Localization of erythrocyte membrane antigens by immune electron microscopy

Torus (T) protein was prepared by differential centrifugation of hemoglobin-free membranes after freezing, thawing and dialysis against water. Ferritin-labeled antibody to T protein failed to agglutinate erythrocytes or to sensitize them to complement lysis. Anti-T bound specifically to the internal aspect of ghosts and to the exterior of inverted vesicles prepared from them. The findings lend further support to the supposition that native T protein exists only on the inner aspect of intact erythrocyte membranes; its possible relation to spectrin is uncertain. In contrast, ferritin-labeled antibody to purified erythrocyte glycoprotein (virus receptor substance, VRS) agglutinated erythrocytes, sensitized them to complement lysis and by its specific reactivity with surface antigen afforded a basis for the electron microscopic quantitation of receptor sites on plasma membranes examined in thin section as well as with freeze-etching.     

Surface carbohydrates of aged erythrocytes

Human erythrocytes, fractioned into populations of different density by ultracentrifugation in albumin gradients were examined to determine what changes in cell surface carbohydrates occur during their lifespan. In addition to changes occurring in $\text{N}$-acetylneuraminic acid ageing was accompanied by reduction in the $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and galactose content of erythrocyte membranes. These results show that extensive heterogeneity exists in the cell surface carbohydrate of the circulating population of erythrocytes and suggest clearance of neuraminidase treated erythrocytes may not be an adequate model for the removal of aged cells.     

Inhibitory effect of iron on the uptake of lead by erythrocytes.

It is well known that more than 90% of the lead found in blood is associated with the erythrocytes. The present $\text{in vitro}$ experiments show that the uptake of lead-203 by rabbit erythrocytes is inhibited by the presence of non-radioactive lead or iron or by reduction of the incubation temperature. The inhibitory effect of iron on radioactive lead uptake by erythrocytes is also demonstrable $\text{in vivo}$.When lead-203 is incorporated into erythrocytes $\text{in vitro}$, about 10% of the radioactivity is attached to the membrane and the remainder is found in the cytoplasm associated with hemoglobin and an unidentified low molecular weight intracellular component. In the presence of 25 μg/ml of added iron (Fe⁺⁺⁺) the uptake of radioactive lead by erythrocytes is reduced to 21.7±5.1% and membrane binding accounts for approximately 5% of this total. Chromatographic analyses of hemolysates show that the reduction in cytoplasmic labeling is directly related to decreased lead binding to the low molecular weight component, since hemoglobin binding remains unchanged.This work suggests that in addition to the interaction between iron and lead which occurs during the biosynthesis of heme, these metals may directly compete for specific erythrocyte binding sites.     

Avidin-induced lysis of biotinylated erythrocytes by homologous complement via the alternative pathway depends on avidin's ability of multipoint binding with biotinylated membrane.

It was reported that avidin and streptavidin induce lysis of prebiotinylated red blood cells via the alternative pathway of both homologous and heterologous complement. Both of these proteins have four biotin-binding sites, providing a polyvalent interaction with biotinylated components of the erythrocyte membrane. We have compared the effects of mono- and multipoint avidin attachment on the sensitivity of biotinylated erythrocytes to lysis by the complement system. In the presence of anti-avidin antibody, avidin-bearing biotinylated erythrocytes were rapidly lysed by heterologous serum. This lysis was independent from the mode of avidin attachment, implying that complement activation by the classical pathway triggered by interaction between C1 and avidin-bound antibody on the erythrocyte surface is independent from the avidin's ability of polyvalent (multipoint) binding with biotinylated membrane components. In the absence of anti-avidin antibody, biotinylated erythrocytes bearing polyvalently attached avidin were lysed by homologous complement better than cells bearing avidin, which possesses reduced ability for multipoint binding with biotinylated erythrocyte. Two independent approaches to reduce avidin's ability of multipoint binding were used: decrease in surface density of biotin on the erythrocyte membrane and blockage of biotin-binding sites of avidin. Both methods result in reduced lysis of avidin-bearing erythrocytes as compared with erythrocytes bearing an equal amount of polyvalent-bound avidin. Thus the activation of homologous complement via the alternative pathway depends on avidin's ability to 'cross-link' to the biotinylated components of the erythrocyte membrane.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe³⁺, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 °C, while that for phosphatidylserine spin label had only one transition at 30 °C.When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogeneous fluidity. Mg²⁺ or $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ prevented the hemolysis-induced spectral changes. Ca²⁺ did not prevent the homogenization and acted antagonistically to Mg²⁺. The heterogeneity preservation by Mg²⁺ was nullified by trypsin, pronase or $\text{N-}\text{ethylmaleimide}$ added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg²⁺ was dependent on hemolysis temperature, starting to decrease at 18 °C and vanishing at 40 °C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Membrane transport in resealed haemoglobin-containing human erythrocyte 'ghosts' prepared by a dialysis procedure

