Furosemide-sensitive Na+ and K+ transport and human erythrocyte volume

The relationship between cation transport and cell volume in human erythrocytes was investigated by measuring ouabain-sensitive K⁺ influx, ouabain-resistant, furosemide-sensitive K⁺ influx, and ouabain + furosemide-resistant K⁺ influx, and maximal ouabain binding in microcytic, normocytic and macrocytic red cells. A significant correlation was found between the mean corpuscular volume and furosemide-sensitive K⁺ influx normalized either to cell number (r = 0.636, P < 0.001) or to cell volume (r = 0.488, P < 0.001). No relationship was seen between mean corpuscular volume and ouabain-sensitive K⁺ influx, and the number of ouabain-binding sites per cell was only weakly correlated with mean corpuscular volume (r = 0.337, P < 0.05). A slight, negative relationship existed between mean corpuscular volume and ouabain + furosemide-resistant K⁺ influx expressed per volume of cells (r = −0.359, P < 0.01), and an apparent relationship between furosemide-sensitive K⁺ influx and mean corpuscular hemoglobin concentration (r = 0.446, P < 0.01) disappeared when microcytic samples were excluded from analysis. Furosemide-sensitive transport, including Na⁺ influx and K⁺ and Na⁺ efflux, was completely absent in microcytic cells from one patient with α-thalassemia minor. In addition, these cells exhibited a furosemide-resistant, Cl⁻-dependent K⁺ influx. Exposure of normal erythrocytes to hypotonic conditions (196 mosM) increased furosemide-sensitive K⁺ influx by a mean of 45% (P < 0.05), while exposure to hypertonic conditions (386 mosM) had no significant effect. The results indicate that furosemide-sensitive transport and cell volume are interrelated in human erythrocytes. However, the inability to fully recreate this relationship with in vitro manipulation of cell volume suggests that this relationship is established prior to red cell maturation.     

Evaluation of genetic risks of alkylating agents IV. Quantitative determination of alkylated amino acids in haemoglobin as a measure of the dose after-treatment of mice with methyl methanesulfonate.

The present study explores the possibilities of using specific amino acids in haemoglobin for tissue dosimetry of alkylating agents. The well-known directly alkylating compound methyl methanesulfonate has been used as a model compound.In one experiment ³H-labelled methyl methanesulfonate was given to mice intraperitoneally at three dose levels. The degree of alkylation of haemoglobin exhibited a linear dependence on the quantity of methyl methanesulfonate injected. The degree of alkylation of guanine-N-7 in DNA indicated a slight positive deviation from linearity at high doses.After a single injection the degree of alkylation of cysteine-S and histidine-N-3 in haemoglobin decreased linearly with time reaching the value zero after about 40 days (the life-time of the erythrocytes in the mouse). This demonstrates a stability of these alkylated products, which is fundamental to their use as integral dose monitors.In a second experiment mice were treated with methyl methanesulfonate once a week over a period of 8 weeks. The experiment demonstrated an accumulation of alkylated groups in haemoglobin in agreement with expectation.A method for the quantitative determination of S-methylcysteine in a protein hydrolysate by gas chromatography was developed.     

Does diamide treatment of intact human erythrocytes cause a loss of phospholipid asymmetry?

Diamide-treated human erythrocytes have been compared with native red cells as to the accessibility of their amino phospholipids to both phospholipase A₂ hydrolysis and fluorescamine labeling. In agreement with observations by others (Haest, C.W.M., Plasa, G., Kamp, D. and Deuticke, B. (1978) Biochim. Biophys. Acta 509, 21–32), treatment of intact human erythrocytes with diamide resulted in considerably enhanced degradation of amino phospholipids upon subsequent incubation of the cells with bee venom phospholipase A₂. The hydrolysis of phosphatidylethanolamine (PE) in control cells reached a plateau value at 5% after 10 min. In diamide-treated cells, on the other hand, PE hydrolysis did not level off. Contrastingly, dose-response curves recorded for the labeling of PE with the very fast reacting NH₂-group-specific reagent, fluorescamine, showed identical results for both native and diamide-treated erythrocytes. In each of these two cases, a plateau was reached after approx. 15% of the PE had been labeled. These results strongly suggest that the enhanced phospholipase-A₂-induced hydrolysis of amino phospholipids in diamide-treated erythrocytes may reflect a destabilization of the lipid bilayer, rather than an in situ loss of phospholipid asymmetry.     

