Density separation of human red blood cells on self forming PercollR gradients: correlation with cell age

Lutz, Hans; Stammler, P; Fasler, Stephan; Ingold, Marlis; Fehr, Jörg

doi:10.1016/0304-4165(92)90120-j

Cited by 109 publications

(93 citation statements)

References 38 publications

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“…During the course of its 120-day life span, the erythrocyte undergoes a time-dependent increase in cell density. This has been the basis of the widely used method of density-dependent separation of young and old erythrocytes (34,39,40). In order to test our hypothesis that caspase 3 activation could play a role in red cell senescence, young and old human red blood cells were separated on a Percoll gradient.…”

Section: Resultsmentioning

confidence: 99%

Caspase 3-mediated Proteolysis of the N-terminal Cytoplasmic Domain of the Human Erythroid Anion Exchanger 1 (Band 3)

Mandal

Baudin‐Creuza

Bhattacharyya

et al. 2003

Journal of Biological Chemistry

117

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The N-terminal cytoplasmic domain of the anion exchanger 1 (AE1 or band 3) of the human erythrocyte associates with peripheral membrane proteins to regulate membrane-cytoskeleton interactions, with glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase and aldolase, with the protein-tyrosine kinase p72 syk , with hemoglobin and with hemichromes. We have demonstrated that the N-terminal cytoplasmic domain of band 3 (CDB3) is a substrate of the apoptosis executioner caspase 3 (1). CDB3 has two non-conventional caspase 3 cleavage sites, TATD 45 and EQGD 205 (2). In vitro treatment of recombinant CDB3 with caspase 3 generated two fragments, which could be blocked by pretreatment with the caspase 3 inhibitor Z-DEVD-fmk Apoptosis is required to maintain the balance between cell proliferation and cell death. Nuclear collapse and DNA fragmentation are universal features of apoptosis in nucleated cells. However, the nucleus itself is not required for apoptosis, since apoptotic stimuli induce apoptotic morphological features in anucleate cells (1-3). The signaling events that culminate in some of the key plasma membrane changes associated with cell death in vivo, such as externalization of PS, 1 operate independently of the nucleus (3). While the process of apoptosis has been studied in-depth in nucleated cells, the role of apoptosis regulatory molecules in anucleate cells is poorly understood. The anucleate mature circulating human erythrocyte is cleared by macrophages after its 120-day life span. The senescent erythrocyte shares at least one feature in common with nucleated cells undergoing apoptosis. The externalization of PS on the outer leaflet of the red cells results in recognition by macrophages probably through a receptor that engages PS (4). We hypothesized that the circulating human erythrocyte potentially shares part of the apoptotic machinery of nucleated cells, and that this machinery may be triggered as a consequence of red cell senescence, pathology such as in diseases like sickle cell anemia, thalassemia, and oxidative insult to red cells. Proteases play a critical role in the expression of mammalian apoptosis. The caspases are a specialized family of cysteine-dependent aspartate-directed proteases (5-7). The caspases recognize a tetrapeptide sequence on their substrates with an aspartate residue at the fourth position. Caspase activation is a critical event in the onset of apoptosis (5). Human caspase 3 is perhaps the most universal apoptosis mediator. It is present in most mammalian cells (8). Its deletion by gene knockout blocks neuronal death during brain development with consequent lethality (9). Studies in our laboratory have demonstrated that caspase 3 is present in mature human erythrocytes and activated by oxidative stress (10). Berg et al. (11) have also demonstrated that caspases 3 and 8 are present in mature human erythrocytes.The cytoskeleton of an apoptotic cell undergoes profound changes. Underlying these changes is the cleavage of many cytoskeletal proteins by caspases. Thes...