Resealed haemoglobin-containing erythrocyte ‘ghosts’ have been proposed as $\text{in}\text{vivo}$ carriers for enzyme replacement therapy. Transport of substrates and metabolites into and out of the ‘ghost’ has been suggested to be a limiting factor in such therapy. Studies of the transport of L-phenylalanine and of uric acid in normal human erythrocytes and prepared ‘ghosts’, in which the transport of sodium ions and D-glucose was intact, have shown that transport characteristics of ‘ghosts’ are identical to those of normal erythrocytes with transport not being a quantitatively limiting factor.     

Inhibition by zinc of the binding of C1 to antigen-antibody complexes

Zinc sulphate in the range of 10^?4 to 2×10^?5 M prevents the binding of $\text{C}\text{1}$ to antigen antibody complexes, and the initation of the cascade of events in the classical complement pathway leading to cell lysis. Other heavy metals, Co⁺⁺, Cd⁺⁺, Cu⁺⁺, or Mn⁺⁺ were without effect in this concentration range. Zinc was ineffective when added after $\text{C}\text{1}$ was bound and failed to displace $\text{C}\text{1}$ which was already bound to antigen antibody complexes. The ability of zinc to regulate the binding of the zymogen or activated form of $\text{C}\text{1}$ to antigen-antibody complexes represents a new method of controlling the initiation of the classical complement pathway.     

Changes in nucleotide concentrations in the erythrocytes of man, rabbit and rat during short-term storage

The $\text{ATP}\text{ADP}$ ratio, measured by high performance liquid chromatography, has been used as an indicator of stability of erythrocyte nucleotides. The nucleotides from human, rabbit and rat whole blood, but not separated erythrocytes were stable for maximum periods of 40, 20 and 15 min respectively after venepuncture. The ratios then declined rapidly from 9 to 5, 12 to 4 and 9 to 1 respectively during 2h storage at room temperature. Similar changes occurred in $\text{GTP}\text{GDP}$ ratios. The relevance of these observations to metabolic studies in intact cells, nucleotide analyses in the clinical situation and comparative studies in other species is discussed.     

Rheological characteristics of desialylated erythrocytes in relation to fibrinogen-induced aggregation

The effect of fibrinogen and sialic acid content of erythrocytes on the aggregation of erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. (1) The electrophoretic mobility of erythrocytes was proportional to the sialic acid content of erythrocytes (the surface potential of erythrocytes could be expressed by the sialic acid content). (2) The aggregation of erythrocytes was accelerated by increasing fibrinogen concentration in the medium (due to the increased bridging force among erythrocytes) or by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes). (3) An empirical equation expressing the velocity of aggregate formation (ν, in μm²/min) by the concentration of fibrinogen ($\text{F}$, in g/dl) and the sialic acid content ($\text{S}$, in μmol/ml red blood cells), log $\text{ν = ?0.065 F}{}^{\mathrm{?1.2}}\text{S + 2.2 F}{}^{0.35}$, was deduced. (4) The contribution of the bridging force of fibrinogen to the erythrocyte aggregation was much greater than that of the electrostatic repulsive force produced by sialic acid on the surface of erythrocytes.     

Comparison of monocyte and alveolar macrophage antibody-dependent cellular cytotoxicity and Fc-receptor activity

The cytotoxic potential of rabbit peripheral blood monocytes and alveolar macrophages in antibody-dependent cellular cytotoxicity (ADCC) toward both erythrocyte (RBC_ox) and tumor cell (CEM T-lymphoblast) targets was examined. ADCC was measured in a 4-hr ⁵¹Cr-release assay. Alveolar macrophages were more efficient at killing the tumor cell targets (optimally sensitized with rabbit antisera) than monocytes at similar effector cell/target cell ($\text{E}\text{T}$) ratios. Tumor cell targets sensitized with seven different antisera (anti-CEM) were lysed by alveolar macrophages but not by the monocytes. In marked contrast, the monocytes were more effective at lysing the sensitized erythrocyte target cells. The degree of cytolysis of RBCox and CEM was dependent on the $\text{E}\text{T}$ ratio and the degree of sensitization of these target cells. It was demonstrated that the effector cell selectivity in ADCC was directly related to their ability or inability to bind the sensitized target cells as determined by Fc-receptor rosette formation. The transition from monocyte to macrophage may, therefore, have resulted in an alteration in the criteria necessary for Fc-receptor binding to antibody-sensitized target cells and subsequent ADCC.     

