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PubMed1980

Preservation of bilayer structure in human erythrocytes and erythrocyte ghosts after phospholipase treatment. A 31P-NMR study.

1. Fresh human erythrocytes were treated with lytic and non-lytic combinations of phospholipases A2, C and sphingomyelinase. The 31P-NMR spectra of ghosts derived from such erythrocytes show that, in all cases, the residual phospholipids and lysophospholipids remain organized in a bilayer configuration. 2. A bilayer configuration of the (lyso)phospholipids was also observed after treatment of erythrocyte ghosts with various phospholipases even in the case that 98% of the phospholipid was converted into lysophospholipid (72%) and ceramides (26%). 3. A slightly decreased order of the phosphate group of phospholipid molecules, seen as reduced effective chemical shift anisotropy in the 31P-NMR spectra, was found following the formation of diacyglycerols and ceramides in the membrane of intact erythrocytes. Treatment of ghosts always resulted in an extensive decrease in the order of the phosphate groups. 4. The results allow the following conclusions to made: a. Hydrolysis of phospholipids in intact red cells and ghosts does not result in the formation of non-bilayer configuration of residual phospholipids and lysophospholipids. b. Haemolysis, which is obtained by subsequent treatment of intact cells with sphingomyelinase and phospholipase A2, or with phospholipase C, cannot be ascribed to the formation of non-bilayer configuration of phosphate-containing lipids. c. Preservation of bilayer structure, even after hydrolysis of all phospholipid, shows that other membrane constitutents, e.g. cholesterol and/or membrane proteins play an important role in stabilizing the structure of the erythrocyte membrane. d. A major prerequisite for the application of phospholipases in lipid localization studies, the preservation of a bilayer configuration during phospholipid hydrolysis, is met for the erythrocyte membrane.

Ceramides↗

PubMed1993

Carrier erythrocytes from white-tailed deer: morphology, osmotic fragility and survival of circulating sickled erythrocytes.

Carrier erythrocytes are used to disseminate drugs in the circulatory system of animals. Carrier erythrocytes prepared from white-tailed deer (Odocoileus virginianus) do not circulate well in vivo. Although carrier cells were prepared from sickle and non-sickle cells with no apparent differences, their 24-hour survival was only 10 per cent. Osmotic fragility of carrier cells was increased over that of normal deer erythrocytes. Unlike erythrocytes from other ruminants, deer carrier erythrocytes are extremely fragile. Scanning electron micrographs of deer erythrocytes (sickle or non-sickle) in different stages of carrier cell preparation revealed no morphological differences. These data suggest that carrier cells from deer would not be amenable for use in long-term dissemination of drugs.

Animals↗

PubMed1988

Acidic phosphoproteins associated with the host erythrocyte membrane of erythrocytes infected with Plasmodium berghei and P. chabaudi.

New phosphoproteins appear on the host erythrocyte membrane during Plasmodium berghei and P. chabaudi infection. Distinct proteins having similar properties and all distinguished by isoelectric points of less than 4.0 are identified. Associated with the erythrocyte membranes of P. berghei infected erythrocytes are two proteins with molecular masses of 65 and 46 kDa, whereas 93, 90 and 76 kDa proteins are observed during P. chabaudi infection. These new erythrocyte membrane associated proteins are all of parasite origin as indicated by metabolic labeling with proline and are synthesized during the ring stage of the asexual replicative cycle. Three of these proteins, the 93 kDa P. chabaudi protein and both P. berghei proteins, have been purified and the amino acid composition determined. All three are characterized by a relatively high proportion of aspartate and glutamate residues. Mono-and polyclonal antibodies were also raised against the same three purified proteins. No cross reactivity between these three proteins is observed, but one monoclonal antibody against the 65 kDa P. berghei crossreacts with a 27 kDa mouse erythrocyte protein. Immunofluorescence using the antibodies in combination with subcellular fractionation studies clearly shows that these phosphoproteins are associated with the host erythrocyte membrane and not the parasite.

Animals↗

PubMed1983

An assay of malaria parasite invasion into human erythrocytes. The effects of chemical and enzymatic modification of erythrocyte membrane components.

