Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.     

Inhibition of invitro sickling by liposome-mediated transport of amino acids into intact human red blood cells

To investigate the role of phenylalanine and tryptophane as potential antisickling agents in intact human SS-red blood cells a liposomal transport system was employed to transfer phenyl-alanine or tryptophane into intact SS-red blood cells. Aromatic amino acids and short peptides containing phenylalanine have been demonstrated to increase the minimum gelling concentration and solubility of deoxy-hemoglobin S in aqueous solution. However, these compounds do not cross the red blood cell membrane under usual incubation conditions. Incorporation of phenylalanine or tryptophane into intact SS-red blood cells $\text{via}$ liposomal transport system markedly inhibited the $\text{in}\text{vitro}$ sickling of deoxy-hemoglobin S. These findings raise the possibility that a nontoxic liposomal transport system which facilitates incorporation of antisickling agents into intact SS-RBC may have significant therapeutic implications in the treatment of sickle cell disease.     

Mathematical model of red cell sickling

A mathematical model of red cell sickling without crisis was established in vitro as a function of the parameters on which it depends: the oxygen partial pressure, the abnormal hemoglobin S concentration, the temperature and the pH. This model was verified both for the homozygotic sickling SS and heterozygotic sickling SC. Such a model allows systematic investigation of patients. Moreover, it opens up the way for rational comparison in the testing of drugs which are effective in the treatment of red cell sickling.     

Single cell laser light scattering spectroscopy in a flow cell: Repeated sickling of sickle red blood cells

We performed dynamic laser light scattering measurements of hemoglobin aggregates in single, sickle erythrocytes. Sickle erythrocytes were attached to the poly-(l-lysine)-coated surface of a flow cell. They were exposed to several oxygenation-deoxygenation cycles by repeatedly changing the flowing solution. The rate of cycling was found to be a determining factor for the formation of irreversible morphologic alterations as well as irreversible hemoglobin aggregates. In slow cycling, the sickle erythrocytes took an irreversible, irregular, rounded shape, and hemoglobin aggregates were observed even in the oxygenated state after 20 cycles. In the fast cycling, however, these changes did not take place even after 60 cycles.     

Nylon-fiber affinity selection of red blood cells and tissue culture cells on the basis of cell surface determinants

Nylon fibers coated with various lectins were used for the specific selection from mixed populations of erythrocytes or tissue culture cells with lectin receptors. Binding of human group O red blood cells to fibers treated with Ulex europaeus lectin I (H-specific) or of human group A red cells to fibers treated with Helix pomatia lectin (A-specific) was proportional to lectin concentration in the solution used to adsorb lectin to the fibers. Binding was blood group specific and increased with increasing concentrations of red cells applied to the fibers. Most adsorption of lectin to the fibers occurred within minutes; cell binding to lectin-coated fibers was almost complete within 30 min. Blood group negative Chinese hamster tissue culture cells bound non-specifically to Helix-coated fibers with a frequency of less than 10⁻⁴ input cells; the yield of viable, colony-forming cells bound to PHA-coated fibers was about 1%. Epithelial cells from cultures of amniotic fluid or fetal kidney contained 1–30% cells positive for the ABO blood group of the donor; blood group positive cells from these cultures were poorly bound to fibers coated with blood group specific lectins, though they bound readily to PHA-coated fibers, suggesting that presence of appropriate surface determinants may be necessary but not sufficient for lectin: cell binding in this system.     

The migration of bone marrow cells to thymic culture supernatants is inhibited by stress

The effect of immobilization stress on precursor cell migration from bone marrow to the thymus was studied in C57BL/6 mice. The in vitro migration assays, using Nuclepore chambers, showed that precursor cell migration to thymus supernatants was strongly inhibited in stressed animals. This inhibition of migration seemed to be cell-associated what can explain the thymic involution observed in mice under stress conditions. The migration of precursor cells from bone marrow may be one of the mechanisms by which the thymus gland is involuted by stress.     