An electron cytochemical study of 6-phosphogluconate dehydrogenase activity in infected erythrocytes during malaria

The distribution of 6-phosphogluconate dehydrogenase (6-PGD) was examined by an electron microscopic technique in erythrocytes of rodents infected with $\text{Plasmodium berghei}$, chickens with $\text{P. gallinaceum}$ and rhesus monkeys with $\text{P. knowlesi}$. Unlike glucose-6-phosphate dehydrogenase, which is restricted to the host erythrocyte, 6-PGD was found to be present in the parasite as well as the host erythrocyte in all infections studied. The implications of these findings are discussed in relation to the metabolism of malaria parasites.     

MEMBRANE LESIONS IN IMMUNE LYSIS : Surface Rings, Globule Aggregates, and Transient Openings

It is known that there are 100 Å-wide circular structures associated with the erythrocyte membrane in immune lysis. To determine whether these structures were functional holes extending through the membrane, freeze-etch electron microscopy was carried out. Sheep erythrocytes incubated with either rabbit complement or rabbit antibody (anti-sheep erythrocyte antibody) did not hemolyze and did not reveal any abnormalities in freeze-etch or negative-stain electron microscopy. Erythrocytes incubated with both complement and antibody revealed rings on the extracellular surface (etch face) of the cell membrane. Allowing for the 30 Å-thick Pt/C replica, the dimensions of the surface rings were similar to those seen by negative staining. The ring's central depression was level with the plane of the membrane; some rings were closed circles, others were crescent shaped. The cleavage face of the extracellular leaflet revealed globule aggregates, each aggregate appearing to be composed of about four fused globules. The cleavage face of the cytoplasmic leaflet was normal. When immune lysis was carried out in the presence of ferritin, ferritin was subsequently detected in all lysed erythrocytes. If ferritin was added after immune lysis was complete, only 15% of the cells were permeated by ferritin, indicating that transient openings exist in the cell membrane during immune lysis. No abnormal structures were detected when C6-deficient rabbit serum was used as a source of complement. It is concluded that antibody and complement produce surface rings, prelytic leakage of K⁺, colloid osmotic swelling, membrane disruption, and membrane resealing; the surface rings persist after these events.     

The relation between Krogh and compartmental transport models

The meaning of compartmental concentration is intuitively simple in well-stirred compartment models with uniform concentration but not so in the general case. This meaning may be better understood by relating the general compartmental model to a spatially explicit one such as the Krogh cylinder model. We show that compartmental equations result directly by spatial averaging of the Krogh partial differential equations. Intercompartmental transport is usually stated as flux is proportional to the capillary-tissue concentration difference or $\text{J = ? (}\text{C}{}_{\mathrm{c}}\text{-}\text{C}{}_{\mathrm{T}}\text{)}$. We verify this simple equation by showing that flux in the Krogh model is also approximately first order and then derive the transport coefficient, ?, in terms of the geometry and diffusivity in the Krogh model. A relation between capillary and venous concentration or, for a gas, partial pressure is needed. For the highly diffusable, metabolic gas C0₂ this relation is $\text{P}{}_{\mathrm{c}}\text{= P}{}_{\mathrm{v}}\text{?}\text{M}\text{20K}{}_{\mathrm{c}}$ that is, capillary and venous partial pressures differ by one half the metabolic generation.     

A kinetic model for the quantitative analysis of complement

A simple model has been developed which accurately predicts the time course of complement mediated lysis of sensitized red cells. The model assumes that the one hit theory of immune hemolysis is applicable and that the rate of lysis is directly proportional to the concentration of a complement component present in rate limiting amounts. It also assumes that the rate of lysis is dependent on the fraction of cells lysed. The model can be related to the classical von Krogh equation for end point complement analyses and can be used to estimate the rate constant for the critical step in hemolysis, as well as the efficiency of the critical complement component in the rate limiting step. Parameters derived from the model can be quantitatively related to complement concentration and can be used as the basis for a quantitative assay of complement activity. The model can also be used to calculate, for a particular sample, the concentration at which complement activity becomes undectable, the complement activity of the pure, undiluted sample, and the time required for the sample to produce complete lysis of the available cells.     

Anestrus in cycling beef heifers experimentally inoculated with Anaplasmamarginale

Normal estrous cycles were established for twenty beef heifers. Ten heifers were inoculated with blood from a known $\text{Anaplasma}$$\text{marginale}$ carrier. The inoculated heifers experienced anemia, anorexia, and positive $\text{Anaplasma}$$\text{marginale}$ complement-fixing antibody titers, and $\text{A}$. $\text{marginale}$ was observed on the erythrocytes while uninoculated control heifers remained normal. Further observation following inoculation revealed that five of ten inoculated heifers experienced anestrus while controls continued to cycle normally. Anestrus coincided with clinical signs of acute anaplasmosis. Normal estrus patterns returned following treatment and recovery. This study provides evidence that acute anaplasmosis in beef heifers may cause anestrus and therefore lead to reproductive inefficiency.     

