Mouse Erythrocyte Membrane Research Articles

An antigen of Plasmodium yoelii that translocates into the mouse erythrocyte membrane upon entry into the host cell.

Monoclonal antibodies against the rodent malaria parasite, Plasmodium yoelii, have been prepared and characterized by indirect immunofluorescence on acetone-fixed infected mouse erythrocytes. The antibody of clone K2 reacted strongly with late trophozoites and schizonts, whereas it did so weakly and diffusely with ring forms and early trophozoites. Strong fluorescence was confined to granular structures in schizonts and merozoites. Parasites that invaded erythrocytes in vitro lost the strong fluorescence. Instead, immunofluorescence appeared in the membranes of erythrocytes infected in vitro with merozoites. Erythrocytes infected with more than one merozoite had intensified immunofluorescence in their membranes. Staining of the invaded erythrocytes with 4',6-diamidino-2-phenylindole (DAPI) hydrochloride demonstrated that membranes of all the invaded erythrocytes acquired the P. yoelii antigen. These results suggest that the P. yoelii antigen in merozoites is translocated into erythrocyte membranes upon entry into the host cell. Immunofluorescence continued to appear in membranes of infected erythrocytes throughout the intra-erythrocytic parasite growth. Staining of unfixed infected erythrocytes with the K2 antibody failed to detect the parasite antigen. In contrast, immunofluorescence was present in unfixed membranes of erythrocyte ghosts, which had been spontaneously formed after rupture of schizont-infected erythrocytes by merozoite release. No immunofluorescence appeared in either acetone-fixed or unfixed ghosts of normal erythrocytes. These results suggest the antigenic determinant of the P. yoelii antigen is exposed at the cytoplasmic surface of the infected erythrocyte membrane. Immunoprecipitation has revealed that the K2 antibody recognizes a 160 X 10(3) Mr P. yoelii antigen.

Identification and relative composition of minor fatty acids of mouse erythrocyte membrane phospholipids

Identification and relative composition of minor fatty acids of mouse erythrocyte membrane phospholipids

Peritoneal B cells differentiate without proliferation into autoantibody secretors under the influence of factors released by other cells.

After 3-5 days of in vitro culture, peritoneal cells from untreated C3H mice produce autoantibodies specific for autoantigens in the membranes of mouse erythrocytes. The autoantigens are exposed in vitro by treating mouse erythrocytes with the proteolytic enzyme bromelain. Limiting-dilution cell culture techniques were used to determine the frequency of the autoreactive B cells. Cells were cultured in Terasaki trays at 10-200 cells/well. Autoantibody production was assayed with an in situ plaque-forming cell (PFC) assay. On average, one autoantibody precursor cell was detected for every 150-200 peritoneal cells cultured. This precursor frequency was increased to 1 in 50 by the addition of lipopolysaccharide (LPS) and dextran sulphate (DS) to the culture medium. The addition of a culture supernatant from an EL4 lymphoma subline also induced a high proportion of the peritoneal cells to secrete autoantibodies. However, it was not possible to determine the frequency of PFC accurately because at limiting numbers of peritoneal cells the effects of EL4 affected more than one limiting variable. Significant cell division was observed in cultures to which LPS/DS had been added, in contrast to untreated cultures to which EL4 supernatant was added. The results show that high numbers of autoreactive B cells committed to self antigens are present in the peritoneal cavity and that these cells under the influence of appropriate cytokines can differentiate in vitro, even without proliferation, into autoantibody secretors. The cell type or types releasing the cytokines remain to be identified.

Changes in membrane lipid content after chronic ethanol administration with respect to fatty acyl compositions and phospholipid type

Changes in the relative proportions of the phospholipid fatty acids of erythrocyte membranes in mice after chronic ethanol treatment (4.5 g/kg, i.p. twice daily for one week) were shown to vary with the differing control profiles observed. It is suggested that certain changes in membrane lipid composition after ethanol administration may not be interpreted simply in terms of an adaptation to a disordering effect of the drug. The fatty acid changes were, in addition, distributed asymmetrically within the individual phospholipid classes. Depending on the control profile, the effects varied from being mainly in phosphatidylethanolamine (PE; 80%) and phosphatidylserine-inositol (PS + PI; 10%), phospholipids primarily located on the inner half of the membrane bilayer, to being more evenly distributed between PE and phosphatidylcholine (PC) and probably, therefore, between the two halves of the bilayer. Changes in the monounsaturated acid remained primarily with PE, suggesting a specific functional role for this species. The remaining results are discussed in the light of possible effects on cell morphology and their potentially similar consequences of increasing cell volume.