Antioxidant effect of red wine polyphenols on red blood cells

The protective effect of red wine polyphenols against hydrogen peroxide (H(2)O(2))-induced oxidation was investigated in normal human erythrocytes (RBCs). RBCs, preincubated with micromolar amounts of wine extract and challenged with H(2)O(2), were analyzed for reactive oxygen species (ROS), hemolysis, methemoglobin production, and lipid peroxidation. All these oxidative modifications were prevented by incubating the RBCs with oak barrel aged red wine extract (SD95) containing 3.5 mM gallic acid equivalent (GAE) of phenolic compounds. The protective effect was less apparent when RBCs were incubated with wines containing lower levels of polyphenols. Furthermore, resveratrol and quercetin, well known red wine antioxidants, showed lower antioxidant properties compared with SD95, indicating that interaction between constituents may bring about effects that are not necessarily properties of the singular components. Our findings demonstrate that the nonalcoholic components of red wine, mainly polyphenols, have potent antioxidant properties, supporting the hypothesis of a beneficial effect of red wine in oxidative stress in human system.     

Illuminating Plasmodium falciparum-infected red blood cells

The malaria parasite undergoes a remarkable series of morphological transformations, which underpin its life in both human and mosquito hosts. The advent of molecular transfection technology coupled with the ability to introduce fluorescent reporter proteins that faithfully track and expose the activities of parasite proteins has revolutionized our view of parasite cell biology. The greatest insights have been realized in the erythrocyte stages of Plasmodium falciparum. P. falciparum invades and remodels the human erythrocyte: it feeds on haemoglobin, grows and divides, and subverts the physiology of its hapless host. Fluorescent proteins have been employed to track and dissect each of these processes and have revealed details and exposed new paradigms.     

Heterogeneity among dog red blood cells

A phthalate density-separation technique has been used to study the heterogeneity of dog red blood cells that becomes manifest when they are suspended in KCl media. It is demonstrated that the proportions of cells that separate into light and dense fractions can be varied by altering the tonicity of the KCl medium. This results from the fact that the Na and K permeabilities of each cell are continuous functions of cell volume. It was found that quinidine inhibits selectively the volume dependence of Na permeability. In the presence of this drug, the heterogeneity demonstrated by KCl incubation disappears. The notion that dog red blood cells are heterogeneous in their permeabilities to Na and K is thus upheld, but the heterogeneity is not an abruptly discontinuous one, as has been claimed. A sample of dog blood does not contain two discrete populations of red cells.     

Proteases in malaria-infected red blood cells

The discrimination between erythrocyte and Plasmodium proteases is now made easier by using synthetic fluorogenic substrates, high-pressure liquid chromatography, reliable methods of cell preparation, as well as radiolabeled extracts from in vitro cultures of P. falciparum. The reinvasion process of an erythrocyte by a merozoite involves specific proteinases, which were recently identified using fluorogenic peptidyl-AEC substrates and by analysis of schizont and merozoite extracts with the gelatin-SDS-PAGE method. The biological targets of both host and parasite proteinases are not yet well characterized because Plasmodium-infected red blood cells contain at least four compartments with different pH values, which could modulate the proteinase activities according to their pH range activity. The processing of the precursor for the major merozoite surface antigens involves cleavage of very specific peptidic bonds by, so far unknown, proteinases. The depletion of the erythrocyte cytoskeleton could depend on a 37 kD proteinase, which cleaves spectrin and the 4.1 component, as shown in P. berghei and P. falciparum species. In contrast to leupeptin, which inhibits the merozoite release from schizont-infected erythrocytes, the structural inhibitor analogous to the Val-Leu-Gly-Lys (or Arg) P. falciparum neutral proteinase substrates appears to block the invasion step of erythrocytes by merozoites and may open new trends in chemotherapeutical strategies.     

Shape memory of human red blood cells

The human red cell can be deformed by external forces but returns to the biconcave resting shape after removal of the forces. If after such shape excursions the rim is always formed by the same part of the membrane, the cell is said to have a memory of its biconcave shape. If the rim can form anywhere on the membrane, the cell would have no shape memory. The shape memory was probed by an experiment called go-and-stop. Locations on the membrane were marked by spontaneously adhering latex spheres. Shape excursions were induced by shear flow. In virtually all red cells, a shape memory was found. After stop of flow and during the return of the latex spheres to the original location, the red cell shape was biconcave. The return occurred by a tank-tread motion of the membrane. The memory could not be eliminated by deforming the red cells in shear flow up to 4 h at room temperature as well as at 37 degrees C. It is suggested that 1). the characteristic time of stress relaxation is >80 min and 2). red cells in vivo also have a shape memory.