Reduced water exchange in sickle cell anemia red cells: a membrane abnormality

We have measured the diffusional water permeability of sickle cell anemia red blood cells under isotonic conditions using pulsed nuclear magnetic resonance (NMR) techniques. We have found that the equilibrium diffusional permeability for sickle cells is about 1.61.10(-3) cm/s, or about 60% of the value measured for normal cells. This abnormality is not related to the heterogeneity generally found in cell populations in sickle red cells with different mean corpuscular hemoglobin concentrations. We speculate that the abnormality of water exchange under isotonic conditions in sickle cells reflects an alteration of membrane proteins responsible for water exchange, possibly caused by oxidation of Band 3 proteins.     

Drosophila sickle is a novel grim-reaper cell death activator

The Drosophila genes reaper, head involution defective (hid), and grim all reside at 75C on chromosome three and encode related proteins that have crucial functions in programmed cell death (reviewed in ). In this report, we describe a novel grim-reaper gene, termed sickle, that resides adjacent to reaper. The sickle gene, like reaper and grim, encodes a small protein which contains an RHG motif and a Trp-block. In wild-type embryos, sickle expression was detected in cells of the developing central nervous system. Unlike reaper, hid, and grim, the sickle gene is not removed by Df(3L)H99, and strong ectopic sickle expression was detected in the nervous system of this cell death mutant. sickle very effectively induced cell death in cultured Spodoptera Sf-9 cells, and this death was antagonized by the caspase inhibitors p35 or DIAP1. Strikingly, unlike the other grim-reaper genes, targeted sickle expression did not induce cell death in the Drosophila eye. However, sickle strongly enhanced the eye cell death induced by expression of either an r/grim chimera or reaper.     

A Ca2+-refractory state of the Ca-sensitive K+ permeability mechanism in sickle cell anaemia red cells

Simultaneous measurements of Ca content and ⁴²K⁺ influx in sickle cell anaemia red cells confirm predictions from earlier data in the literature that the increased Ca content of sickle cell anaemia cells which are not metabolically depleted does not cause a quinine-sensitive increase in K⁺ permeability.It is shown that the ionophore, A23187, can cause the Ca contained inside sickle cell anaemia cells to activate the quinine-sensitive K⁺-permeability mechanism. This demonstrates the existence of a Ca²⁺-refractory state of the K⁺ channel in sickle cell anaemia cells and a direct stimulatory effect of the ionophore A23187 on its Ca sensitivity.     

Kinetics of increased deformability of deoxygenated sickle cells upon oxygenation

We have examined the kinetics of changes in the deformability of deoxygenated sickle red blood cells when they are exposed to oxygen (O(2)) or carbon monoxide. A flow-channel laser diffraction technique, similar to ektacytometry, was used to assess sickle cell deformability after mixing deoxygenated cells with buffer that was partially or fully saturated with either O(2) or carbon monoxide. We found that the deformability of deoxygenated sickle cells did not regain its optimal value for several seconds after mixing. Among density-fractionated cells, the deformability of the densest fraction was poor and didn't change as a function of O(2) pressure. The deformability of cells from the light and middle fraction increased when exposed to O(2) but only reached maximum deformability when equilibrated with supraphysiological O(2) concentrations. Cells from the middle and lightest fraction took several seconds to regain maximum deformability. These data imply that persistence of sickle cell hemoglobin polymers during circulation in vivo is likely, due to slow and incomplete polymer melting, contributing to the pathophysiology of sickle cell disease.     

Increased membrane-associated phorbol-12,13 dibutyrate (PDBu) receptor function in sickle red cells

A four-fold increase in the binding of 3H-PDBu by red cell membrane ghosts isolated from sickle red cells compared to that from normal controls is presented. Phosphorylation studies with gamma-32P-ATP indicate a similar (two to three-fold) increase in the radiolabelling of the acid-precipitable membrane proteins in sickle red cells. When red cells were loaded with Ca2+ using Ionophore A23187, both normal and sickle red cells enhanced their phosphorylation and sickle red cells to a greater extent than normal red cells. Polyacrylamide slab gel electrophoretic separation of the phosphoproteins and autoradiography also reveal phosphorylation, predominantly of protein bands 3, 4.1 and 4.9 which are known in the red cells as specific substrates for the PDBu receptor, protein kinase C. These results indicate that membrane association of protein kinase C in sickle red cells is increased, possibly as a consequence of the pathological change in their ability to accumulate intracellular calcium.     