Correlation of Ultrastructure in Azotobacter vinelandii with Nitrogen Source for Growth

Azotobacter synthesizes an extensive internal membranous nework when grown with air (N₂), i.e., under conditions when these bacteria fix nitrogen. Very slight quantities of internal membrane, concentrated mainly about the cell periphery, are formed when Azotobacter grows with fixed nitrogen, i.e., ammonia and amino acids. Compared to cells growing with ammonia, cells utilizing atmospheric nitrogen as the sole nitrogen source are smaller in size and volume, grow one-third slower, and lack detectable poly-β-hydroxybutyrate.     

Methionine transport in the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na⁺ and H⁺. Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin.     

The effect of age on beta-adrenergic receptors and adenylate cyclase activity in rat erythrocytes.

Beta-adrenergic receptors and catecholamine-sensitive adenylate cyclase activity were studied in erythrocytes obtained from rats 6 weeks, 6 months, and 15 months of age. Intact erythrocytes from 6 week old rats contained significantly more beta receptors (411 ± 31 sites/cell) than 6 month (328 ± 21) or 15 month old rats (335 ± 16), as determined by binding of [¹²⁵I] iodohydroxybenzylpindolol. Erythrocytes from 6 week old rats also contained significantly greater isoproterenol-sensitive adenylate cyclase activity (95.0 ± 9.4pmoles/10⁹ cells) than erythrocytes from 6 month (27.9 ± 3.3) or 15 month old rats (23.7 ± 3.6). The erythrocyte population of 6 week old rats was bigger (mean corpuscular volume = 62 ± 2μ^3/cell) than the older rat erythrocytes (47 ± 1μ³ and 48 ± 1μ³). When the data were expressed relative to a unit of cell volume, there was no difference in the density of beta receptors among all three populations but a progressive and significant fall in hormone-sensitive adenylate cyclase activity. In the rat erythrocyte, the age-related loss of adenylate cyclase activity is not accompanied by changes in β-receptor density.     

Theoretical model of thalassemic erythrocyte shape transformation

Our earlier model of reticulocyte shape transformation [Pawlowski, P.H., Burzynska, B., Zielenkiewicz, P., 2006. Theoretical model of reticulocyte to erythrocyte shape transformation. J. Theor. Biol. 243, 24-38] was applied to explain the morphological properties of thalassemic erythrocytes. Modification of the standard set of parameters of the model, describing minimal cell volume, membrane bending rigidity, and membrane tension, allowed for simulation of development of α- and β-thalassemic cells from splenectomized and nonsplenectomized individuals. This resulted in observation of thin rim discocytes, tailed erythrocytes and oval forms, as well as in differentiation of time of the cell shape metamorphosis. A comparative analysis of the susceptibility of thalassemic and normal erythrocytes to undergo deformation as well of their stability was performed.     

Transport of neutral amino acids by human erythrocytes

The transport of several neutral amino acids by human erythrocytes in vitro was studied. The measurements made included steady-state distributions, kinetics of initial rates of uptake, effects of monovalent cations and anions, general mutual inhibitory interactions, kinetics of inhibitions, effluxes, ability to produce accelerative exchange diffusion, and the inhibitory action of the thiol reagent N-ethylmaleimide. The results are interpreted as showing that the human erythrocyte membrane possesses several distinct transport systems for these amino acids, including one Na⁺-dependent system and one dependent on both Na⁺ and a suitable anion, that are qualitatively similar to those systems previously described in pigeon erythrocytes and mammalian reticulocytes. Quantitatively, however, the systems differ among the different kinds of red cell and a major difference lies in their abilities to produce accelerative exchange diffusion.     

