Erythrocytes In Solutions Research Articles

Receptor-based identification of an inhibitory peptide against blood stage malaria

Plasmodium falciparum uses multiple host receptors to attach and invade human erythrocytes. Glycophorins have been implicated as receptors for parasite invasion in human erythrocytes. Here, we screened a phage display cDNA library of P. falciparum (FCR3, a sialic acid-dependent strain) using purified glycophorins and erythrocytes as bait. Several phage clones were identified that bound to immobilized glycophorins and contained the same 74bp insert encoding the 7-amino acids sequence ETTLKSF. A similar screen using intact human erythrocytes in solution identified additional phage clones containing the same 7-amino acids sequence. Using ELISA and immunofluorescence, direct binding of ETTLKSF peptide to glycophorins and erythrocytes was confirmed. Pull-down and protease treatment assays suggest that ETTLKSF peptide specifically interacts with glycophorin C. The synthetic ETTLKSF peptide partially blocks merozoite invasion in human erythrocytes. Further characterization of ETTLKSF peptide could lead to the development of a novel class of inhibitors against the blood stage malaria.

An interfacial instability approach to erythrocyte adhesion by macromolecules

Models of cell plasma membrane interactions in the presence of adhesive macromolecules often follow the general principles initially formalised by Derjaguin, Landau, Verwey and Overbeek for colloidal particles in suspension. In this model the parallel membranes of mutually adhering cells are considered to lie in an energy minimum at an equilibrium separation distance which is determined by the relative size and range of attractive and repulsive interactions. In contrast, the interfacial instability model considered here directs attention to the dynamic processes which can result in deformation of a thin aqueous intermembrane film to give localised spatially periodic contact points. The contact seam of erythrocytes suspended in polycation, polysaccharide or lectin solutions has been examined in this work by light and electron microscopies. The seams formed by erythrocytes in solutions of the lectins concanavalin A and wheat germ agglutinin show spatial periodicity of contacts (range 0.8-0.5 μm). In normal cells the polycation-induced seam is always spatially periodic while it is usually continuous and parallel in dextran solutions. In the latter case, pretreatment of the cells by the protease pronase results in spatially periodic contacts on subsequent exposure of the cells to dextran. For both polycation- and polysaccharide-induced contact the experimental changes which increase or decrease membrane attraction consistently lead to a decrease or increase respectively in the lateral contact separation distance (range 3.4–0.8 μm). These changes in lateral contact separation distances are qualitatively consistent with the predictions of interfacial instability theory. The changes are discussed as particular examples of a general consequence of the form of membrane interaction profiles.

Investigation of monovalent cation influxes of diamide-treated human erythrocytes in solutions of different ionic strenght

Total and residual i.e. (ouabain + bumetanide + EGTA)-insensitiv K + as well as Na + influxes were investigated in human erythrocytes before and after treatment with diamide (5 mM). In physiological and in low ionic strength solution these influxes were increased after diamide treatment. Diamid-treated cells do not exhibit significant differences between the total and residual influxes for both Na + and K +. The diamide-induced cation fluxes in low ionic strength solution are significantly higher compared with the fluxes in physiological ionic strength solution. The diamide-induced K + influx is not chloride-dependent, and replacement of NaCl by sodium methylsulfate does not significantly reduce this flux. A subsequent incubation of diamide-treated erythrocytes with dithioerythritol which restores the cellular glutathione level to its original value only partly decreases the enhanced K + influx. From these results it can be concluded that electrodiffusion and K/Cl cotransport are not involved in the diamide-induced stimulation of the residual K + influx of human erythrocytes.

Studies on the cytoskeletal and nuclear architecture of Xenopus erythrocytes.

A proteinaceous cytoskeletal network is present in nucleated erythrocytes, which is obscured ultrastructurally in whole cells due to the presence of haemoglobin. Lysis of Xenopus erythrocytes in solutions containing Triton X-100 reveals a cytoskeleton that contains a centrally positioned nucleus, which is linked to the cell surface-associated cytoskeleton by intermediate filaments. The marginal band microtubules are also preserved in these structures. In addition, a single or a pair of perinuclear centrioles is frequently observed in thin sections. These structures are surrounded by a mass of intermediate filaments and fibrogranular material. In contrast to the centrioles in invertebrate erythrocytes those in Xenopus erythrocytes are not associated with the marginal band. Cytonuclear skeletons were obtained by DNase I digestion and subsequent high-salt extraction of cytoskeletons. The resulting structures were chromatin-depleted and consisted of a nuclear lamina that was maintained in the same overall shape and position as that of intact nuclei. With the exception of the marginal band, the remaining cytoskeletal elements persisted after these treatments. Although marginal bands were not detectable by electron microscopy, the cytonuclear skeletons contained roughly the same amount of tubulin as cytoskeletons, as indicated by immunoblotting with affinity-purified anti-tubulin antibodies. When intact erythrocytes were exposed to the ionophore A23187 in the presence of calcium, the cell shape and centric nuclear position were altered. Nuclear dislodgement may be attributable to the disruption of intermediate filament associations with the subsurface cytoskeletal shell. Indirect immunofluorescent staining of cytoskeletons lysed in buffers containing either EGTA or calcium indicates that in the absence of calcium, the intermediate filament network extends to the cell periphery. In structures lysed in calcium, however, the filaments are restricted to the vicinity of the nucleus.

11] Preparation of human factor [formula omitted] of the alternative pathway of complement

Publisher Summary This chapter presents the procedure for purification and assaying of human Factor D. Outdated human plasma can be used to prepare satisfactory yields of Factor D but it is preferable to use fresh or flesh-frozen plasma because material having a similar molecular weight and similar ion-exchange properties may be generated on prolonged storage of plasma or even of the partially purified fractions during the purification procedure. Addition of protease inhibitors in the early stages of the procedure did not appear to increase the recovery of Factor D. Plasma or serum may be used in the method in this chapter, the yield and final specific hemolytic activity of factor D being the same in both cases. When serum was used it was prepared from the plasma by clotting overnight at 4°C on the addition of 1.0 M CaC1 2 to give a final concentration of 20 mM. The clot was removed by centrifugation and filtration through muslin. The hemolytic activity of factor D involves the alternative pathway dependent lysis of rabbit erythrocytes in solution, or by alternative pathway-dependent lysis of guinea pig erythrocytes on an agarose plate. The factor D hemolytic activity is determined by the extent of hemolysis at 412 nm.

Time Progression of Hemolysis of Erythrocyte Populations Exposed to Supraphysiological Temperatures

Populations of erythrocytes in solution were heated “instantaneously” to and maintained at temperatures in the range of 44 to 60°C on a microscope stage specifically designed for this purpose. Simultaneously, the visually observed hemolysis-time history of these cells was measured. The results were successfully correlated on the basis of two models: 1) a kinetic scheme assuming two sequential, first-order reactions by which the cells are first reversibly altered and then irreversibly damaged; and 2) a statistical model for which the number of cells that are damaged at each instant is assumed to be normally distributed. From the experimental data the rate constants for the two reactions in the kinetic model were determined and were found to have an Arrhenius dependence on temperature. By applying the statistical model to the data, we were able to determine the mean and standard deviation of the distribution curve for this model. The logarithms of these latter two parameters vary with temperature in a linear fashion.

Regulation of Sugar Transport in Avian Erythrocytes

Abstract Inhibition of oxidative metabolism by cyanide, dinitrophenol, arsenate, or simple anoxia accelerated the carrier-mediated transport of monosaccharides in the erythrocytes of several avian species. This conclusion was supported by demonstrations of saturation kinetics, specificity of transport, competition between pairs of sugar, and counter-transport. Accelerated transport was shown for both the entry and exit of nonmetabolized sugars. The major kinetic effect was an increase in the maximal rate of transport. Treatment of avian erythrocytes with sulfhydryl blocking agents, such as HgCl2 and N-ethylmaleimide, resulted in an increased rate of transport of nonmetabolized sugars. The accelerated transport rate observed in cells treated with these agents was competitively inhibited by the addition of glucose. The increased transport rate induced by HgCl2 was immediately reversed by the addition of sulfhydryl compounds. HgCl2 treatment did not result in an increased level of 5'-AMP in the cells, suggesting that the acceleration of transport was not the result of an inhibition of oxidative metabolism. These observations suggest that the regulation of sugar transport in these cells may involve a sulfhydryl group. A partial reversal of the anoxic stimulation of transport by dithiothreitol strengthens this possibility. Hemolysis of erythrocytes in solutions of low osmotic strength followed by restoration of membrane integrity in solutions containing 300 milliosmoles per liter resulted in increased rates of transport in aerobic cells, suggesting that some inhibitor of transport present in aerobic cells had been lost. The accelerated rate of transport observed in cells subjected to hemolysis and restoration could not be inhibited by dithiothreitol.

Behavior of Erythrocytes in Various Solvent Systems II: Effect of Temperature and Various Substances on Water—Glycerin and Water–Propylene Glycol Solutions

Hemolytic behavior of rabbit and human erythrocytes in water-glycerin and water-propylene glycol solutions was studied at 25 and 37°, and the effect of various added substances to these systems was investigated. Human <i>i</i> values obtained for sodium chloride in aqueous glycerin or propylene glycol solutions at 37° were slightly greater than the corresponding <i>i</i> values at 25°. Increase in temperature from 25 to 37° decreased the concentrations of propylene glycol in 0.9 per cent saline solution needed to cause hemolysis of erythrocytes. Mono-monovalent salts, sugars and sugar alcohols, magnesium chloride, and sulfate and sodium succinate afforded essentially the same degree of protection as sodium chloride against hemolysis by propylene glycol. Isotonic concentrations of sodium or potassium sulfate, potassium sodium or disodium tartrate, or trisodium citrate afforded greater protection to erythrocytes than 0.9 per cent sodium chloride. The order in which the anions of the above salts appeared to protect human erythrocytes against propylene glycol hemolysis is citrate>tartrate>gluconate>sulfate; for rabbit erythrocytes the order is sulfate, tartrate>citrate. Much lower concentrations of propylene glycol were required to hemolyze erythrocytes in solutions containing isotonic concentrations of calcium chloride.

Isotonic Solutions IX: The Permeability of Red Corpuscles to Some Monohydric- and Polyhydhc Alcohols

The degree of hemolysis of rabbit and human erythrocytes in solutions of a series of monohydric and polyhydric alcohols was investigated. When possible, these data were used to calculate van't Hoff i values. The monohydric and polyhydric alcohols which had a molecular weight less than that of erythritol caused complete hemolysis in concentrations calculated to be isosmotic. Certain concentrations of the monohydric alcohols caused hemolysis even in the presence of 0.6 per cent (0.1 M ) sodium chloride. The low molecular weight diols and triol investigated did not cause hemolsis in the presence of 0.6 per cent sodium chloride. The olyhydric alcohols of higher molecular weight prevented hemolysis. It appeared that the phenomena occurring at the erythrocyte membrane in the case of the alcohols might be influenced by molecular weight and the number of hydroxyl groups present; the ratio of hydroxyl groups to the number of carbon atoms in the molecule might be important.

PHYSIOLOGIC HYPERBILIRUBINEMIA AND HEMOLYSIS OF ERYTHROCYTES BY HYDROGEN PEROXIDE SOLUTION IN THE NEWBORN: INVESTIGATION OF A POSSIBLE RELATIONSHIP

Forty-three full-term infants have been studied with respect to hemolysis of erythrocytes in solutions of hydrogen peroxide and concentrations of bilirubin in the serum. Mean values for concentration of bilirubin in the serum and percentage of hemolysis followed similar patterns in the first few days of life. However, statistical analysis of the data in individual cases showed no significant correlation between the degree of hemolysis in solutions of hydrogen peroxide and the concentrations of bilirubin in the serum. Administration of vitamin E prevented an increase in hemolysis of erythrocytes in solutions of hydrogen peroxide but failed to produce any significant change in concentrations of bilirubin as compared with the control group. The evidence suggests that the relative deficiency of vitamin E which exists in most newborn infants does not play a part in the causation or maintenance of physiologic hyperbilirubinemia. The clinical significance of increased hemolysis of the erythrocytes of the newborn infant in solutions of hydrogen peroxide remains a mystery. Possible approaches to the clarification of this problem are suggested.

THE ROLE OF CARBONIC ANHYDRASE IN CERTAIN IONIC EXCHANGES INVOLVING THE ERYTHROCYTE

1. The acceleration by bicarbonates of the swelling and hemolysis of erythrocytes in solutions of ammonium salts, first reported by Ørskov, is strikingly dependent upon carbonic anhydrase, being almost abolished by inhibitors of this enzyme such as KCN and sulfanilamide, and under suitable conditions being enhanced by its addition to the external solution. This behavior gives support to the theory of "catalyzed diffusion" as an explanation of the Ørskov effect. 2. The inhibitory effects of both sulfanilamide and KCN seem to be capable of complete reversal on washing the erythrocytes in isotonic salt solutions. The full effect of KCN appears almost instantly; that of sulfanilamide requires a period measured in seconds, or possibly even in minutes, to reach its maximum, the delay presumably being due to the slower penetration of the erythrocyte by this substance. Under favorable conditions the effect of concentrations of sulfanilamide of a few hundredths of a milligram per cent can be demonstrated. No similar effects have been obtained with sulfapyridine. 3. Bicarbonates also have a "catalytic" effect on the response of the internal pH of erythrocytes to changes in that of their surroundings. The resulting volume changes of the cell, which otherwise frequently require many minutes for their completion, may take place within a few seconds in the presence of low concentrations of bicarbonates. At a given pH value the effect of the latter substances is chiefly on the rate of the change and only to a minor extent on its magnitude. It may be further accelerated under appropriate conditions by the addition to the cell suspension of carbonic anhydrase, and can be almost abolished by KCN and by sulfanilamide. 4. Volume changes of erythrocytes associated with exchanges of Cl' for SO(4)'' ions are greatly accelerated by low concentrations of bicarbonates, this effect being likewise dependent upon carbonic anhydrase. There is some evidence that in this case the exchange takes place, at least in part, in two steps: Cl' for HCO(3)' and HCO(3)' for SO(4)''.

Sedimentation of Erythrocytes in Solutions of Albumin, Fibrinogen and Peptone

Beef-blood serum-albumin, when suspeiidecl in Locke's solution or in plasma, prolonged the sedimentation time of human erythrocytes. A solution of human-blood serum-albumin suspended in Locke's solution and in plasma prolonged the sedi mentat ion time. A sample of powdered human-blood serum-albumin shortened the sedimentation time when it was suspended in Locke's solution, and prolonged it when suspended in plasma. A solution of bleef -blood serum-fibrinogen suspended in Locke's solution and in plasma did not significantly affect the sedimentation time. Witte's peptone suspended in Locke's solution or in plasma prolonged the sedimentation time insignificantly. Talc or kaolin were without effect on the sedimentation time, Dog-bile suspended in plasma prolonged the Sedimentation time.

Suspension Stability of Erythrocytes in Solutions of Globulin.

SummaryAn analysis of the effects of 6 different types of globulin, dissolved in Locke's solution, upon the sedimentation time of erythrocytes, reveals that in 12 experiments 4 of the types prolonged the sedimentation time, and that in 12 experiments the remaining 2 types shortened the sedimentation time. In 8 experiments in which 2 of the types of globulin were dissolved in plasma, the sedimentation time was prolonged.

Suspension Stability of Erythrocytes in Solutions of Gum Acacia.

SummaryThe factors responsible for alterations in the suspension stability of erythrocytes is independent of the corpuscles. Solutions of gum acacia reduce the suspension stability of the blood. A solution of 3 or 4% of acacia is the minimal amount that will produce the maximal instability of erythrocyte sedimentation. The rapidity of formation and the size of agglutinated erythrocyte masses progresses parallel to the increase in concentration of acacia in solution. The curve of sedimentation for rapidly sedimenting bloods is not a rectilinear function.