Sickle hemoglobin instability: a mechanism for malarial protection

Heterozygosity for the mutant sickle hemoglobin confers protection from severe Plasmodium falciparum malaria. It is here proposed that this protection derives from the instability of sickle hemoglobin, which clusters red cell membrane protein band 3 and triggers accelerated removal by phagocytic cells. This explanation requires that sickle trait cells manifest greater hemoglobin instability than normal red cells, something that could derive from their content of sickle hemoglobin. The mechanism also implicates splenic function as a determinant of the protective effect.     

Sickle hemoglobin instability: a mechanism for malarial protection

Abstract

Heterozygosity for the mutant sickle hemoglobin confers protection from severe Plasmodium falciparum malaria. It is here proposed that this protection derives from the instability of sickle hemoglobin, which clusters red cell membrane protein band 3 and triggers accelerated removal by phagocytic cells. This explanation requires that sickle trait cells manifest greater hemoglobin instability than normal red cells, something that could derive from their content of sickle hemoglobin. The mechanism also implicates splenic function as a determinant of the protective effect.     

Kinetics of water transport in sickle cells

This paper reports the results of stopped-flow studies on differences in the kinetics of osmotic water transport of sickle and normal erythrocytes. The kinetics of inward osmotic water permeability are similar in sickle and normal red blood cells. In contrast, the kinetics of outward water flux are significantly (approx. 38%) decreased in sickle cells. Deoxygenation does not modify the water influx kinetics in either type of cells, but accelerates considerably the rate of water efflux in sickle cells. No significant variation of water transport kinetics was observed in density-separated cell fractions of either type. The results suggest that membrane-associated hemoglobin may decrease the outward water permeability and that in deoxygenated sickle cells the fraction of hemoglobin S near the lipid bilayer is lower than in oxygenated conditions.     

Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni

Summary Using the label-fracture technique, an ultrastructural comparison was made of the number and distribution of wheat germ agglutinin (WGA)-binding sites between human normal and sickle red blood cells. The WGA was adsorbed to colloidal gold, and quantitative analysis of the electron micrographs revealed that more binding sites were present on the sickle erythrocytes than on the normal erythrocytes. Moreover, the sites were more clustered on the sickle red cells than on the normal red cells. Use of another lectin, Bandieraea simplicifolia-II, revealed that it did not bind to normal or sickle red cells. Because of the affinity of the WGA for sialic acid residues, it is probable that the WGA is binding to a transmembrane sialoglycoprotein, glycophorin A. The conformation and/or distribution of the glycophorin A molecules may be altered by the sickle hemoglobin that binds to the red cell membrane. Hence, as detected by WGA, new surface receptors, which could play a role in the adhesion of sickle cells to endothelium may be exposed.     

Reduced transglutaminase-catalyzed cross-linking of exogenous amines to membrane proteins in sickle erythrocytes

In order to determine the capacity of sickle cells to undergo transglutaminase-catalyzed cross-linking of membrane proteins, human normal and sickle erythrocytes were incubated with [ring-2-14C]histamine in the presence of Ca2+ and ionophore A23187. The [14C]histamine incorporation into membrane components was observed in freshly prepared erythrocytes. Incorporation of radioactivity into spectrin and Band 3 membrane components was significantly (P less than 0.001) less in sickle erythrocytes than in normal cells. Transglutaminase deficiency was excluded by the finding of increased activity of this enzyme in sickle cells from patients with reticulocytosis. The incorporation of [3H]spermine into red cell membranes was also less in sickle erythrocytes than in normal cells under the same conditions of incubation used for [ring-2-14C]histamine. Sickle erythrocytes were more permeable to these amines than normal cells. It is proposed that the gamma-glutamyl sites of membrane proteins in sickle erythrocytes are less accessible for transglutaminase-catalyzed cross-linking to histamine and polyamines in vitro, perhaps due to prior in vivo activation of this enzyme by the increased calcium in sickle cells and/or shielding secondary to altered membrane organization.     