Purification and properties of a lectin from lonchocarpus capassa (apple-leaf) seed

A lectin has been purified from L. capassa seed by ammonium sulphate fractionation and affinity chromatography on a column of D-galactose-derivatized Sepharose. The lectin is a glycoprotein which contains 3.8% neutral carbohydrates comprised of mannose, N-acetylglucosamine, xylose and fucose. The subunit M, of the lectin is 29 000, it has only alanine as N-terminal amino acid and contains 240 amino acids with a high content of acidic and hydroxy amino acids, single residues of methionine and histidine and the absence ofcystine. The lectin of L. capassa seed is a metalloprotein in that it contains 0.8 mol Ca²⁺ and 0.4 mol Mn²⁺ per mol. It agglutinates untreated human A, O and B type erythrocytes and rabbit erythrocytes. N-Acetyl-D-galactosamine was the best inhibitor. D-Galactose and various carbohydrates containing this sugar inhibit the hemagglutinating activity of the lectin. The lectin is also inhibited by D-glucose. The amino-terminal sequence of the lectin from L. capassa seed shows a significant degree of homology with many lectins from leguminous plants and is related to concanavalin A by a circularly permuted sequence homology.     

A lactose specific lectin from the sponge Cinachyrella apion: Purification,characterization, N-terminal sequences alignment and agglutinating activity on Leishmania promastigotes

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca²⁺, Mg²⁺ and Mn²⁺ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 °C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.     

Purification and characterization of arcelin seed protein from common bean

Arcelin, a seed protein originally discovered in wild bean accessions, was purified, characterized, and compared to phaseolin, the major seed protein of common bean, and to phytohemagglutinin (PHA), the major bean seed lectin. Arcelin and PHA has several characteristics in common. Both were glycoproteins having similar subunit M_r, deglycosylated M_r, and amino acid compositions. The two proteins were related antigenically and they had the same developmental timing of accumulation. Arcelin also had some hemagglutinating activity, a characteristic associated with lectins. However, several features distinguished arcelin from PHA. Arcelin had a more basic isoelectric point than PHA, greater numbers of basic amino acid residues, additional cysteine residues, and one methionine residue, which PHA lacks. Native PHA protein is a tetramer of subunits, and although a small component of native arcelin protein was also tetrameric, most of the arcelin preparation was dimeric. The hemagglutinating activity of arcelin was specific only for some pronase-treated erythrocytes. It did not agglutinate native erythrocytes, nor did it bind to thyroglobulin or fetuin affinity resins as did PHA. Although arcelin has lectin-like properties, we believe the distinctions between arcelin and PHA warrant the designation of arcelin as a unique bean seed protein.     

Isolation and properties of N-acetyllactosamine-specific lectins from nine erythrina species

Lectins from seeds of nine species of Erythrina have been purified by affinity chromatography on columns of lactose coupled to Sepharose and their properties compared with those of the lectin from Erythrina cristagalli. All lectins are glycoproteins of M, ca 60 000 composed of two identical or nearly identical subunits. They contain between 3–10% carbohydrates comprised of N-acetylglucosamine, mannose, fucose and xylose. The amino acid composition of all Erythrina lectins is very similar. The N-terminal amino acid is valine, with the exception of the lectin from E. flabelliformis in which it is alanine. To the extent tested, identities or near identities have been found in the N-terminal sequences (up to 15 residues in some cases) of the lectins. Hapten inhibition experiments of agglutination have shown that the lectins are specific for N-acetyllactosamine, this disaccharide being 10–30 times more inhibitory than D-galactose and 10–20 times more than N-acetyl-D-galactosamine. All lectins agglutinate human erythrocytes equally well, irrespective of blood type, at minimal concentrations of 5–20 μg/ml. Six of the lectins are also very effective in agglutinating rabbit erythrocytes and are mitogenic for human peripheral blood lymphocytes, whereas three of them are considerably weaker hemagglutinins for rabbit erythrocytes, and two of these are also very weak mitogens. Our results, while demonstrating striking similarities in the molecular properties and sugar specificity of all Erythrina lectins studied, suggest the existence of differences at or close to the carbohydrate-binding site.     

