Structural research on the membranes and adjacent layers of erythrocytes during the production of erythrocyte ghosts

Using the polarization microscopy and X-ray crystal analysis it has been shown that the ordered structure is destroyed in membranes and adjacent layers of erythrocytes in the course of erythrocyte ghost preparation. These changes are accompanied by quenching of the birefringence which completely disappears in erythrocytes ghosts. The intensities of reflections in X-ray patterns are essentially decreased and the interlayer distances are changed.     

Polarized light microscopy in the study of the molecular structure of collagen and reticulin

Although collagen structure has been studied by polarized light microscopy since the early 19th century and continued since, modern studies and reviews failed to correlate the conclusions based on data obtained by the techniques with those of polarized light microscopy. Collagen I is intensely positively birefringent in respect to length of the fibres; the positive intrinsic birefringence indicates a quasi-crystalline alignment parallel to the fibre and molecule axis of the amino acid residues of the polypeptide chains. This would not have been compatible with a helical structure but has been achieved by similar tilt angles and opposite directions of the coiling and supercoiling. Birefringence characteristics of collagen are also affected by chemical treatments, extractions and staining procedures. Attachment of chemical groups to the anionic charges present on the surface of collagen molecules results in increased positive birefringence in the case of bipolar molecules attached to two or more anionic residues. Unipolar attachment to the same groups, or to the cationic groups of the associated proteoglycans, as well as sulfation or acetylation of hydroxyls of the protein and/or the carbohydrate, reduced or reversed the sign of birefringence. Increased birefringence caused by stretching cannot be due to intramolecular events and is caused by intermolecular changes. The same applies to changes in collagen during aging. Reticulin is a group of different substances which mostly contain collagen III. The pliability and deformability of this collagen is related to its weakly negative birefringence due to large side chains and presence of different and greater amounts of interstitial proteoglycans and other molecules. The so-called reticulin of healing wounds differs in its constitution from other reticulins but is also rich in intermolecular carbohydrate components.     

The effect of cholesterol level in erythrocyte membranes on the sedimentation rate, electrophoretic motility, and the rate of potassium ferricyanide reduction

It has been found that with an increase of the molar ratio cholesterol/phospholipids in human erythrocyte membranes in vitro their sedimentation rate in diluted suspensions increases and the reduction rate of potassium ferricyanide decreases, while the electrophoretic mobility of erythrocytes is not changed. The effect of cholesterols discussed in terms of the mechanism of transmembrane transfer of reduction equivalents and non-equilibrium in the double electric layer of erythrocytes surface.     

The kinetics of drug-membrane interactions in human erythrocytes from neonates

The kinetics of drug-membrane interactions of erythrocytes from neonates were compared with those from adults by monitoring the time course of the shape transformations and vesicle release caused by drugs, using a light microscope--video recording technique. Both crenation caused by lysophosphatidylcholine (LPC) and cupping caused by chlorpromazine (CPZ) took place more slowly in the neonatal cells than in the cells from adults. The equilibrium concentrations of LPC and CPZ in erythrocytes did not differ significantly between the neonates and adults, however. The slower responses of the neonatal erythrocytes can be explained by the presence of negatively charged phosphatidylethanolamine and phosphatidylserine in the outer layer of the erythrocyte membrane, which may reduce the rate of incorporation of amphipathic LPC and attract cationic CPZ to remain in the outer membrane layer, lowering the rate of inward bending of the membrane normally caused by CPZ.     

Increased myosin orientation during muscle contraction: a measure of cardiac contractility

Myosin form birefringence has been studied in cryostat sections of left ventricular myocardium from the dog and human. The muscle in such sections has been shown to demonstrate the sliding filament phenomenon. The sarcomere length of canine myocardium agreed with that found in comparable electron micrographs. Unexpectedly, it was found that glycerol, normally used as an inert and optically ideal mountant, caused profound change in myosin birefringence. This apparently invalidates results obtained with this mountant. The absolute birefringence found in these sections, whether mounted in glycerol or in an ATP-calcium buffer, corresponded to values found by other workers with skeletal muscle and isolated myosin. However, the birefringent properties (optical path difference: o.p.d.) of well functioning muscle was found to be low, the o.p.d. increasing when exposed to ATP and calcium. Poorly functioning muscle could be distinguished from well functioning muscle on the basis of its higher 'in air' o.p.d. This difference correlated well with physiological assessments of myocardial function or with clinical assessments of cardiac failure. Evidence is presented indicating that changes in apparent birefringence, caused by ATP-calcium or by anoxia, are due to altered orientation of the myosin micelles and can be inhibited by agents that inhibit myosin ATPase activity.     

