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.     

Erythrocyte membrane permeability for univalent cations (Na+, K+) and their transformation in patients with hypertension and symptomatic (renal) hypertension

The form and surface architectonic of erythrocytes studied in 18 teenagers and men with hypertensive disease of I stage (HD) and in 7 men with symptomatic (renal) hypertension (SH). Simultaneously permeability of erythrocyte membranes for Na+ and K+ was studies. The change in the form and surface architectonics was found in the erythrocytes of the patients with hypertensive disease, I stage. The same was true for the patients with renal hypertension but the difference was not so prominent. Rapid shift of correlation of erythrocyte morphological varieties has been revealed during the study of Na+ and K+ ions' transport rate after the treatment by p-chloromercuribenzoate acid. Increase of irreversible transformation of erythrocytes was discovered in patients with HD. Besides in some cases there was decrease in the size of echinocytes. The transformation of erythrocytes into echinocytes is more prominent in healthy subjects and in patients with SH. Our data suggest that the change in erythrocyte form is related to the change of erythrocyte membrane permeability to Na+ and K+ ions and the alteration of membrane structure is the basis for these disturbances.     

The characteristics of the structural-functional state of the erythrocytes from homoiothermic and heterothermic animals during hypothermic storage]

Haemoglobin leakage and permeability for 86Rb and K ions during storage at normal and hypothermic conditions have been investigated in the erythrocytes of the ground squirrel Citellus undulatus in hibernating, arousing and awake animals, as well as in rats. During hibernation, stabilization of the barrier properties and a decrease in passive ionic permeability of erythrocyte membrane were observed. Preservation of ionic homeostasis of the erythrocytes in hibernating animals is favoured by activation of Na(+)-pump. By means of radioautography of electrophoregrams of the blood serum proteins, appearance of a rapidly labeling low-molecular protein was noted at the beginning of the baut and its disappearance before arousal. The data obtained are discussed in relation to the role of the blood plasma components in modification of erythrocyte membranes in hibernating animals.     

Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-<Superscript>19</Superscript>F-NMR

The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using ¹⁹F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Effect of some nickel compounds on red blood cell characteristics

The effect of certain inorganic and coordinated nickel compounds on the resistance to different destructive substances, rheological properties, and functional activity of healthy human red blood cells (RBC), was investigated. It is shown that nickel compounds affect the erythrocyte membrane lipid bilayer, as well as membrane proteins to various extents, depending on the type of compounds used. In general, the acceleration of erythrocyte aging was observed to be more pronounced in young erythrocytes. The observed results suggest that nickel compounds decrease water permeability across erythrocyte membranes. Almost all the investigated nickel compounds decrease erythrocyte thermostability, deformability, and the rate of O₂ release by erythrocytes.     

Antibody-mediated targeting of liposomes to erythrocytes in the whole blood

F(ab′)₂ fragments derived from anti-rat erythrocyte antibody or normal rabbit serum IgG were covalently attached to the surface of liposomes consisting of equimolar amounts of egg phosphatidylcholine and cholesterol. These liposomes were interacted with rat, monkey or mouse blood, and their binding to both red and white blood cells was determined. Results of these studies show that coupling of liposomes to anti-rat erythrocyte F(ab′)₂ considerably enhances their binding to erythrocytes in rat blood. However, no such increase in the binding was observed with rat leukocytes or monkey and mouse erythrocytes. Besides, the interactions between the liposomes and target cells did not affect the permeability properties of the liposome bilayer. These observations indicate that liposomes coupled to cell-specific antibodies may serve as highly useful carriers for homing of drugs/enzymes to specific cells in biophase.     

Changes in blood viscosity after administration of vitamin E in the rabbit

Vitamin E is an essential factor to maintain biological membranes stability and its lack may affect membranes structures and reduce erythrocyte life-span. Vitamin E also play a role in the maintenance of a normal platelet aggregation. A.A. studied the effects of a ten days supply of d-1-alpha tocopherol acetate (50 mg/Kg/die) on blood viscosity in 8 rabbits. Results obtained show a significant reduction of blood viscosity on 6th day of treatment in the male rabbits and a progressive reduction of values from the 6th till the 10th day in female rabbits. The most significant decrease of blood viscosity were obtained at the lowest shear-rates, due to an increased red cells deformability to the antioxidative action of vitamin E on the erythrocytes membrane and to a reduced red cells aggregation. Such modifications on the red blood cells caratheristics can be determined by vitamin E through different mechanism: a) inhibiting red cell membrane's polyunsaturable fatty acids oxidation; b) by removal of abnormal lipids from erythrocyte membrane; c) physical and chemical stabilization of membrane's surface.     