Agglutination of mouse erythrocytes by binding of non-choline phospholipids to A 70 000-Dalton protein

The structure of some phospholipids that cause agglutination of mouse erythrocytes has been studied. Haemagglutination is a property of non-choline-containing phospholipids; the phosphate group is essential and unsaturated fatty acids optimal. A protein of M r 70 000 was isolated from mouse erythrocyte membranes which completely inhibited phospholipid-mediated erythrocyte agglutination. It is proposed that this protein is the phospholipid binding site on mouse erythrocytes and the ligand for the human B-lymphocyte receptor for mouse erythrocytes. Preliminary investigations suggest that a similar inhibitor of phospholipid-mediated agglutination is found in serum. Agglutination of mouse erythrocytes by phospholipid and specific inhibition by the 70 kDa membrane protein constitute a simple system for studying the interaction of phospholipid with protein.

Isolation and characterization of plasma membranes from friend erythroleukaemic cells. A study with sphingomyelinase C

Plasma membranes have been prepared from Friend erythroleukaemic cells using a Dounce homogenization technique followed by differential and sucrose gradient centrifugations. (1) A plasma membrane fraction was obtained which showed a 20- to 30-fold enrichment in 5′-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and in 32P-labeled (poly)phosphoinositides. About 1% of the total protein, 6–7% of phospholipid, 8–9% of cholesterol and 12–15% of each of the above markers were recovered in the plasma membrane fraction with an average yield of 15–20%. The plasma membrane was characterized by a high cholesterol to phospholipid molar ratio (0.626), a 2-fold enrichment in sphingomyelin and in phosphatidylserine as compared to the whole cell and by the complete absence of diphosphatidylglycerol. (2) When compared to the phospholipid composition of the mature mouse erythrocyte membrane, the plasma membrane of the Friend cell only differs by a higher phosphatidylcholine and a lower phosphatidylethanolamine content, whereas the levels of sphingomyelin and phosphatidylinositol plus phosphatidylserine are similar. (3) Friend cells were treated with sphingomyelinase C ( S. aureus) under non-lytic conditions and subsequently submitted to subcellular fractionation. The results showed that the plasma membrane accounted for 38.5% of the total phospholipid, 64.1% of the total cholesterol and about 4.4% of the total protein content of Friend cells. (4) Sphingomyelin appeared to be asymmetrically distributed in the plasma membrane of Friend cells, with about 85% of this phospholipid being present in the outer monolayer.

Effects of chronic ethanol administration on the composition of membrane lipids in the mouse

The relative proportions of the phospholipid fatty acids of erythrocyte membranes in mice were changed by chronic ethanol treatment and were not related to effects of the drug on nutrition, body temperature or experimental stress. Similar changes were observed using two different routes of ethanol administration and they did not reflect the metabolic effects of ethanol seen in the phospholipid fatty acids of whole liver. The observed increased content of saturated fatty acids and decreased content of polyunsaturated acids support the concept of adaptive changes taking place in the membrane during tolerance development to compensate for an increased membrane fluidity caused by ethanol. However, an increased content of the mono-unsaturated acid, octadecenoic (oleic), was found and there was no change in the cholesterol/phospholipid ratio. Other contrasting types of plasma membrane in mice showed different patterns of change in their phospholipid fatty acids during chronic ethanol administration. It is suggested that changes in membrane lipid composition could only partly account for an adaptation to ethanol-induced membrane disordering.