Invasion of erythrocytes by malaria parasites is known to be blocked by proteolytic digestion of merozoite receptors allegedly present in red cell membranes. This information was used in the present work to develop a simple and convenient assay for parasite invasion into red blood cells and for evaluating the role played by red cell membrane components in this process. Synchronized in vitro cultures of Plasmodium falciparum containing only ring stages were subjected to either trypsin or pronase digestion, a treatment that neither affected ring development into schizonts nor mature merozoite release. Cells from this culture were not invaded by the released merozoites. However, upon addition of untreated human red blood cells, marked invasion was observed, either microscopically or as [3H]isoleucine incorporation. The new assay circumvents the need for separating schizonts from uninfected cells and provides a convenient means for assessing how chemical and biochemical manipulation of red blood cells affects their invasiveness by parasites. Using this assay, we verified that sheep and rabbit erythrocytes were resistant to invasion, as were human erythrocytes which had been treated with trypsin, pronase or neuraminidase. Chymotrypsin digestion of human erythrocytes was without effect on invasion. Human erythrocytes which were chemically modified with the impermeant amino reactive reagent H2DIDS, or with the crosslinker of spectrin, TCEA, were found to resist invasion. The results underscore the involvement of surface membrane components as well as of elements of the cytoskeleton in the process of parasite invasion into erythrocytes.

Animals↗

PubMed2004

Expression of Babesia equi EMA-1 and EMA-2 during merozoite developmental stages in erythrocyte and their interaction with erythrocytic membrane skeleton.

In the present study, we investigated the cellular localizations and expression patterns of equi merozoite antigens (EMA) -1 and -2 of Babesia equi during its asexual erythrocytic-developmental cycle using anti-EMA-1t or -2t mono-specific mouse serum. Indirect fluorescent antibody tests demonstrated that EMA-1 and EMA-2 were not expressed in all the erythrocytic-developmental stages of the merozoites and that these two antigens were co-expressed during the early developmental stages. Additionally, it was shown that EMA-1 and EMA-2 were mutually expressed on the surface of extra-erythrocytic merozoites and also that the intra-erythrocytic merozoites shed only EMA-2 antigen in the infected erythrocytic cytoplasm or inside the membrane surface. The specific binding of EMA-2 to a Triton X-100-insoluble horse erythrocyte membrane fraction was also demonstrated. These findings facilitate our understanding of the biological roles of merozoite surface proteins of B. equi and our investigation for new drug targets.

Animals↗

PubMed2000

Peripheral blood erythrocyte parameters in hemochromatosis: evidence for increased erythrocyte hemoglobin content.

We studied peripheral blood erythrocyte parameters and HFE genotypes in 94 hemochromatosis probands and 132 white, normal control subjects. Mean red blood cell counts in probands and control subjects were not significantly different. However, mean values of hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were significantly higher in C282Y/C282Y probands (n = 60) than in wild-type control subjects (n = 65). Probands with other HFE genotypes also had increased mean erythrocyte parameters (other than red blood cell count). Peripheral blood smears prepared before therapeutic phlebotomy revealed that erythrocytes in many probands had increased diameters and were well filled with hemoglobin. Erythrocyte parameters were similar in C282Y/C282Y probands with and without hepatomegaly, elevated serum concentrations of hepatic enzymes, hepatic cirrhosis, diabetes mellitus, arthropathy, or hypogonadism. Among C282Y/C282Y probands, significantly greater values of MCV (but not other erythrocyte parameters) occurred among those who had transferrin saturation values of 75% or greater or iron overload at diagnosis. After iron depletion, the mean MCV, MCH, and MCHC values of C282Y/C282Y probands decreased but remained significantly greater than values in wild-type control subjects. Mean values of prephlebotomy MCH and MCHC concentrations were lower in HLA-A3-positive than in HLA-A3-negative C282Y/C282Y probands. We conclude that increased values of mean hemoglobin, hematocrit, MCV, MCH, and MCHC in hemochromatosis probands are caused primarily by increased iron uptake and hemoglobin synthesis by immature erythroid cells. Mechanisms of iron uptake by erythrocytes that could explain these results are discussed.