Determination of the content of nonfilterable cells in erythrocyte suspensions as a function of the medium osmolality

The results of most filtration assays for deformability of erythrocytes do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple filtration method for determination of the percentage of nonfilterable cells in erythrocyte suspension using membrane filters with mean pore diameter of 3.1 microns. This method makes it possible to detect even minor abnormal subpopulations in erythrocyte suspensions. The flow rate of buffer depends on the number of free pores of a filter. The plot of the number of pores clogged by nonfilterable cells vs the total number of erythrocytes that were allowed to pass through the filter had a linear portion, with a slope representing the relative content, Z%, of nonfilterable cells in the suspension. We determined Z% for various medium osmolalities u and used the data to derive the distribution of erythrocytes in ucr (ucr is the maximum value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations). The distribution of ucr in suspension of normal erythrocytes has a maximum of about 200 mOsm/kg and a half-width of about 20 mOsm/kg. The distributions of ucr are altered in normal erythrocyte suspensions at decreased pH values, in cryopreserved and ATP-depleted erythrocyte suspensions and in erythrocytes from a xerocytosis patient.     

Flow cytometric analysis of erythrocyte populations in Tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen.

Flow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, Gal-NAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu-NAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc alpha 1-O-Ser(Thr) tetrasaccharide or to fixed neuraminidase-digested cells presenting the Gal-GalNAc disaccharide. The percentages of Tn-positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A-specific monoclonal antibodies showed that the erythrocytes composing the Tn-negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn-positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 x 10(7) erythrocytes, suggesting that the frequency of such cells in normal individuals is less than 1 x 10(-6).     

Superoxide anion generation, erythrocytes superoxide dismutase activity, and lipid peroxidation during hemoperfusion and hemodialysis in chronic uremic patients

In 11 chronic uremic patients superoxide anion generation in whole blood, both without and with opsonized zymosan stimulation, was lower than that in 11 healthy controls, while erythrocyte superoxide dismutase (SOD-1) activity and erythrocyte and plasma malonyldialdehyde (MDA) concentrations were elevated. During hemoperfusion (HP) and hemodialysis (HD) superoxide anion generation transiently significantly increased. Changes in the erythrocyte SOD-1 activity and plasma and erythrocyte MDA concentrations during HP suggested that this procedure exerted beneficial effects on lipid peroxidation. On the other hand, during HD erythrocyte membrane lipid peroxidation seemed to be enhanced even further; this phenomenon took place mainly within the dialyzer and a decrease in the erythrocyte SOD-1 activity seemed to be one of the contributing factors. Results of in vitro experiments with cross-incubation of erythrocytes and blood plasma and incubation of whole blood with cuprophan membrane suggest existence of an SOD-1 activator in the uremic blood plasma, which is possibly eliminated during HD.     

Possible basis for membrane changes in nonparasitized erythrocytes of malaria-infected animals

Previous studies (Gupta et al. (1982) Nature 299, 259-261) have shown that nonparasitized erythrocytes of Plasmodium knowlesi-infected monkeys contain the procoagulant phospholipid phosphatidylserine (PS) in the outer-half of their membrane bilayer. A reinvestigation of this problem has now revealed that in acute P. knowlesi infection, at least 30% of the infected animals do not have this abnormality. However, PS externalization was a consistent feature in the uninfected red cells of chronically infected animals. Also, a similar membrane change was observed in the red cells of uninfected splenectomized monkeys. These results strongly suggest that spleen plays an important role in maintaining the exclusive inner distribution of PS in the normal erythrocyte membrane, and that partial migration of this lipid to the outer monolayer in nonparasitized erythrocytes could be attributed to an abnormal physiology of this organ in malarial infection.     

Rhnull human erythrocytes have an abnormal membrane phospholipid organization.

Rhnull human erythrocytes lack the antigens of the Rhesus blood group system, have an abnormal shape and an increased osmotic fragility, and are associated with mild chronic haemolytic anaemia. Studies with phospholipase A2 and sphingomyelinase C show that the asymmetric distribution of phosphatidylethanolamine (PtdEtn) in the membrane of these cells differs from that found in control cells. The amount of PtdEtn which can be hydrolysed by phospholipase A2 in the presence of sphingomyelinase C in intact Rhnull cells is twice as high as that in normal erythrocytes. In intact Rhnull cells all of the phosphatidylcholine (PtdCho) present in the membrane can be readily exchanged with a PtdCho-specific exchange protein, whereas in control cells 75% is readily exchanged and 25% at a much lower rate. This indicates that PtdCho experiences a relatively fast transbilayer movement in the Rhnull cells. The observation that the loss of two membrane polypeptides in the Rhnull cells leads to abnormal shape, increased osmotic fragility, abnormal PtdEtn distribution and enhanced transbilayer mobility of PtdCho strongly suggests that one or both polypeptides are essential for the maintenance of a proper membrane-membrane skeleton interaction.     

