A dynamic and stationary rheological study of erythrocytes incubated in a glucose medium

A higher than normal glucose concentration in a suspending medium may produce non-enzymatic glycosylation of erythrocyte proteins. This process can modify the viscoelastic properties of erythrocytes. In this paper, we studied the possible relationship between glucose concentration in a suspending medium and erythrocyte rheological parameters. Human venous blood was obtained from the antecubital veins of 10 healthy volunteers. Blood samples were anticoagulated with EDTA and centrifuged. Red blood cells (RBCs) were washed and subsequently divided in aliquots, which were incubated in vitro with glucose solutions of different concentrations. Dynamic and stationary viscoelastic parameters of RBCs were determined by laser diffractometry in an Erythrodeformeter. Aggregate shape parameter (ASP) of the RBCs was determined by digital image processing. Significant changes were observed both in ASP and in rheological parameters when the glucose concentration in the medium was increased, demonstrating that a glucose concentration as low as 1% induces alterations in the mechanical properties of RBCs.     

Biorheological Action of Ascaris lumbricoides Larvae on Human Erythrocytes

Previous studies have shown that A. lumbricoides extracts capture sialic acid (SA) from human red blood cells (RBC). The aim of this work was to study hemorheological alterations in vitro caused by parasite larvae. The biorheological action of three larva concentrates of first and second larval stage on group O erythrocytes was analyzed by incubating the erythrocyte packed together with an equal volume of larvae (treated RBC) and PBS (control RBC). Distribution and parameters of aggregation (digital image analysis), aggregation kinetics (erythroaggregameter), and viscoelasticity (erythrodeformeter) were measured. The digital image analysis showed that all the larvae diminished the isolated cells percentage and increased the size of the formed aggregates. The aggregate formation velocity was lower in the treated than in the control. The deformability index (ID) values of treated RBC did not present variations with respect to those of the control, but a decrease in the erythrocyte elastic modulus (μ_m) and membrane surface viscosity (η_m) values was observed, indicating that the larvae not only induced a diminution in the membrane surface viscosity of RBC but also altered the dynamic viscoelasticity of the membrane. Experiments carried out in vitro support the conclusion that the contact between larvae and RBC produces hemorheological alterations.     

Effect of polyene antibiotics on the lectin-induced agglutination of transformed and untransformed cell lines.

Treatment of transformed Py3T3, SV101-3T3, and L1210 cells, as well as mitotic and Pronase-treated untransformed 3T3 cells, with the polyene antibiotics filipin, nystatin, and amphotericin B inhibited agglutination by wheat germ agglutinin. The effect of polyene antibiotic treatment was lectin and cell specific. Concanavalin A induced agglutination was not inhibited, wheat germ agglutination induced agglutination of untransformed 3T3 interphase cells was not influenced, and other aggregation phenomena, including those of erythrocytes with blood group specific antibodies or divalent cations, were unaffected by polyene treatments. This suggests that the formation of polyene-cholesterol complexes in transformed and erythrocyte cell membranes may specifically affect wheat germ agglutinin receptors and/or secondary events necessary for wheat germ agglutinin induced agglutination. Fluorescence studies of membrane filipin-cholesterol complexes showed that pretreating the cells with wheat germ agglutinin, but not concanavalin A, perturbed the fluorescence properties of filipin. Electron spin resonance studies with spin-labeled fatty acids revealed at best only a slight decrease in fatty acyl chain flexibility following filipin treatment. These studies indicate that there are not only quantitative differences between the agglutinability of transformed and untransformed cells with wheat germ agglutinin but that qualitative differences exist as well.     

Effects of wheat germ agglutinin on membrane transport

(1) Low concentrations of wheat germ agglutinin are cytotoxic toward several tissue culture lines, including Chinese hamster ovary cells, Swiss 3T3 cells, mouse L cells and baby hamster kidney cells. The LD50 ranged from 1 to 5 microgram wheat germ agglutinin per ml. Similar concentrations of the lectin inhibited the transport of the non-utilizable amino acids alpha-aminoisobutyric acid and cycloleucine and inhibited the uptake of thymidine. In contrast, 2-deoxy-D-glucose uptake was not altered and colchicine uptake was enhanced. (2) The inhibition of alpha-aminoisobutyric acid uptake occurred within minutes after lectin addition and was maximal by 1 h. Maximal inhibition ranged from 50 to 70% of control values. Studies of the kinetics of the uptake demonstrated that wheat germ agglutinin decreased the V of the uptake by 70% without affecting the apparent Km. Ovomucoid, a haptene inhibitor of wheat germ agglutinin-binding to cell surface receptors, prevented the wheat germ agglutinin-induced inhibition of alpha-aminoisobutyric acid transport. Three other lectins (Concanavalin A, Phaseolus vulgaris E-phytohemagglutinin and L-phytohemagglutinin) inhibited the uptake by 20% or less at doses up to 50 microgram/ml. (3) We propose that the cytotoxicity of wheat germ agglutinin probably results in part, if not totally, from membrane alterations which impair multiple membrane transport systems.     

Isolated erythroglycans have a high-affinity interaction with wheat germ agglutinin but are poorly accessible in situ

The sugar and cell specificities of wheat germ agglutinin have been studied extensively. In particular, it is well established that wheat germ agglutinin will interact with highly sialylated glycoconjugates of the type carried by the erythrocyte glycoprotein, glycophorin (Adair, W.L. and Kornfeld, S. (1974) J. Biol. Chem. 249, 4696-4704). We have found that polylactosamines isolated from adult and fetal erythrocytes can have a high-affinity interaction with immobilized wheat germ agglutinin. In fact, this interaction is much stronger than the sialic acid-dependent interaction. Using flow microfluorimetry in conjunction with various serological and enzymatic pretreatments, we have measured the extent to which polylactosamines contribute to wheat germ agglutinin binding. We have found that most of the neuraminidase-resistant receptors on erythrocytes are polylactosamine in nature. However, this residual binding of wheat germ agglutinin to neuraminidase-treated erythrocytes is of much lower apparent affinity than the sialic acid-dependent interaction. The lower reactivity of polylactosamines at the erythrocyte surface suggests that these large glycans are actually poorly accessible.     

Changes in surface architecture during murine erythroleukemia cell differentiation as detected by lectin binding and agglutination

Cell surface alterations occurred during murine erythroleukemia cell (clone 745) differentiation that were detected by both agglutination and lectin binding. Agglutination of erythroleukemia cells was produced by wheat germ agglutinin; whereas, concanavalin A, Ricin, soybean agglutinin and fucose-binding protein were either ineffective or much less efficacious. Treatment of leukemia cells with the inducer of erythroid differentiation dimethylsulfoxide (DMSO) caused a progressive accumulation of hemoglobin-containing cells in culture and a decrease in the rate of agglutination by wheat germ agglutinin, which began at 24 h after exposure to the polar solvent, reached a nadir at 48 h, and remained essentially constant thereafter. The binding of radioactive wheat germ agglutinin by untreated control erythroleukemia cells increased with time in culture, reaching a maximum value at 48 h, and decreased progressively thereafter. Although an increase in ³H-labeled wheat germ agglutinin binding also occurred in DMSO-treated cells, the level bound was significantly lower than that observed in control cells at 24–96 h. The treatment of erythroleukemia cells with various concentrations of DMSO resulted in a decrease in the number of wheat germ agglutinin receptor sites. Other inducers of differentiation (i.e., dimethylformamide, bis(acetyl)diaminopentane) also inhibited the rate of wheat germ agglutinin-induced agglutination of erythroleukemia cells while, in contrast, the inducer tetramethylurea did not. These studies indicate that membrane changes occur during differentiation and suggest that there may be more than one mechanism involved in the initiation of maturation which ultimately leads to the common pathway of erythroid development.     

Determination of red blood cell shape recovery time constant in a couette system by the analysis of light reflectance and ektacytometry

Red blood cell (RBC) shape change under shear is generally reversible, with the time course of shape recovery a function of the elastic and viscous properties of the RBC membrane. RBC shape recovery can be investigated, using several different techniques, to provide information about the membrane material properties that are not directly accessible by frequently used methods to assess RBC deformability (e.g., micropore filtration). In the present study, RBC shape recovery was studied in a Couette system after abrupt cessation of shear, either by analyzing the time course of laser light reflection or by serial measurements of elongation indexes from laser diffraction patterns. The time course of shape recovery monitored with both techniques can be described with an exponential equation. Calculated time constants for normal human RBC were 119 ± 17 msec and 97 ± 15 msec as measured by light reflection and ektacytometry, respectively. Treatment of RBC with glutaraldehyde resulted in dose-dependent decreases in the shape recovery time constant. Heat treatment (48 ° C, 20 min), which is known to increase mainly the shear elastic modulus of the membrane, decreased the time constant by 65%. In contrast, wheat germ agglutinin treatment increased the shape recovery time constant by 22%, presumably by increasing membrane surface viscosity. Our results indicate that the shape recovery time constant of RBC can be measured easily and accurately by computerized light reflection analysis.     

Agglutination of Staphylococcus saprophyticus: a structural and cytochemical study

Abstract Staphylococcus saprophyticus was shown to be agglutinated by wheat germ agglutinin, wheat germ agglutinin-biotin and bovine serum albumin- p -aninophenyl- N -acetyl-β-d-glucosaminide (GlcNAc-BSA), and sheep red blood cells. In these agglutinations, filamentous or amorphous structures radiating from the surface of S. saprophyticus were demonstrated by electron microscope observation. Cytochemical analyses of the agglutination revealed the binding sites of wheat germ agglutinin in S. saprophyticus and the binding sites of GlcNAc in the sheep red blood cells and S. saprophyticus . Since GlcNAc-BSA contains N -acetylglucosamine to which wheat germ agglutinin can bind, it is most likely that an interaction between a wheat germ agglutinin-bindable substance in S. saprophyticus and an N -acetylglucosamine-bindable substance in sheep red blood cells is involved in the agglutination.     

Protein translocation across the endoplasmic reticulum. I. Detection in the microsomal membrane of a receptor for the signal recognition particle

Thin-section and critical-point-dried fracture-labeled preparations are used to determine the distribution and partition of glycophorin- associated wheat germ agglutinin (WGA) binding sites over protoplasmic and exoplasmic faces of freeze-fractured human erythrocyte membranes. Most wheat germ agglutinin binding sites are found over exoplasmic faces. Label is sparse over the protoplasmic faces. These results contrast with previous observations of the partition of band 3 component where biochemical analysis and fracture-label of concanavalin A (Con A) binding sites show preferential partition of this transmembrane protein with the protoplasmic face. Presence of characteristic proportions of WGA and Con A binding sites over each fracture face is interpreted to indicate the operation of a stochastic process during freeze-fracture. This process appears modulated by the relative expression of each transmembrane protein at either surface as well as by their association to components of the erythrocyte membrane skeleton.     

Exposed and hidden lectin-binding epitopes at the surface of<Emphasis Type="Italic">Borrelia burgdorferi</Emphasis>

The presence of surface- and subsurface-located lectin-binding epitopes of Borrelia burgdorferi was examined by electron microscopy using a variety of gold-labeled lectins. Concanavalin A reacted predominantly with extracellular material adjacent to the spirochetes. Wheat germ agglutinin bound weakly to the surface of borreliae; however, alterations of the outer membrane by preincubation in 100 ppm Triton X-100 or boiling uncovered numerous periplasmic sites recognized by the lectin. The periplasmic flagella liberated by some cells after detergent treatment were labeled with concanavalin A, wheat germ agglutinin and Ulex europaeus agglutinin UEA-I. No surface-exposed or periplasmic epitopes for the lectins from Glycine max, Dolichos biflorus or Helix pomatia were detected.     

Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.     

Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents.

Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.     

Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents

Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.     

The chromaffin granule surface Localization of carbohydrate on the cytoplasmic surface of an intracellular organelle

Intact chromaffin granules from bovine adrenal medulla are shown to have complex carbohydrates on their external (cytoplasmic) surface. This is demonstrated by the facts (1) that granules can be agglutinated by wheat germ agglutinin, and (2) that significant amounts of sialic acid can be removed from the granule surface with neuraminidase. Glycoproteins located in the granule membrane, and not glycolipids, are the molecules that mediate wheat germ agglutinin agglutination. The possible involvement of granule surface carbohydrate in the process of exocytosis is discussed.     

Imaging morphological details and pathological differences of red blood cells using tapping-mode AFM

The surface topography of red blood cells (RBCs) was investigated under near-physiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but non-invasive attachment of the cells. Using tapping-mode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circular-shaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane.     

Expression of lectin receptors on the surface of granulocyte-macrophage progenitor cells (CFU-gm)

It has been emphasized that specific bindings between membrane glycoproteins and membrane lectin-like substances are important in cell-to-cell interactions. We explored the surface of granulocyte-macrophage precursor cells (CFU-gm) by the differential agglutination technique. Enrichment of CFU-gm in the agglutinated fraction, containing the cells which have lectin receptors, from marrow treated with soybean agglutinin (SBA), peanut agglutinin (PNA) and concanavalin A (Con A), suggests the presence of reactive galactosyl and mannosyl residues on the surface of CFU-gm. On the other hand, wheat germ agglutinin (WGA), phytohemagglutinin (PHA) and ulex europaeus agglutinin (UEA), which bind to reactive N-acetylglucosamine, N-acetylgalactosamine and fucose, respectively, did not specifically agglutinate CFU-gm. Thus, reactive groups containing galactosyl and mannosyl structures on the surface of CFU-gm may possibly play a role in the process of cell-to-cell interactions between CFU-gm and marrow stromal cells.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Effects of wheat germ agglutinin and concanavalin A on parameters of highly purified sodium-potassium adenosine triphosphatases from Squalus acanthias and Electrophorus electricus.

The effects of two lectins, wheat germ agglutinin and concanavalin A, were studied on a variety of parameters of two highly purified (Na+ + K+)-ATPases (ATP phosphohydrolase, EC 3.6.1.3), from the rectal salt gland of Squalus acanthias and from the electroplax of Electrophorus electricus. Both lectins agglutinated the rectal gland enzyme equally, but wheat germ agglutinin inhibited (Na+ + K+)-ATPase activity much more. The electroplax enzyme was only marginally agglutinated and inhibited by the lectins. Neuraminidase treatment of the rectal gland (Na+ + K+)-ATPase had no effect on germ agglutinin inhibition. The inhibition of the rectal gland (Na+ + K+)-ATPase by wheat germ agglutinin could be reversed by N,N'-diacetylchitobiose, which has a high affinity for wheat germ agglutinin. Neither ouabain inhibition nor ouabain binding to the rectal gland enzyme was affected by wheat germ agglutinin. The p-nitrophenylphosphatase activity of the rectal gland enzyme was not inhibited by wheat germ agglutinin. Na+-ATPase activity, which reflects ATP binding and phosphorylation at the substrate site was inhibited by wheat germ agglutinin and this inhibition was reversed by potassium. Evidence is cited (Pennington, J. and Hokin, L.E., in preparation) that the inhibition of the (Na+ + K+)-ATPase by wheat germ agglutinin is due to binding to the glycoprotein subunit.     

Similarities in the membrane fluidity of 3T3 and SV101-3T3 cells and its relation to concanavalin A- and wheat germ agglutinin-induced agglutination.

Intact, viable ultransformed 3T3 and transformed SV101-3T3 cells were labeled with fatty acid spin labels and with 2,2,6,6-tetramethylpiperidine-1-oxyl in order to measure the fluidity properties of membrane lipids. Both cell types were grown in regular calf serum and in a lipid-depleted serum supplemented with either oleate or elaidate. The temperature dependence of the spectra obtained revealed inflections that correlate with the temperature below which agglutination with concanavalin A is inhibited, and another inflection that correlates with the temperature below which agglutination with wheat germ agglutinin is inhibited, suggesting that (a) the lipid phase(s) in the vicinity of the receptor(s) for these two lectins differ, and (b) a fluid membrane in the vicinity of the lectin receptor(s) is necessary for agglutination with either concanavalin A or wheat germ agglutinin. Studies with a partially characterized plasma membrane fraction suggest that the plasma membrane fluidity parameters closely resemble those of the intact cell. 3T3 and SV101-3T3 cells show virtually identical fluidity profiles by all of the tests we have applied.