Adult↗

PubMed1999

Membrane-protease interactions. III: A consideration of the difference in binding potential of pancreatic proteases to erythrocytes and erythrocyte ghosts.

Trypsin and chymotrypsin readily bind to human erythrocyte ghosts and to resealed right-side-out ghosts, but not to intact erythrocytes, as followed with [3H]trypsin and [3H]chymotrypsin and with cold proteases in a caseinolytic assay. The proteases freely reacted with casein in the presence of intact cells. Trypsin activated trypsinogen over an 8-hr time course at a faster rate in the presence of erythrocytes than in the absence thereof, after a slight initial delay. Trypsinogen did not bind to intact erythrocytes, thereby behaving comparably to trypsin. These results suggest that different microenvironments exist about the erythrocyte ghosts and the intact erythrocytes, thereby permitting the proteases to bind to the former but not to the latter. Hence, in the absence of considerable ghosts in circulating blood, which may mask the binding site of the proteases, the proteases may be more readily accessible for interaction with circulating serpins, leading to inactivation of the proteases and protection from their degradative potential. The presence of the serpins in circulating blood may assist in the control of the degradative power of the pancreatic proteases in pancreatitis and may negatively modulate such processes as thrombosis, activation of the complement system, and vascular remodeling.

Binding, Competitive↗

PubMed1993

Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion and non-enzymic binding to membrane proteins.

We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.

Adenine Nucleotides↗

PubMed1995

Effect of erythrocyte membrane on extracellular development of the erythrocytic cycle of Plasmodium falciparum.

We have earlier reported conditions that support the axenic development in vitro of a complete asexual erythrocytic cycle of Plasmodium falciparum. Up to 30% of merozoites showed initial differentiation into trophic forms (rings) viable at 14 hr. However, only approximately 1% of the merozoites would develop further into trophozoites and early schizonts viable at 36 hr. In efforts to increase the number of late stage parasites, we have now found a significant favorable effect of the addition of erythrocyte ghosts. Doubling the quantity of erythrocyte membrane in the erythrocyte sonicate medium resulted in approximate doubling of the number of trophozoites and early schizonts. These results indicate that components of the erythrocyte membrane are essential for the complete development of the erythrocytic cycle.

Animals↗

PubMed2001

O(2) release from erythrocytes flowing in a narrow O(2)-permeable tube: effects of erythrocyte aggregation.

The effects of erythrocyte aggregation on O(2) release were examined using O(2)-permeable fluorinated ethylenepropylene copolymer tubes (inner diameter, 25 microm; outer diameter, 100 microm). Measurements were performed using an apparatus built on an inverted microscope that contained a scanning-grating spectrophotometer with a photon count detector connected to two photomultipliers and an image processor through a video camera. The rate of O(2) release from the cells flowing in the narrow tube was determined based on the visible absorption spectrum and the flow velocity of the cells as well as the tube size. When the tube was exposed to nitrogen-saturated deoxygenated saline containing 10 mM sodium dithionite, the flowing erythrocytes were deoxygenated in proportion to the traveling distance, and the deoxygenation at a given distance increased with decreasing flow velocity and cell concentration (hematocrit). Adding Dextran T-70 to the cell suspension increased erythrocyte aggregation in the tube, which resulted in suppressed cell deoxygenation and increased marginal cell-free-layer thickness. The deoxygenation was inversely proportional to the cell-free-layer thickness. The relation was not essentially altered even when the medium viscosity was adjusted with Dextran T-40 to remain constant. The rate of O(2) release from erythrocytes in the tube was discussed in relation to the O(2) diffusion process. We conclude that the diffusion of O(2) from erythrocytes flowing in narrow tubes is inhibited primarily by erythrocyte aggregation itself and partly by thickening of the cell-free layer.

Diffusion↗

PubMed1977

The effect of pentoxifylline on erythrocyte deformability and on phosphatide fatty acid distribution in the erythrocyte membrane.