Relationship between choline derivatives and mouse erythrocyte membrane antigens revealed by mouse monoclonal antibodies. I. Anticholine activity of anti-mouse erythrocyte monoclonal antibodies

Different clones of mouse hybridomas, derived from the fusion of unstimulated mouse peritoneal cells with mouse myeloma cells, producing IgM monoclonal antibodies directed against the membrane of bromelain-treated mouse erythrocytes (MRBC(Br)) have been previously established. We have recently shown that one of these hybridomas produce, in ascites, antibodies cross-reacting with phosphorylcholine derivatives (trimethylammonium (TMA) derivatives). In this work the cross-reactivity for TMA derivatives of the monoclonal antibodies produced by 4 anti-MRBC(Br) hybridomas have been studied at the cell level (plaque-forming cells). Phosphorylcholine, choline bromide and p-aminophenyl-trimethylammonium were found to be potent specific inhibitors of plaque formation (anti MRBC(Br)). The hemolytic activities of ascites and tissue culture supernatants were studied and their inhibition by TMA derivatives was determined. Immunoglobulins from ascites purified on TMA immunoadsorbant column were analyzed by two-dimensional gel electrophoresis, their spectrotype was compared to the spectrotype of immunoglobulins from tissue culture supernatants from the same hybridoma radioactively tagged by internal incorporation of [ 14C]leucine. It could be shown without ambiguity that the PTMA column retained an IgM with the same characteristics as the IgM secreted in vitro.

An immunological analysis of the effects of bromelain on the mouse erythrocyte membrane

The technique of crossed immunoeletrophoresis was used to analyse the effects of bromelain on the antigens contained within the membranes of mouse erythrocytes. Immune sera were obtained from goats that had been injected with membranes prepared from either normal erythrocytes or bromelain-modified erythrocytes. Precipitation patterns obtained by crossed immunoelectrophoresis showed that treatment of erythrocytes with bromelain changed the electrophoretic mobility of a major protein antigen and increased the solubility of two membrane glycoproteins. The possibility that enzymic modification of the erythrocytes led to the creation of entirely new antigenic determinants was not supported by the precipitin patterns obtained using sera adsorbed with various membrane preparations.

Vanadate-stimulated NADH oxidation in plasma membrane

The rate of NADH oxidation with oxygen as the acceptor is very low in mouse liver plasma membrane and erythrocyte membrane. When vanadate is added, this rate is stimulated 10- to 20-fold. The absorption spectrum of vanadate does not change with the disappearance of NADH. The reaction is inhibited by superoxide dismutase, and there is no activity under an argon atmosphere. This indicates that oxygen is the electron acceptor and the reaction is mediated by superoxide. The vanadate stimulation is not limited to plasma membrane. Golgi apparatus and endoplasmic reticulum show similar increase in NADH oxidase activity when vanadate is added. The endomembranes have significant vanadate-stimulated activity with both NADH and NADPH. The vanadate-stimulated NADH oxidase in plasma membrane is inhibited by compounds, which inhibit NADH dehydrogenase activity: catechols, anthracycline drugs and manganese. This activity is stimulated by high phosphate and sulfate anion concentrations.

Matrix control of protein diffusion in biological membranes.

Lateral diffusion coefficients of fluorescently labeled lipids and integral membrane proteins were determined in the membranes of normal and spectrin-deficient spherocytic mouse erythrocytes by the technique of fluorescence redistribution after photobleaching. The results were used to generate a mathematical description of a matrix-control model of membrane protein diffusion. In the spherocytic cells, which lack the principal components of the cytoskeletal matrix of normal cells, the diffusion coefficients of lipid (1.5 +/- 0.5 X 10(-8) cm2/s) and protein (2.5 +/- 0.6 X 10(-9) cm2/s) differ only by a factor of 6, close to the difference predicted on the basis of size by the two-dimensional bilayer continuum model of Saffman and Delbrück [Saffman, P. G. l& Delbrück, M. (1975) Proc. Natl. Acad. Sci. USA 72, 3111-3113]. In contrast, the membranes of normal cells show a lipid diffusion coefficient (1.4 +/- 0.5 X 10(-8) cm2/s) that is some 300-fold greater than that of the membrane proteins (4.5 +/- 0.8 X 10(-11) cm2/s). Analysis of these results, based on the hypothesis that protein diffusion in normal membranes is sterically hindered by a labile matrix, yields an effective matrix surface viscosity consistent with the viscoelastic mechanical properties of the membranes. Thus, a relationship is established between the deformation characteristics of the membrane and the lateral mobility of proteins suspended in the membrane.