Small fragment homologous replacement-mediated modification of genomic beta-globin sequences in human hematopoietic stem/progenitor cells

An ultimate goal of gene therapy is the development of a means to correct mutant genomic sequences in the cells that give rise to pathology. A number of oligonucleotide-based gene-targeting strategies have been developed to achieve this goal. One approach, small fragment homologous replacement (SFHR), has previously demonstrated disease-specific genotypic and phenotypic modification after introduction of small DNA fragments (SDFs) into somatic cells. To validate whether the gene responsible for sickle cell anemia (beta-globin) can be modified by SFHR, a series of studies were undertaken to introduce sickle globin sequences at the appropriate locus of human hematopoietic stem/progenitor cells (HSPCs). The characteristic A two head right arrow T transversion in codon 6 of the beta-globin gene was indicated by restriction fragment length polymorphic (RFLP) analysis of polymerase chain reaction (PCR) products generated by amplification of DNA and RNA. At the time of harvest, it was determined that the cells generally contained 相似文献   

Sickle cell trait human erythrocytes are significantly stiffer than normal

Atomic force microscopy (AFM) allows for high-resolution topography studies of biological cells and measurement of their mechanical properties in physiological conditions. In this work, AFM was employed to measure the stiffness of abnormal human red blood cells from human subjects with the genotype for sickle cell trait. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that Young's modulus of pathological erythrocytes was approximately three times higher than in normal cells. Observed differences indicate the effect of the polymerization of sickle hemoglobin as well as possible changes in the organization of the cell cytoskeleton associated with the sickle cell trait.     

Membrane components in the red cells of patients with sickle cell anemia. Relationship to cell aging and to irreversibility of sickling

Cholesterol, phospholipid and sialic acid were measured in red cells from patients with sickle cell anemia to determine whether the cells had abnormal concentrations of these components and whether the amounts of these compounds differed in irreversibly sickled cells as compared to non-irreversibly sickled cells. Sickle cells had significantly higher levels of both lipids than similar populations of normal cells, however, comparisons to populations of young control cells showed that the differences were generally not significant. Sialic acid levels in sickle cells were not significantly different from normal cells. Irreversibly sickled cells had lower lipid and sialic acid concentrations than those not irreversibly sickled, but the differences were either not significant or did not occur when compared to young control cells. The studies show that the increased lipid concentrations in the membrane of sickle cells are not abnormal but are related to cell age and that the decrease in membrane components in irreversibly sickled cells is no greater than would be predicted for similarly aged populations of cells.     

Sickle cells show low expansibility and high resistance to saponin

The expansibility under hypotonic condition and the susceptibility to saponin under hypotonic and deoxygenated conditions were determined. The experiments were carried out using a conventional blood cell counter and comparable volume changes were displayed on a chart as a graphic presentation. The sickle cells showed low expansibility and strong resistance to saponin, where the resultant curve of sickle cells showed prolonged hemolysis time and characteristic volume change. Thus, the methods would be useful for detecting sickle cell disease during routine laboratory procedures.     

Massive sequestration of human sickle cells after transfusion to a baboon

A baboon was exchange-transfused with sickle cell anemia patients' blood. The animal died suddenly, and postmortem examination showed widespread red cell sequestration, particularly in the spleen and liver. The clinical and pathological findings were similar to those in children with sickle cell anemia who die of acute splenic sequestration syndrome. A control animal, exchange-transfused with normal human blood, tolerated the procedure without difficulties for a period of 4 days, when a delayed transfusion reaction occurred. Thus the baboon can be used as a model for the abnormal circulatory behavior of sickle cells and for the sickle cell sequestration syndrome.     

Membrane components in the red cells of patients with sickle cell anemia Relationship to cell aging and to irreversibility of sickling

Cholesterol, phospholipid and sialic acid were measured in red cells from patients with sickle cell anemia to determine whether the cells had abnormal concentrations of these components and whether the amounts of these compounds differed in irreversibly sickled cells as compared to non-irreversibly sickled cells. Sickle cells had significantly higher levels of both lipids than similar populations of normal cells, however, comparisons to populations of young control cells showed that the differences were generally not significant. Sialic acid levels in sickle cells were not significantly different from normal cells. Irreversibly sickled cells had lower lipid and sialic acid concentrations than those not irreversibly sickled, but the differences were either not significant or did not occur when compared to young control cells. The studies show that the increased lipid concentrations in the membrane of sickle cells are not abnormal but are related to cell age and that the decrease in membrane components in irreversibly sickled cells is no greater than would be predicted for similarly aged populations of cells.     