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Section: Resultsmentioning

confidence: 99%

Caspase 3-mediated Proteolysis of the N-terminal Cytoplasmic Domain of the Human Erythroid Anion Exchanger 1 (Band 3)

Mandal

Baudin‐Creuza

Bhattacharyya

et al. 2003

Journal of Biological Chemistry

117

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“…Aliquots were taken for ektacytometry or gradient centrifugation. 18,19 In the latter case aliquots containing 4×10 10 RBC were centrifuged and the cells resuspended with 36 mL Percoll solution containing exclusively either Na + or K + ions. Where indicated the Percoll solutions were supplemented with 10 µM DIDS or 100 µM SITS.…”

Section: Ektacytometry and Density Gradientsmentioning

confidence: 99%

“…These suspensions were centrifuged, the gradients photographed, and fractionated as outlined. 18 Even RBC from the lightest fraction could be washed and re-centrifuged on a density gradient, implying that a significant portion if not all swollen cells did not lyse during centrifugation in Percoll gradient. Osmoscans were obtained as described previously with an ektacytometer (Technikon Products, Bayer, Germany).…”

Section: Ektacytometry and Density Gradientsmentioning

confidence: 99%

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Bogdanova

Goede²,

Weiss³

et al. 2009

Haematologica

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BackgroundCryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky. Design and MethodsCold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient's erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient's erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed. ResultsIn the cold, the cryohydrocytosis patient's erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO3-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na + /K + exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K + uptake. Unidirectional K + influx measurements showed that the patient's cells have abnormally high activities of the cation-proton exchanger and the K + ,Cl -co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient's erythrocytes differed from that of healthy donors. ConclusionsThese results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.

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“…Since the discovery that the ratio of protein 4.1a to protein 4.1b, which are a part of the red cell membrane cytoskeleton, is correlated with erythrocyte life span in mammals [7][8][9], several authors have adopted the 4.1a/4.1b ratio as a parameter to characterize the absolute age of density-fractionated human erythrocytes [10][11][12]. Results obtained so far have shown that the population of circulating red cells is in fact very heterogeneous, with respect to membrane surface, cell volume, cation and water content, and cell deformability [12,13].…”

Section: Introductionmentioning

confidence: 99%

Cellular properties of human erythrocytes preserved in saline–adenine–glucose–mannitol in the presence of L‐carnitine

Arduini¹,

Minetti

Ciana

et al. 2006

American J Hematol

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L-Carnitine (LC) in the preservation medium during storage of red blood cells (RBC) can improve the mean 24-hr percent recovery in vivo and increase RBC life-span after reinfusion. The purpose of the study was to investigate the differences in the biochemical properties of RBCs stored in the presence or absence of LC, and the cell-age related responses to storage conditions and to LC. RBC concentrates in saline-adenine-glucose-mannitol (SAG-M) were stored in the presence or absence of 5 mM LC at 48C for up to 8 weeks. RBC subpopulations of different densities were prepared by centrifugation on Stractan density gradient. Cells were sampled at 0, 3, 6, and 8 weeks, and hematological and cellular properties analyzed (MCV, MCHC, 4.1a/4.1b ratio as a cell age parameter, intracellular Na + and K + ). After 6 weeks, MCV of RBC stored in the presence of LC was lower than that of controls (6 weeks MCV: controls 95.4 ± 1.8 fl; LC 91.5 ± 2.0 fl; n = 6; P < 0.005). This was due to swelling of control cells, and affected mainly older RBCs. LC appeared to reduce or retard cell swelling. Among the osmotically active substances whose changes during storage could contribute to cell swelling, only intracellular Na

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Density separation of human red blood cells on self forming PercollR gradients: correlation with cell age

Cited by 109 publications

References 38 publications

Caspase 3-mediated Proteolysis of the N-terminal Cytoplasmic Domain of the Human Erythroid Anion Exchanger 1 (Band 3)

Caspase 3-mediated Proteolysis of the N-terminal Cytoplasmic Domain of the Human Erythroid Anion Exchanger 1 (Band 3)

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Cellular properties of human erythrocytes preserved in saline–adenine–glucose–mannitol in the presence of L‐carnitine

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