol or depleted of cholesterol in vitro, the same analyses were performed. Changes in the cholesterol/phospholipid ratio of erythrocytes had marked effects on PS exposure upon cell activation. Excess cholesterol profoundly inhibited PS exposure, whereas cholesterol depletion led to increased PS exposure. The activity of the ATP‐dependent flippase was not changed, suggesting a major influence of cholesterol on the outward translocation of PS. The effects of cholesterol were not accompanied by eminent changes in cytoskeletal and membrane proteins. These findings emphasize the importance of cholesterol exchange between circulating plasma and the erythrocyte membrane as determinant for phosphatidylserine exposure in erythrocytes.     

Comparative analysis of phospholipid composition in erythrocytes of mouse-like rodents of different species

Comparative analysis of phospholipid quantitative composition of blood erythrocytes has been performed in white (laboratory mice and rats) and wild (tundra voles) mouse-like rodents. A non-characteristic of mammals low relative content of sphingomyelin is revealed in erythrocyte phospholipids in tundra voles. A hypothesis is put forward that the unique composition of erythrocyte lipids is a peculiar evolutionary developed strategy of adaptation aimed at survival under condition of constant circulation of agents of leptospirosis in populations of this species.     

Phosphorus nuclear magnetic resonance studies of lipid-protein interactions: human erythrocyte glycophorin and phospholipids

Human erythrocyte glycophorin containing four molecules of phospholipid tightly bound to the protein was isolated from human red cell ghosts. This protein preparation was reconstituted into a digalactosyl diglyceride bilayer. The 31P NMR spectrum of this reconstituted membrane produced an axially symmetric powder pattern arising exclusively from the phospholipids bound to glycophorin. The width of the powder pattern, about 90 ppm, is about twice as broad as that normally exhibited by a phospholipid bilayer. The chemical shift tensor is perturbed relative to phospholipids in a bilayer. The spin-lattice relaxation rate of these protein-bound phospholipids is found to be nearly an order of magnitude faster than phospholipids in a bilayer. The results are consistent with phospholipids tightly bound to the membrane protein and undergoing rotational diffusion, perhaps as a complex of phospholipid and protein.