The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells. In 38 cases we observed an increase and in 11 cases a decrease in content in the sickle cell membranes. The proteins of interest were identified by in-gel tryptic digestion followed by liquid chromatography in line with tandem mass spectrometry. From 38 analyzed gel spots, we identified 44 protein forms representing different modifications of 22 original protein sequences. The majority of the identified proteins fall into small groups of related proteins of the following five categories: actin accessory proteins--four proteins, components of lipid rafts--two proteins, scavengers of oxygen radicals--two proteins, protein repair participants--six proteins, and protein turnover components--three proteins. The number of proteins whose content in sickle RBC membrane is decreased is noticeably smaller, and most are either components of lipid rafts or actin accessory proteins. Elevated content of protein repair participants as well as oxygen radical scavengers may reflect the increased oxidative stress observed in sickle cells.     

Ubiquitination of spectrin regulates the erythrocyte spectrin-protein-4.1-actin ternary complex dissociation: implications for the sickle cell membrane skeleton.

It has been demonstrated by our laboratory that the irreversibly sickled cell (ISC) spectrin-4.1-actin complex dissociates slowly as compared to ternary complexes formed out of control (AA) and reversibly sickle cell (RSCs) core skeletons. These studies indicated that the molecular basis for the inability of irreversibly sickled cells (ISCs) to change shape is a skeleton that disassembles, and therefore reassembles, very slowly. The present study is based on the following observations: a) alpha-spectrin repeats 20 and 21 contain ubiquitination sites, and b) The spectrin repeats beta-1 and beta-2 are in direct contact with spectrin repeats alpha-20 and alpha-21 during spectrin heterodimer formation, and contain the protein 4.1 binding domain. We demonstrate here that alpha-spectrin ubiquitination at repeats 20 and 21 increases the dissociation of the spectrin-protein-4.1-actin ternary complex thereby regulating protein 4.1's ability to stimulate the spectrin-actin interaction. Performing in vitro ternary complex dissociation assays with AA control and sickle cell SS spectrin (isolated from high-density sickle cells), we further demonstrate that reduced ubiquitination of alpha-spectrin is, in part, responsible for the locked membrane skeleton in sickle cell disease.     

Decrease of transport of some polyols in sickle cells

This paper reports the results of kinetic studies on the inward net-flux of small non-electrolytes (ethylene glycol, glycerol and erythritol) in sickle cells as compared to normal erythrocytes. Net transport rates were evaluated by turbidimetric measurements for ethylene glycol and glycerol and by hematocrit monitoring for erythritol. A 2-fold and 4-fold reduction in the permeability coefficient for ethylene glycol and glycerol, respectively, were found in sickle cells as compared to normal erythrocytes. In contrast, no significant changes in erythritol transport kinetics were observed. The dependence of glycerol permeability on temperature, pH and oxygenation is the same in both types of cells. A significant correlation was observed between glycerol permeability and cell density only for sickle cells. The results indicate that irreversible modifications of membrane proteins, responsible for the glycerol and ethylene glycol transport, do occur in sickle cells.     

Inhibition of the in vitro formation of dense cells and of irreversibly sickled cells by charybdotoxin, a specific inhibitor of calcium-activated potassium efflux

Charybdotoxin, a specific inhibitor of the calcium-activated potassium channel, was found to inhibit the in vitro formation of irreversibly dehydrated cells and of irreversibly sickled cells, which occur as a result of repeated cycles of sickling and unsickling of sickle red blood cells. The degree of formation of dense cells was measured by Percoll-renografin density gradient centrifugation. 50% inhibition of the formation was achieved at a concentration of 30 nM of charybdotoxin. The approximate half-life of this compound in the circulation of the guinea pig was determined to be 4 h. Charybdotoxin did not inhibit the sickling of sickle cells under deoxygenation. The effects of charybdotoxin in preventing the irreversible changes of sickle cell membranes may be related to the inhibition of calcium-activated potassium efflux in sickle red blood cells.