Distinct lectin activities from six species of cellular slime molds.

Extracts of cohesive cells of four species of cellular slime mold, D. mucoroides, D. purpureum, D. rosarium and P. violaceum agglutinate erythrocytes in a manner that is similar to that previously observed with extracts of D. discoideum and P. pallidum. We determined inhibitory activity of a series of sugars on the agglutination activity of each of these extracts, using both semiquantitative and quantitative agglutination assays. The inhibitory potency of this series of sugars was distinct for each extract, although only slight differences were found between several species, especially D. discoideum and P. violaceum. A possible role of these agglutinins in species-specific cell cohesion is considered.     

Uric acid in nucleic and amino acid synthesis in the boll weevil, Anthonomus grandis

When uric acid-2-¹⁴C was injected into the boll weevil, Anthonomus grandis, it was metabolized to RNA, DNA, amino acid, and ¹⁴CO₂ at the end of 2 hr. The free amino acids, lipoamino acids, and protein amino acids were all labelled with the free amino acids showing the highest specific activity. Incorporation in DNA was slight, but it was extensive in RNA; cytidylic acid showed the greatest amount of incorporation.     

Purification and characterization of sepiapterin reductase from rat erythrocytes

Sepiapterin reductase from rat erythrocyte hemolysate was purified 2000-fold to apparent homogeneity with 30% yield. The specific activity of the purified enzyme was 18 units/mg protein, and its molecular weight was 55 000. The enzyme consists of two identical subunits, each of which has a molecular weight of 27 500. The enzyme showed a single peak by isoelectric focusing with a pI of 4.9 and partial specific volume of 0.73 cm³/g. The amino acid composition was determined. pH optimum of the enzyme was 5.5. The equilibrium constant of 2.2·10⁹ of the enzyme showed that the equilibrium lies much in favor of dihydrobiopterin formation from sepiapterin in rat erythrocytes. From steady-state kinetic measurements, ordered bi-bi mechanism was proposed to the reaction of sepiapterin reductase in which NADPH binds to free enzyme and sepiapterin binds next. NADP⁺ is released after the release of dihydrobiopterin. The K_m values for sepiapterin and NADPH were 15.4 μM and 1.7 μM, respectively, and the V_max value was 21.7 μmol/min per mg.     

Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells

Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca²⁺]_i by Ca²⁺ ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7^−/−) than in erythrocytes from wild type mice (anxA7^+/+). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7^−/− and anx7^+/+-mice following increase of [Ca²⁺]_i by Ca²⁺ ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7^−/− than in anx7^+/+-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7^−/− than in anx7^+/+-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia.     

Schistosoma mansoni: L-lysil residues in agglutination of mouse erythrocytes by acid phospholipids of parasite membranes

The agglutination of mouse erythrocytes caused by acid phospholipids of Schistosoma mansoni membranes is strongly inhibited by l-lysine; other amino acids do not interfere with the reaction. The inhibitory effect observed after treatment of mouse erythrocytes with low concentrations of glutaraldehyde confirms that l-lysil residues are present on the surface of the erythrocytes and that, probably, protonated ?-amino groups are responsible for the susceptibility of these cells to the agglutination by worm membranes in physiological conditions. There is a possible role for lysil-phospholipid interactions in the host-parasite relationships.     

Impact of the large-scale deployment of artemether/lumefantrine on the malaria disease burden in Africa: case studies of South Africa,Zambia and Ethiopia

Background

Azathioprine triggers suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and exposure of phosphatidylserine at the erythrocyte surface. Eryptosis may accelerate the clearance of Plasmodium -infected erythrocytes. The present study thus explored whether azathioprine influences eryptosis of Plasmodium -infected erythrocytes, development of parasitaemia and thus the course of malaria.

Methods

Human erythrocytes were infected in vitro with Plasmodium falciparum (P. falciparum) (strain BinH) in the absence and presence of azathioprine (0.001 – 10 μM), parasitaemia determined utilizing Syto16, phosphatidylserine exposure estimated from annexin V-binding and cell volume from forward scatter in FACS analysis. Mice were infected with Plasmodium berghei (P. berghei) ANKA by injecting parasitized murine erythrocytes (1 × 10⁶) intraperitoneally. Where indicated azathioprine (5 mg/kg b.w.) was administered subcutaneously from the eighth day of infection.

Results

In vitro infection of human erythrocytes with P. falciparum increased annexin V-binding and initially decreased forward scatter, effects significantly augmented by azathioprine. At higher concentrations azathioprine significantly decreased intraerythrocytic DNA/RNA content (≥ 1 μM) and in vitro parasitaemia (≥ 1 μM). Administration of azathioprine significantly decreased the parasitaemia of circulating erythrocytes and increased the survival of P. berghei -infected mice (from 0% to 77% 22 days after infection).

Conclusion

Azathioprine inhibits intraerythrocytic growth of P. falciparum, enhances suicidal death of infected erythrocytes, decreases parasitaemia and fosters host survival during malaria.     

Morphological alterations in erythrocytes treated with silver nanoparticles biomineralized by marine sediment-derived Bacillus sp. VITSSN01

Biomineralization-inspired preparation of nanoparticles by marine microorganisms is in the limelight of modern nanotechnology. In recent years, the use of marine microorganisms for the synthesis of nanoparticles has been gaining importance due to the simplicity and eco-friendliness of the approach. Here we describe the synthesis of silver nanoparticles using halotolerant Bacillus sp. isolated from the southern coastal waters of India. Our selective and enriched isolation technique resulted in the isolation of a silver nitrate-resistant novel marine Bacillus sp. isolated from sediments collected at Ennore Port, Chennai, India. The strain was characterized by the polyphasic taxonomic approach, and phenotypic and phylogenetic analysis identified the strain as Bacillus sp. VITSSN01. The resistant strain was further assayed for the synthesis of silver nanoparticles and its biological activity evaluated. Nanoparticles were synthesized under optimized nutritional and cultural conditions with shaking and the production continuously monitored. The nanoparticles thus produced were then characterized by atomic force microscopy, X-ray diffraction, Fourier transform-infrared spectrophotometer and transmission electron microscopy. The mean particle size was 46 nm. Hemotological toxicity of nanoparticles is very severe form and less studied. We therefore checked the synthesized silver nanoparticles for toxicity against erythrocytes and found that the silver nanoparticles exhibited moderate hemolytic activity against human erythrocytes, with a half maximal effective concentration (EC₅₀) value of 60 μg/ml. Microscopic studies of the treated erythrocytes showed slight structural perturbations. The results of our study strongly suggest that marine microorganisms could be a potential source for the rapid and eco-friendly synthesis of nanoparticles.     

Prevention of in vitro sickling of human erythrocytes by monofunctional amidination

Treatment of sickle erythrocytes with the monoimidate, ethylacetimidate, prevented anoxia-induced sickling in vitro. Hemoglobin isolated from amidated erythrocytes exhibited an increase in the minimum gelling concentration compared to untreated preparations. Although the prevention of sickling did appear to involve chemical modification of hemoglobin S, an increase in the oxygen affinity was not a requisite for the antisickling activity of the imidates. Our results suggest that substitution of amidino for amino groups prevents the aggregation of deoxyhemoglobin S. Furthermore, comparison of monoimidates with bifunctional reagents indicates that molecular crosslinking is not essential for antisickling activity.