A differential interaction of daunomycin,adriamycin and their derivatives with human erythrocytes and phospholipid bilayers

Drug-membrane association of daunomycin, adriamycin and three of its derivatives, adriamycin-14-octanoate (AD-14-OCTA), adriamycin-14-acetate (AD-14-ACE) and $\text{N-}\text{trifluoroacetyladriamycin-14-valerate}$ (AD32), was studied using phospholipid bilayers and human erythrocytes. The various drugs exhibited a differential affinity to membrane-lipid domains.Lipid-incorporated drugs exhibit a marked change in the shape of the emission spectrum which was utilized for the evaluation of the apparent dielectric constant, ?, of the environment surrounding the anthracycline moiety, as well as for the determination of the partitioning constant. By measuring the fluorescence polarization and the fluorescence lifetime of the incorporated drugs, rotational relaxation times of 4–8 ns were derived. These parameters provide a supportive evidence for the association of the fluorophore of the drugs with membrane-lipid domains.The anthracycline derivatives interact to a different degree with dipalmitoyl phosphatidylcholine and phosphatidylserine as reflected by changes in their thermotropic properties assessed by differential scanning calorimetry. Daunomycin was the most effective in decreasing the temperature of the phase transition and brought about a comparable reduction in the enthalpy of melting as AD32 and AD-14-OCTA. Adriamycin was the least potent of the series.AD-14-ACE and AD32 protected erythrocytes against hypotonic lysis, adriamycin and daunomycin had no significant effect on the susceptibility to hypotonic lysis, whereas AD-14-OCTA proved to be hemolytic even at low concentration (approx. 10^?7 M).The interaction of erythrocytes with daunomycin, AD-14-ACE and Ad-14-OCTA resulted in a shape change from biconcave discs to cups. Adriamycin and AD32 did not affect erythrocyte shape.The differential drug-membrane interactions may be an important determinant in the antitumor differential efficiency of the drugs, especially in view of the fact that derivatives that do not intercalate into the DNA (AD32) are at least as potent as those that do.     

Permeability changes of erythrocytes and liposomes by 5-(n-alk(en)yl) resorcinols from rye

$\text{5-(n-}\text{Alk(en)yl}\text{)}$ resorcinols can induce potassium release from liposomes and erythrocytes. The results suggest that $\text{5-(n-}\text{pentyl)resorcinol}$ can induce a specific permeability to protons as well as to potassium and other small molecules. The highest permeability changes were found in the presence of $\text{5-(n-}\text{pentadecyl)resorcinol}$ and alkenyl resorcinols. Orcin and resorcin were without effect. The size of permeant as investigated by turbidity measurements indicated that Ca²⁺ and Mg²⁺ cannot pass through the alkyl resorcinol-modified membrane but can pass through the alkenyl resorcinol-modified membrane. It was observed that alkenyl resorcinol at a concentration of 15 μM induced not only potassium release but also lysis of erythrocytes.     

Fluorescence polarization study of human erythrocyte membranes with 1-phenyl-3-(2-naphthyl)-2-pyrazoline as orientational probe

The emission and polarization spectra of 1-phenyl-3-(2-naphthyl)-2-pyrazoline (PNP) in various environments were studied. Compared to the widely used orientational membrane probe 1,6-diphenylhexatriene (DPH), PNP is five times less photolabile and since its fluorescence emission maximum is at longer wavelengths $\text{(γ}{}_{\max }\text{≈ 445}\text{nm}\text{)}$, it is more suitable for use with intact erythrocytes. The limiting fluorescence anisotropy of PNP is 0.385. In erythrocyte ghosts, the steady-state emission anisotropy of PNP is a decreasing function of wavelength and its temperature dependence parallels that of DPH, dropping from 0.298 at 2°C to 0.185 at 38°C when averaged between 420 and 470 nm.     

The fate of SV40 DNA in infected cells after treatment with benzo(a)pyrene diol epoxide

Treatment of SV40-infected monkey kidney cells with $\text{anti}$-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide results in the alkylation of viral DNA. The specific infectivity of viral DNA isolated from diol epoxide-treated cells is markedly lower than that of viral DNA from untreated cells. Within 5–6 hours following treatment, approximately half of the alkylated viral DNA disappears; during this same period, single-stranded regions in SV40 DNA are detected. These data indicate that, in infected cells, a significant amount of the alkylated viral DNA is removed from the cellular DNA pool while the remainder stays intact. Whether BP-modified SV40 DNA undergoes repair is still uncertain.