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[ 3H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[ 3H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.

Lateral diffusion in biological membranes. A normal-mode analysis of diffusion on a spherical surface

A new approach is described for the analysis of lateral diffusion in biological membranes. It is shown that a suitably defined first moment of the concentration distribution on a spherical surface decays as a single exponential with a relaxation rate proportional to the diffusion coefficient and inversely proportional to the square of the radius of the sphere. The approach is illustrated with an example of fluorescence redistribution after photobleaching of membrane proteins in a spectrin-deficient spherocytic mouse erythrocyte membrane.

Effects of bromelain and pronase on erythrocyte membranes

The effects of the proteolytic enzymes, bromelain and pronase, on mouse erythrocyte membranes have been examined using the techniques of polyacrylamide gel electrophoresis, surface labelling and crossed immunoelectrophoresis. The results show first that bromelain treatment digested protein bands, I, II and III of mouse erythrocyte membranes while pronase only digested band III. Secondly, both bromelain and pronase removed glycoprotein and protein components from the outer surface of mouse erythrocytes. Thirdly, at least one antigen found on the surface of bromelain-modified erythrocytes. is present within the membranes, but is not detectable on the surface of normal erythrocytes. On the basis of these results it is concluded that the proteases digest outer structures made up of protein and glycoprotein thereby exposing antigens that are normally hidden. It was observed that digestion, probably by endogenous proteases, persisted after bromelain and pronase-treated cells were washed. The possibility that this digestion may have contributed towards the revelation of target antigens was investigated. The inhibition of the persisting digestion did not reduce the number of antibody-producing cells detected.

Demonstration of MHC-specific haemolytic plaque-forming cells

IT would be convenient to be able to measure the antibody response to alloantigens of the major histocompatibility complex (MHC) at a cellular level. As MHC alloantigens are expressed on erythrocyte membranes in mice one might suppose that a conventional Jerne plaque assay with enumeration of haemolytic plaque-forming cells (allo-PFC) against a lawn of suitable allogeneic erythrocytes would be straightforward. In fact such assays have proved difficult, with very few plaques being detected1,2. We have found the same problem in detecting allo-PFC in the spleens of alloimmunised rats. Although alloantisera raised in appropriate strain combinations have a high titre in a direct complement dependent haemolytic assay (Fig. 1) emphasising the potential value of this convenient assay for alloantibody in the rat3,4, very few allo-PFC were found in spleen cell suspensions from hyperimmunised donors. We show here that the discrepancy may be due to the homogeneity of the antibody product of a single PFC compared with the heterogeneity of serum antibody.

Fluidity and Lipid Composition of Mouse Biomembranes during Adaptation to Ethanol

Showing that ethanol added in vitro increased the fluidity of mouse erythrocyte and synaptosomal membranes and that the same membranes isolated from mice after chronic treatment with ethanol had increased cholesterol and were resistant to ethanol in vitro, the study suggests that the animals had adapted to the chronic presence of the drug by changing their membrane lipid composition to offset the fluidizing effects of ethanol.

Lymphocyte Subpopulations in Lymphoproliferative Diseases

In analogy with the data of Henkart and Henkart for normal persons, lymphocytes with parallel tubular structures (PTS or the macrotubular bundle) of patients with Hodgkin’s disease were all found to bear an Fc receptor on their cell membrane (EA-RFC). An explanation for this correlation of EA-RFC and PTS or for the increase of EA-RFC in some patients with Hodgkin’s disease was not yet found. Comparison of groups of 22 normal subjects, 20 patients with Hodgkin’s disease, 20 with chronic lymphatic leukemia, and 20 with non-Hodgkin lymphoma showed the EA-RFC to be increased in the Hodgkin group. They were decreased in CLL patients, while mouse erythrocyte rosette-forming cells and membrane surface immunoglobulins in this group were increased. Surface Ig and EA-RFC varied widely in the non-Hodgkin lymphoma group of patients.

Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice

Mice were treated with ethanol for eight or nine days, using a liquid diet regimen known to produce physical dependence. In previous experiments, synaptosomal plasma membranes and erythrocyte ghosts from such ethanoltreated animals were found to be resistant to the fluidizing effects of ethanol in vitro, as measured by electron paramagnetic resonance. In the present experiments, corresponding membranes were analysed for phospholipid and cholesterol. The ratio of cholesterol to phospholipid was found to be significantly increased in both types of membrane after chronic ethanol treatment. The changed ratio was produced by an increase in cholesterol. There was little or no change in phospholipid content of the membranes. Increased cholesterol may explain the previously observed alteration of physical properties of the membranes.

Erythrocyte membrane antigen expression during Friend cell differentiation: analysis of two non-inducible variants.

AbstractErythrocyte membrane antigens have been detected on induced Friend erythroleukemic cells with a rabbit antiserum raised against mouse erythrocyte membranes. The antibody specificities of this antiserum have been quantitatively analyzed using a cellular radioimmunoassay. After absorption with thymocytes, the rabbit anti‐erythrocyte membrane serum bound to dimethylsulfoxide (DMSO)‐induced Friend erythroleukemic cells and to mouse erythrocytes but not to uninduced Friend cells or thymocytes. Reciprocal inhibition studies demonstrated that, following complete thymocyte absorption, the antiserum detected similar antigenic specificities, termed erythrocyte membrane antigens (EMA), on both mature erythrocytes and induced Friend cells.The expression of these erythrocyte membrane antigens was also induced on Friend cells by other agents, such as ouabain and dimethylacetamide (DMA). In contrast, exogenous hematin, which did not induce hemoglobin synthesis in the Friend cell clones used in this study, also did not induce erythrocyte membrane antigen expression.Two independently derived variant clones which do not produce hemoglobin in reponse to DMSO were analyzed for their ability to produce erythrocyte membrane antigens in response to various inducers of Friend cell differentiation. Clone TG‐13 is not inducible by DMSO or hematin but is weakly induced by DMA for both hemoglobin production and erythrocyte membrane antigen expression. Another variant clone, M18, was also analyzed. This clone does not synthesize detectable hemoglobin when grown in either DMSO or hematin alone, but undergoes extensive hemoglobin synthesis when grown in medium containing both DMSO and hematin. M18 does, however, express erythrocyte membrane antigens when grown in DMSO alone: the presence of hematin and DMSO together in the growth medium does not enhance expression of these antigens. Thus M18 appears to be defective for hemoglobin inducibility, and this defect can be overcome by exogenous hematin; however, the expression of erythrocyte membrane antigens is not affected by this block in hemoglobin synthesis. The results with the variant clones are discussed in terms of a program for Friend cell differentiation in which the induction of hemoglobin synthesis and erythrocyte membrane antigen expression are under both co‐ordinate and separate controls.

Erythrocyte membrane abnormality in muscular dystrophy

We found that erythrocyte membranes of dystrophic mice were considerably altered as measured by the technique of whole cell aqueous polymer partitioning. The difference was directly linked to the sex of the animal. Littermates of the diseased animals (genetic carriers or 'expressers') also had erythrocytes that were dramatically different from the sexand age-matched normals. The data show conclusively absolute differences in erythrocyte membranes from dystrophic animals. This observation has two implications: 1) expression of muscular dystrophy is not confined to muscle, and 2) the disease may be a general membrane defect instead of an abnormality of other cellular organelles. The prime defect in the group of diseases classified as muscular dystrophies is unknown except descriptively from the disease name--l.e., dystrophy of muscles (1,2). Furthermore, no curative therapy is known. Many investigators have been interested in whether the disease is manifest in organs other than muscle, and the idea of a systemic membrane defect has been advanced (3,4). Using aqueous polymer partitioning we are able to show definite alterations in erythrocyte membranes in dystrophic mice. Aqueous polymer partitioning is a sensitive technique for measuring properties of surface membranes (5,6). The technique is so sensitive that it is possible to identify subtle differences between membranes. However, the significance of these differences in partitioning ratios is uncertain-it can be shown only that a difference exists--but best speculations are that the technique