Birefringence of Protein Solutions and Biological Systems. I

The quantitative interpretation of birefringence of biological structures such as muscle requires a knowledge of intrinsic birefringence of the components. The intrinsic birefringence of fibrous structures as determined by variation of solvent index is positive while the intrinsic birefringence of proteins in solution is negative as calculated by the Peterlin-Stuart theory. As a first step in clarifying this discrepancy the basis of the Peterlin-Stuart theory has been re-examined. The theory has been recalculated from the standpoint of light scattering and extended to particles whose length is not small compared to the wavelength. The birefringence of a system of particles possessing a shell with index different from the bulk solvent has been obtained in order to interpret measurements in mixed solvents.     

Electrostatic repulsion among erythrocytes in tube flow, demonstrated by the thickness of marginal cell-free layer

Electrostatic repulsion among erythrocytes in flow was evaluated through measurement of the thickness of the marginal cell-free layer in narrow glass tubes of 20-50 microns in inner diameter. To reduce the electrostatic repulsive force, due mainly to sialic acid of the membrane glycoproteins, human erythrocytes were treated with neuraminidase. The surface negative charge of the erythrocytes, as determined from the electrophoretic mobility using free-flow electrophoresis, was found to be proportional to the sialic acid content. When erythrocytes with decreased sialic acid content flowed through narrow tubes, the thickness of cell-free layer determined using an image processor increased even in the absence of erythrocyte aggregation in the suspension. The effect was more pronounced at acidic pH. The addition of Dextran T-70 (70,400 Mol. Wt.) further increased the cell-free layer thickness due to erythrocyte aggregation. Thus, reducing the negative charge density on the erythrocyte surface by itself accelerates the axial accumulation of erythrocytes in flow due to the decreased electrostatic repulsive force between the cells, even in the absence of erythrocyte aggregation.     

Ribosomal proteins S2, S6, S10, S14, S15 and S25 are localized on the surface of mammalian 40 S subunits and stabilize their conformation. A study with immobilized trypsin

Trypsin immobilized on collagen membranes has been used to digest accessible ribosomal proteins of rat liver 40 S subunits. Six proteins (S2, S6, S10, S14, S15 and S25) have been found to be highly exposed on the surface of 40 S particles. They appear to be in close physical contact and localized in the same region of the subunit, most likely protruding at its surface. Electric birefringence reveals that digestion of these proteins results in unfolding of subunits: the birefringence of 40 S particles becomes negative, like that of RNA, the relaxation time undergoes a 15-fold decrease and the mechanism of orientation is drastically modified.     

STRUCTURE OF THE PEAR LEAF CUTICLE WITH SPECIAL REFERENCE TO CUTICULAR PENETRATION

Study of the pear leaf cuticle (Pyrus communis L. ‘Bartlett‘), in both intact and enzymatically isolated forms, has revealed that the cuticular membrane is separated from the underlying epidermal cell wall by a layer of pectic substances which extend into but not through the membrane. A layer of embedded birefringent waxes occurs towards the outer surface of the cuticular membrane. Platelet-like epicuticular waxes are deposited on the outer surface. The upper cuticular membrane is astomatous. The lower epidermis is stomatous, and the outer cuticular membrane is continuous with that lining the substomatal cavity. The lower cuticular membrane is also generally thicker than the upper, and both the upper and lower cuticular membranes are thicker over veinal than over mesophyll tissue. The birefringence frequently is discontinuous over anticlinal walls and over veinal tissue. The lower cuticle appears to contain fewer embedded waxes (as indexed by birefringence) than the upper. Enzymatic isolation of the cuticular membrane from the underlying tissues does not appear to cause any discernible change in structure as viewed with a light microscope. These findings are discussed in light of current knowledge concerning penetration of foliar applied substances into the leaf.     

Comparison of membrane structure,osmotic fragility,and morphology of multiple sclerosis and normal erythrocytes

Erythrocyte membranes from multiple sclerosis (MS) patients and normal individuals were studied by electron spin resonance spectroscopy, osmotic fragility tests, scanning electron microscopy (SEM) and fatty acid analysis of membrane lipids. There was no significant difference in the membrane fluidity between MS and normal erythrocytes using fatty acid spin labels with the nitroxide moiety on carbons 5, 12, or 16 from the carboxyl group. Linoleic acid, which has been reported to decrease the absolute electrophoretic mobility of only MS erythrocytes, increased the fluidity of MS and normal erythrocyte membranes to a similar extent. The osmotic fragility of MS erythrocytes obtained from outpatients was similar to normal control cells but the osmotic fragility of erythrocytes obtained from hospitalized MS patients was greater than normal. Scanning electron microscopy of MS erythrocytes revealed no gross abnormalities. Cells incubated with linoleic acid had transformed from discocytes into sphero-echinocytes with prominent membrane surface indentations but MS and normal erythrocytes appeared identical. Of the fatty acid content of the total lipid extract, erythrocytes from most, but not all, MS hospitalized patients and some patients with other demyelinating diseases had relatively less (P<.001) 18:2 than the normal cells. These results indicate that at least some of the abnormalities reported in MS erythrocytes may only be found in hospitalized patients and may be due to other complications of the disease. They also indicate that the reported abnormal effects of linoleic acid on the electrophoretic mobility of MS erythrocytes may be caused by some other mechanism than an effect on the fluidity of the bilayer.     

An experimental test of new theoretical models for the electrokinetic properties of biological membranes. The effect of UO2++ and tetracaine on the electrophoretic mobility of bilayer membranes and human erythrocytes

For a large smooth particle with charges at the surface, the electrophoretic mobility is proportional to the zeta potential, which is related to the charge density by the Gouy-Chapman theory of the diffuse double layer. This classical model adequately describes the dependence of the electrophoretic mobility of phospholipid vesicles on charge density and salt concentration, but it is not applicable to most biological cells, for which new theoretical models have been developed. We tested these new models experimentally by measuring the effect of UO2++ on the electrophoretic mobility of model membranes and human erythrocytes in 0.15 M NaCl at pH 5. We used UO2++ for these studies because it should adsorb specifically to the bilayer surface of the erythrocyte and should not change the density of fixed charges in the glycocalyx. Our experiments demonstrate that it forms high-affinity complexes with the phosphate groups of several phospholipids in a bilayer but does not bind significantly to sialic acid residues. As observed previously, UO2++ adsorbs strongly to egg phosphatidylcholine (PC) vesicles: 0.1 mM UO2++ changes the zeta potential of PC vesicles from 0 to +40 mV. It also has a large effect on the electrophoretic mobility of vesicles formed from mixtures of PC and the negative phospholipid phosphatidylserine (PS): 0.1 mM UO2++ changes the zeta potential of PC/PS vesicles (10 mol % PS) from -13 to +37 mV. In contrast, UO2++ has only a small effect on the electrophoretic mobility of either vesicles formed from mixtures of PC and the negative ganglioside GM1 or erythrocytes: 0.1 mM UO2++ changes the apparent zeta potential of PC/GM1 vesicles (17 mol % GM1) from -11 to +5 mV and the apparent zeta potential of erythrocytes from -12 to -4 mV. The new theoretical models suggest why UO2++ has a small effect on PC/GM1 vesicles and erythrocytes. First, large groups (e.g., sugar moieties) protruding from the surface of the PC/GM1 vesicles and erythrocytes exert hydrodynamic drag. Second, charges at the surface of a particle (e.g., adsorbed UO2++) exert a smaller effect on the mobility than charges located some distance from the surface (e.g., sialic acid residues).     

A simple and effective method for hemolysis with a hypoxanthine-xanthine oxidase system and alteration of erythrocyte phospholipid composition during the hemolysis

A very rapid hemolysis was found to be caused by active oxygen species produced by a hypoxanthine-xanthine oxidase system with very low concentrations of hypoxanthine. The addition of superoxide dismutase or catalase inhibited the hemolysis, indicating that O2- and H2O2 participate in this system. The extent of erythrocyte hemolysis was found to depend on the sex of the human donor. The change in phospholipid composition before and after hemolysis in human erythrocytes from donors of each sex was compared by thin layer chromatography. A significant decrease in phosphatidylethanolamine content and a concomitant increase in altered phospholipid fraction were observed in erythrocytes from male donors, suggesting that these erythrocytes were easily attacked by active oxygen species to produce modified phosphatidylethanolamine.     

Influence of green tea on surface charge density and phospholipids composition of erythrocytes membrane in ethanol intoxicated rats

Ethanol is oxidized to acetaldehyde and then to acetic acid and these processes are acompanied by free radical generation. This paper reports the effect of green tea on electric charge and phospholipids composition of erythrocytes membrane from rats intoxicated with ethanol. Electrophoresis technique and HPLC have been applied to above-menthioned studies. Ethanol administration caused increase in erythrocyte membrane surface charge density and phospholipid composition. Ingestion of green tea with ethanol partially prevented changes in structure and function of membrane caused by chronic ethanol intoxication.     

A quantitative description in three dimensions of oxygen uptake by human red blood cells

Oxygen uptake by human erythrocytes has been examined both experimentally and theoretically in terms of the influence of unstirred solvent layers that are adjacent to the cell surface. A one-dimensional plane sheet model has been compared with more complex spherical and cylindrical coordinate schemes. Although simpler and faster, the plane sheet algorithm is an inadequate representation when unstirred solvent layers are considered. The cylindrical disk model most closely represents the physical geometry of human red cells and is required for a quantitative analysis. In our stopped-flow rapid mixing experiments, the thickness of the unstirred solvent layer expands with time as the residual turbulence decays. This phenomenon has been quantified using a formulation based on previously developed hydrodynamic theories. An initial 10(-4) cm unstirred layer is postulated to occur during mixing and expand rapidly with time by a (t)0.5 function when flow stops. This formula, in combination with the three-dimensional cylinder scheme, has been used to describe quantitatively uptake time courses at various oxygen concentrations, two different external solvent viscosities, and two different internal heme concentrations.     

Phosphorylation and degradation of ribosomes in starved Tetrahymena pyriformis.

We have characterized an embryonic antigen on the surface of chick erythrocytes using immunochemical electron microscopy. An indirect surface labeling technique (hemocyanin conjugated to goat antirabbit IgG and specific antisera prepared in rabbits) revealed that the antigenic sites, at hatching, nearly saturate the surface of erythrocytes with hemocyanin markers. The number of antigenic sites gradually decreases with age, and the antigen can no longer be detected at 7 months. Further, the antigen has been detected on the very earliest primitive erythrocytes which form in the extra-embryonic mesenchyme before circulation begins. The embryonic antigen appears to be firmly associated with the erythrocyte surface and cannot be removed by extensive washing either with phosphate-buffered saline or with EDTA. Labeling unfixed cells at 37 °C produces clustering of the surface markers, suggesting that the antigen is associated with a membrane component which is fairly free to move in the plane of the membrane. In addition, the erythrocytes from newly hatched chicks were found to agglutinate more readily with several different lectins, particularly Concanavalin A (ConA), than did the erythrocytes from adults. Three times more ConA is bound to chick erythrocytes than to adult erythrocytes, as estimated by electron microscopy. Although this difference in lectin binding suggests that the ConA-binding sites might be related to the embryonic antigen, the sugars known to block lectin-induced hemagglutination had no blocking effect on antiserum-induced agglutination or on antibody binding, as visualized by the electron microscope technique. Also, ConA binding was not inhibited by treatment of the chick erythrocytes with the specific antiserum.     

Surface components of shigellae involved in adhesion and haemagglutination

Strains of Shigella species were studied for their ability to adhere and agglutinate mammalian erythrocytes. Shigella dysenteriae and Sh. flexneri exhibited haemagglutinating (HA) properties when cultured in Casamino Acids-Yeast Extract (CYE) broth in the presence of 1 mmol 1^-1 calcium chloride, but other shigellae did not show this property under the same culture conditions. Repeated subcultivation of Sh. boydii, Sh. sonnei and HA negative strains of Sh. dysenteriae and Sh. flexneri in CYE broth medium induced adhesive and haemagglutinating properties that were inhibited by sodium periodate. HA activities of Shigella spp. were also inhibited by N -acetylneuraminic acid, α₁-glycoprotein and fetuin, but not by protease. Electron microscopy of Sh. dysenteriae 1, Sh. flexneri 2a, Sh. boydii 12 and Sh. sonnei 1 grown in CYE broth showed the presence of an extracellular slime layer that promoted agglutination of erythrocytes. The slime layer extracted from the cell surface of Shigella spp. showed HA properties, whereas lipopolysaccharide (LPS) obtained from the same strains, except Sh. dysenteriae 1, did not agglutinate erythrocytes. This evidence suggests that the cell surface haemagglutinin is a loosely bound slime layer which is expressed in CYE broth medium.     

Electric Birefringence in Solutions of High Molecular Weight Ribonucleic Acid

The electric birefringence of low ionic strength solutions of high molecular weight ribonucleic acids from various sources was studied. RNA preparations with a high helical content were found to have negative electric birefringence as a result of the segment anisotropy of the helical portions of the RNA molecule. For completely unfolded molecules of RNA, the electric birefringence is positive and results from the orientation of the macromolecular coil. In intermediate cases, the observed electric birefringence is the sum of negative and positive birefringence. The negative birefringence is caused by the segment orientation of helical sections, and the positive birefringence is caused by the orientation of the macromolecular coil as a whole. Different relaxation times for the positive and negative birefringence permit the pulsed electric birefringence method to analyze these separate phenomena.     

Dielectro-deformations of erythrocyte: analysis of the ellipsoidal shear model

Using the theoretical analysis within the framework of the proposed ellipsoidal shear electromechanical model of erythrocyte, the main mechanisms and relationships have been established and studied for the deformations of erythrocytes caused by a spatially homogeneous high-frequency electric field. The main types of the stress-strain curves characteristic of stationary and dynamic deformations caused by the rectangular-pulse and harmonic modulations of the field amplitude have been calculated. The relationship has been established between the parameters of essentially nonlinear stress-strain curves and mechanical, electric, and geometric parameters of erythrocyte. The impossibility of unlimited elongation of erythrocyte by the field, due to the conservation of the cell volume and surface area, has been shown, and the dependence of the maximum possible elongation of the cell on its volume has been calculated. It has been shown that the relaxation time of dynamic deformations of erythrocyte in the presence of an electric field considerably differs from that characteristic of the membrane material and sharply decreases with the increase of the initial elongation of the cell.     

Correlation between the oscillatory and adhesion activities in erythroid cells

The attachment kinetics of erythroid cells, such as human erythrocytes, their saponin ghosts, and erythroleukemic cells K562 to a glass surface has been studied in the presence of substances inhibiting spontaneous fluctuations of cell membranes. It has been shown that wheat germ agglutinin (WGA) slows down the attachment kinetics of K562 cells, as is the case in intact erythrocytes. Concanavalin A (Con A), which inhibits the attachment of erythrocytes to glass does not affect the adhesion of K562 cells to glass due to the absence of band 3 proteins in the membranes of K562 cells. Both lectins slow down the adhesion rate of saponin ghosts of human erythrocytes, as it takes place in intact erythrocytes. Suramin and the anionic dye ANS bind specifically to the actin protofilaments of the erythrocyte skeleton and also inhibit cell adhesion to glass. At the same time, these substances do not affect the oscillatory and adhesion activities of intact erythrocytes due to the impermeability of erythrocyte membranes for these drugs. The results obtained allow the conclusion that inhibition of erythrocyte adhesion by lectins is due to lectin binding to different constituents of the erythrocyte membrane--sialic acid moieties of glycophorin in the case of WGA and band 3 proteins in the case of Con A. The most probable mechanism of erythrocyte and K562 cell attachment to glass is the formation of the so-called local contacts between cells and the glass surface. It is also suggested that the cell surface oscillations facilitate the formation of cell contacts.     

Immune complex binding by erythrocytes and its significance in the destruction of these cells during immunization

Immunization of BALB/c mice by sheep red blood cells and Salmonella typhi vaccine has been shown to augment the immune complexes in plasma and erythrocytes in blood fixing the immune complexes on their surface. The inactivation of immune complexes in immunized mice by intravenous injection of the antiserum against aggregated immunoglobulins decreases the hemoglobin in blood serum. The data obtained show that the fixation of immune complexes on erythrocytes is one of the reasons of erythrocytes destruction activation in immunization.