Erythrocyte cytosolic free Ca2+ and plasma membrane Ca2+-ATPase activity in cystic fibrosis

The properties of the Ca2+, Mg2+-ATPase of erythrocyte membranes from patients with cystic fibrosis (CF) were extensively compared to that of healthy controls. Following removal of an endogenous membrane inhibitor of the ATPase, activation of the enzyme by Ca2+, calmodulin, limited tryptic digestion or oleic acid, as well as inhibition by trifluoperazine, were studied. The only properties found to be significantly different (CF cells vs controls) were calmodulin-stimulated peak activity (90 vs 101, P less than 0.02) and trypsin-activated peak activity (92 vs 102, P less than 0.02). No significant difference could be measured in the steady-state Ca2+-dependent phosphorylation of CF and control erythrocyte membranes indicating similar numbers of enzyme molecules per cell. The functional state of Ca2+ homeostasis in intact erythrocytes was investigated by measuring the resting cytosolic free Ca2+ levels using quin-2. Both CF and control erythrocytes maintained cytosolic free Ca2+ between 20 to 30 nM. Addition of 50 uM trifluoperazine resulted in an increase in erythrocyte cytosolic free Ca2+ to about 50 nM in both CF and control cells. Estimates of erythrocyte membrane permeability using the steady-state uptake of 45Ca into intact erythrocytes revealed no differences between CF and control cells. These results confirm that there is a small decrease in the calmodulin-stimulated activity of the erythrocyte Ca2+, Mg2+-ATPase in CF. However, this deficit is apparently not large enough to impair the ability of the CF erythrocyte to maintain normal resting levels of cytosolic free Ca2+.     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

Polyamines--sickling red blood cell interaction.

The binding of polyamine as a function of concentration to normal and sickling rcc'. blood cells is analyzed by Langmuir type binding isotherms, based on the Gouy-Chapman model for an electrical double Iayer, where the zeta potential is a function of only the normal distance coordinate. For normal erythrocytes, the apparent exotropic binding constants are found to be 103, 110, and 130 dl/g at normal distance coordinates of 4, 5, and 6 Å, iezpectively. The esotropic binding constant is determined to be 420 dl/g at a distance of 7 Å. For sickling red blood cells, the apparent exotropic binding constants are 3.3, 3.8, 4.6, and 6-7 dl/g at a distance of 4 to 7 Å. The esotropic binding constant at a distance of 8 Å is found to be 12-9 dl/g. The apparent binding affinity of polyamines to the normal red blood cell. therefore, is approximately 30 times greater than to the sickling erythrocyte.The Praxis pulse nuclear magnetic resonance spectrometer is used to determine the spin-lattice relaxation time (T₁) for water in the presence of normal and sickling red blood cells. The spin-lattice relaxation time is found to be 540 ms for normal erythrocytes and 445 ms for sickling red blood cells in the oxy state. Differences in the spin-spin relaxation time (T₂) for the two types of erythrocyte are negligible, being within the range of normal experimental error.     

Further investigation of methylation on sickle erythrocyte membranes

The methylation of erythrocyte membrane proteins has been investigated with fractionated reversible and irreversible sickle erythrocytes to better understand conflicting results obtained from two laboratories (Green and Kalra (6), Ro et al. (1). When subpopulations of intact erythrocytes obtained by two different separation methods (33% bovine serum albumin and Stractan II gradient centrifugations) were incubated with L-[methyl-3H] methionine at pH 7.2 and 37 degrees C, membranes from both reversible and irreversible sickle erythrocyte populations showed about half the [3H]methyl group incorporation than that observed in normal erythrocytes. In addition, this difference in the level of methylation between normal and sickle cells was maintained during the entire course of a 2-hr incubation utilizing S-adenosyl-L-[methyl-3H]methionine, the immediate in vivo methyl donor.     

A modification of the filtration method for studying the content of nonfilterable cells in erythrocyte suspension

The results of filtration assays provide estimates of the deformability of erythrocytes averaged over the entire suspension. These assays do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple method using a filtrometer to determine the percentage of non-filterable (under given conditions) cells in the erythrocyte suspension. Membrane filters made of a polyethylene terphthalate film had the mean pore diameter of 3.1 microns and the length of cylindrical micropores of 7 microns. The buffer flow rate tb depends on the number of free pores in a filter. The plot of the number of pores clogged by non-filterable cells versus the total number of erythrocytes passed through the filter had a linear portion whose slope represents the relative content Z of non-filterable cells in the suspension. We determined Z for various medium osmolarities u. These data were used to derive the distribution of erythrocytes in ucr, the value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations. The distribution maximum corresponded to 190-200 mOsm/kg for erythrocytes from the normal blood. This means that normal erythrocytes have the median values of their surface area and area-to-volume ratio of 155-151 microns2 and 1.72-1.68 microns-1, respectively. The half-width of the distribution was approximately 30 mOsm/kg. This finding suggests that the normal blood contains a certain fraction of erythrocytes with a decreased area-to-volume ratio. Our results showed that the distribution is altered in various forms of anemia and in ATP-depleted erythrocyte suspensions.     

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

Plasma 125I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the 125I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies.