Mouse Red Cells Research Articles

Endothelial Activation by Sickle Mouse Red Cells and Their Endothelial Adhesion In Vivo is Abolished by Catalase Treatment of Sickle Cells, but Not Quiescent Endothelium, Implying a Role of Heme-Mediated Peroxide Generation in Sickle Cell Adhesion.

Abstract 902Multiple adhesion molecules, expressed on sickle red blood cells (SS RBCs) and activated endothelium, have been implicated in SS RBC adhesion to vascular endothelium. Moreover, intrinsic differences among heterogeneous SS RBC subpopulations, involving differences in red cell adhesion molecules and cell deformability, may contribute to their adhesive and obstructive properties and lead to postcapillary obstruction. However, the role of SS RBCs in endothelium activation and adhesion has not been evaluated despite the insightful studies of Hebbel and coworkers (JCI, 1982) demonstrating that SS RBCs generate excessive amounts of reactive oxygen species due to the presence of unstable hemoglobin S (HbS) and autoxidation of iron in heme. RBCs from transgenic-knockout sickle (BERK) mice similarly show a pronounced increase in heme degradation (Nagababu et. al. Blood Cells Mol Dis, 2008). We hypothesize that hypoxic conditions in venules (oxygen tension,∼30 mm Hg) will accelerate autoxidation of RBC membrane-bound HbS and release H2O2that will be transferred to adjoining endothelium resulting in its activation (i.e., up-regulation of endothelial adhesion molecules) and SS RBC adhesion. To test the hypothesis that HbS-containing red cells from BERK mice will result in activation of quiescent endothelium in normal mice, we infused FITC (fluorescein isothiocynate)-labeled BERK red cells into congenic C57BL mice. BERK mice, expressing exclusively human βS- and α-globins, have been extensively backcrossed onto C57BL background. Intravital observations were made in the cremaster muscle microcirculatory bed. A single bolus of 150 μl of FITC-labeled BERK RBCs (Hct 30%) was infused into the recipient C57BL mouse via the jugular vein over a period of 5 min to avoid any shear related platelet aggregation. Infusion of FITC-labeled control (C57BL) mouse RBCs into C57BL recipient mice resulted in rare or no RBC adhesion, suggesting that there was no activating effect on endothelium. In contrast, infusion of BERK mouse RBCs into C57BL mice resulted in time-dependent increase in adhesion to venular endothelium. Adhesion became discernable after 3 minutes and showed a 3-5 fold increase after 5-min compared with the number of adherent RBCs at 3 min (P<0.01). Next, we investigated if the infusion of BERK mouse RBCs would induce increased endothelial oxidants. To this end, the cremaster preparation was suffused for 15 min with 123 dihydrorhodamine (DHR), a H2O2-sensitive probe (10 μl/L), followed by a bolus infusion of BERK mouse RBCs, and time-dependent changes in DHR fluorescence intensity were monitored in venules, the sites of adhesion. Infusion of BERK mouse RBCs, but not C57BL RBCs, resulted in time-dependent increase in the fluorescence intensity (ΔI) in venular endothelium, with almost 5-fold increase in DHR intensity after 5 min of BERK RBC infusion (P<0.001) compared with ΔI at 1 min. When infusion of catalase (900 U/mouse) into recipient C57BL mice was followed 30 min later by a bolus of FITC-labeled BERK mouse RBCs, BERK RBC adhesion and pronounced DHR fluorescence in endothelium were observed, demonstrating that intravascular infusion of catalase had little effect on oxidant generation by BERK mouse RBCs. In contrast, infusion of BERK RBCs pre-treated with catalase (100 U in 0.2 ml RBC suspension, 9-fold less catalase per mouse) to quench RBC generated H2O2 inhibited endothelial DHR fluorescence and BERK RBC adhesion. These results strongly suggest an obligatory role of heme-mediated peroxide generation by SS RBC in endothelial activation and SS RBC adhesion, and support the notion that heme-mediated oxidant generation may play a vital role in endothelial dysfunction in sickle cell disease. Disclosures:No relevant conflicts of interest to declare.