Ten maturity-onset diabetics with chronic vascular disease were treated with 400 mg pentoxifylline 3-times daily for 14 days. Erythrocyte deformability (using a filtration technique for whole blood) and phosphatide fatty acid distribution in the erythrocyte membrane were measured before and after the treatment period. Statistical analysis of the data showed that the erythrocyte filtration rate had increased significantly by the end of treatment (2alpha = 0.02), and that there were only slight changes in erythrocyte membrane phosphatide fatty acid levels. The drug was well tolerated, and there were no adverse laboratory findings in the parameters measured. On the basis of results described by other investigators, theimprovement in erythrocyte deformability was attributed to an increase in erythrocyte ATP levels. The authors discuss the importance of red cell fluidity for capillary perfusion.

Aged↗

PubMed1999

Relation between erythrocyte reduced glutathione and glutamate concentrations in Korean Jindo dogs with erythrocytes possessing hereditary high activity of Na-K-ATPase and a high concentration of potassium.

The concentrations of sodium, potassium, reduced glutathione (GSH) and free amino acids and Na-K-ATPase activity in erythrocytes were examined in 35 purebred Jindo dogs in Korea. The incidence of Jindo dogs with a high potassium concentration and high activity of Na-K-ATPase in erythrocytes (HK phenotype) was 25.7%. The erythrocyte GSH concentration in HK Jindo dogs varied widely, from 2.45 to 12.38 mmol/l of RBCs, and was positively correlated with the erythrocyte glutamate concentration. These results indicate that HK Jindo dogs have normal to very high levels of erythrocyte GSH, which might result from the varying quantity of Na-dependent glutamate influx in the erythrocytes.

Animals↗

PubMed1989

Erythrocyte insulin receptor characteristics and erythrocyte membrane lipid composition in healthy men.

The cell membrane plays an important role in the mechanism of insulin action. To test whether erythrocyte insulin receptor characteristics are related to the erythrocyte membrane lipid composition, 11 healthy volunteers were studied. The relationship between insulin binding to erythrocytes, the number of receptors per cell and the affinity of receptors to insulin on the one hand and total phospholipid fatty acid (FA) composition and cholesterol/phospholipid molar ratio in the erythrocyte membrane on the other hand were evaluated. 1. We found a significant negative correlation between specific insulin binding and the proportion of n-6 essential FA in erythrocyte membrane phospholipids, especially linoleic acid (r = -0.82, p less than 0.01) and arachidonic acid (r = -0.73, p less than 0.05). On the other hand, a significant positive correlation between insulin binding and the proportion of nonessential FA (r = +0.65, p less than 0.05) was seen. Number of receptors per cell and the affinity of receptors were not significantly related to phospholipid FA composition. 2. There was no significant correlation between insulin receptor characteristics and the cholesterol/phospholipid molar ratio in the erythrocyte membrane. The data presented support the hypothesis that the FA pattern of membrane total phospholipids may modify the properties of insulin receptors.

Adult↗

PubMed1982

Persistent macrocytosis assessed by erythrocyte subpopulation analysis following erythrocyte regeneration in cats.

Serial erythrocyte volume distribution curves were used to characterize changes in erythrocyte subpopulations following the induction of Heinz body hemolytic anemia in cats. Macrocytes produced in response to hemolysis were observed after loss of reticulum. After recovery from hemolysis, a gradual reduction in mean cell size resulted from a combination of macrocyte remodeling and production of normocytic cells. The mean apparent prehemolysis half-survival time for chromium-51-labeled erythrocytes was 13.7 days. There were two components to the posthemolysis survival curve. An initial accelerated loss of label was due to either destruction of posthemolysis macrocytes or loss of cell contents associated with remodeling. The second survival curve component reflected an increased mean apparent half-survival time of 20.2 days. After correction for isotope elution, the latter component reflected cohort-like labeling of relatively young erythrocytes having normal survival for the duration of the experiment. At the beginning for this latter component, 42% of the erythrocytes were macrocytic (mean of 3.3 x 10(6) macrocytes/microliter). Since the number of macrocytes gradually declined during the period of normal survival, it was concluded that these cells entered the normocytic size range. Recognition of persistent macrocytosis is important in the concept of the regenerative response and should be considered in the interpretation of erythrocyte volume distribution curves.

Anemia, Hemolytic↗

PubMed1980

Study of erythrocytes in a hereditary hemolytic syndrome (HHS): comparison with erythrocytes in lecithin:cholesterol acyltransferase (LCAT) deficiency.

Erythrocyte membrane abnormalities in 3 members of a family with a hereditary haemolytic syndrome (HHS) were compared to those previously described in a family with lecithin:cholesterol acyltransferase (LCAT) deficiency. Despite similarities including an increase in membrane phosphatidylcholine, a decrease in phosphatidylethanolamine, stomatocytosis, and a marked decrease in erythrocyte osmotic fragility a number of differences were observed. These included membrane cholesterol content (increased in homozygotes with LCAT deficiency), changes in sodium and potassium content and Na+,k+-ATPase activity (the latter being increased in HHS), changes in acetylcholinesterase and sulfhydryl group latency (present in LCAT deficiency, but not in HHS) and 2,3 DPG content (decreased in HHS, normal in LCAT deficiency. Full compensation of the erythrocyte defect occurred in HHS but the homozygotes for LCAT deficiency were slightly anaemic. It is concluded that, although similar abnormalities in phospholipid composition, osmotic fragility, and erythrocyte morphology exist in these two disorders, the molecular nature of the erythrocyte membrane structural and functional changes in HHS and LCAT deficiency is clearly different.

4-Nitrophenylphosphatase↗

PubMed1991

Alterations in erythrocyte rheology in patients with severe peripheral vascular disease: 1. Cell volume dependence of erythrocyte rigidity.

The erythrocyte rigidity of patients suffering from severe peripheral vascular disease (PVD) was measured by a filtration method using 3 microns pore size filters. Filtration pressures for both PVD patients and normal subjects showed a cell volume dependency, and patient filtration pressures were normalized to cell volume to evaluate intrinsic, ie, nonvolume dependent, abnormalities in erythrocyte deformability. A significant (p less than 0.001) increase in cell rigidity was found in 44 of 54 PVD patients in comparison with volume-matched normal controls. No significant difference was found between patient mean corpusculer hemoglobin (MCH) and normal MCH at any given mean corpuscular volume (MCV), indicating that observed increases in erythrocyte rigidity are not attributable to changes in patient MCH. Therefore, the mechanism of increase in erythrocyte rigidity for PVD patients still needs further investigation into such parameters as levels of adenosine triphosphate,2,3-DPG, and membrane fluidity (calcium- and/or protein-binding membrane, cholesterol and phospholipid content of membrane, etc), as well as other aspects of erythrocyte physiology.

Adult↗

PubMed1983

Effect of gastrointestinal parasite infection in calves on the preparation of carrier erythrocyte ghosts and on erythrocyte survival.

Calves were inoculated with helminths to induce a severe anemia. Increased erythrocyte mean cell volume caused by acute parasitism made erythrocytes more resistant to osmotic stress as reflected in the percentage of encapsulation parameters. Preparation of resealed carrier erythrocytes before and during parasitosis was studied. Low percentage of encapsulation of the marker [14C]sucrose could be corrected by extended dialysis time. Typically, 40% encapsulation was found in erythrocytes from healthy calves, whereas only 20% encapsulation was found for erythrocytes from helminth-infected animals.

Anemia↗

PubMed1977

Prostaglandin E2-hemoglobin AA and SS erythrocyte interaction (prostaglandin-erythrocyte interaction).

Prostaglandin E2 (PGE2), at concentration larger than or equal to 5 x 10(4) ng/ml, induced discocyte leads to echinocyte transformation of saline-suspended hemoglobin (Hb) AA and SS erythrocytes. This erythrocyte transformation is concentration-dependent and is reversible at room temperature after 90-120 min. The Hb SS erythrocytes treated with PGE2 did not exhibit accelerated sickling or increased formation of sickled echinocytes. Erythrocytes suspended in autologous plasma treated with PGE2, 2 X 10(6) ng/ml, did not exhibit echinocytic transformation probably because of drug binding to the plasma proteins. Other in vitro studies showed that PGE2 of concentrations of 10-500 ng/ml had no adverse effects on intact, plasma-suspended Hb SS erythrocytes. These Hb SS erythrocytes were examined for changes in morphology, potassium and calcium flux, and blood viscosity under oxygenated and hypoxic conditions.

Anemia, Sickle Cell↗