Peroxiredoxin-2 (Prx2) Membrane Displacement is A New Additional Factor to the Severity of β-Thalassemic Mouse Red Cell Membrane Damage.

Abstract 2009Poster Board I-1031Peroxiredoxin 2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells (RBCs) and is involved in defence against oxidative stress. Although much is known regarding the structure and function of Prx2 in healthy RBCs, its role in protecting pathological RBCs remains largely unexplored. Here, we studied Prx2 dimerization and membrane translocation in mouse RBCs (wild-type mice, n=20) in response to in vitro oxidative stress by either phenylhydrazine-PHZ (50 μM) or diamide (2mM) or H2O2 (75-100 μM) and in vivo in β thalassemic (β thal) mouse RBCs (Hbbth/th and Hbbth3/+ mouse strains; n=16 from each group).In RBCs lysates we observed (i) slightly increased Prx2 dimerization in PHZ treated RBCs; (ii) complete Prx2 dimerization in presence of diamide; (iii) not apparent Prx2 dimerization in H2O2, suggesting a different sensitivity of Prx2 to the various oxidant agents. In RBCs membrane, Prx2 association was (i) reduced in PHZ treated RBCs; (ii) increased in diamide treated RBCs as both monomers and dimers and (iii) reduced to almost undetectable amount in H2O2 treated RBCs. In order to evaluate whether Prx2 native or from RBCs exposed to oxidative stress possess different degrees of affinity to native or oxidized membrane, we compared Prx2 membrane association in control, diamide, PHZ treated membranes with the cytoplasmic RBC fractions obtained from control, diamide and PHZ treated RBCs. We observed a remarkable loss of interactions only between Prx2 and the membranes obtained from PHZ treated red cells (n=3), suggesting that PHZ may inhibit Prx2 binding to the membrane through the masking of its docking sites. Using multiple techniques (immunofluorescence confocal microscopy, immunoblot and flow cytometric analysis), we showed that Prx2 was displaced from PHZ treated RBC membrane in a dose dependent manner. We then investigated the connection between Prx2 membrane displacement and hemicrome binding in time course experiments with PHZ treated RBCs. Prx2 was displaced from the membrane concomitantly to hemichrome membrane binding, indicating that PHZ blocks the Prx2 binding to the membrane through the masking of its docking sites by hemichromes.Since PHZ membrane oxidative damage mimics that of b thal RBCs we have examined Prx2 in RBCs from two mouse models of b thalassemia. We showed that Prx2 content was higher in b thal mouse RBCs than in controls and correlated with the clinical severity of b thalassemia, but the amount of Prx2 associated to the membrane was markedly reduced as observed in PHZ-treated RBCs. These data suggest that in b thal RBCs the masking of Prx2 membrane binding sites by hemichromes and the accumulation of its oxidized/dimerized state in the cytoplasm may affect Prx2 catalytic efficiency on b thal membrane proteins, therefore contributing to their lack of repair from oxidative insults. Thus, the abnormally low membrane association of Prx2 in b thal mouse RBCs represents a new additional factor amplifying the oxidative damage characterizing b thal RBCs and responsible for their reduced lifespan in the periferal circulation. Disclosures:No relevant conflicts of interest to declare.

Protein 4.1R-dependent multiprotein complex: New insights into the structural organization of the red blood cell membrane

Protein 4.1R (4.1R) is a multifunctional component of the red cell membrane. It forms a ternary complex with actin and spectrin, which defines the nodal junctions of the membrane-skeletal network, and its attachment to the transmembrane protein glycophorin C creates a bridge between the protein network and the membrane bilayer. We now show that deletion of 4.1R in mouse red cells leads to a large diminution of actin accompanied by extensive loss of cytoskeletal lattice structure, with formation of bare areas of membrane. Whereas band 3, the preponderant transmembrane constituent, and proteins known to be associated with it are present in normal or increased amounts, glycophorin C is missing and XK, Duffy, and Rh are much reduced in the 4.1R-deficient cells. The inference that these are associated with 4.1R was borne out by the results of in vitro pull-down assays. Furthermore, whereas Western blot analysis showed normal levels of band 3 and Kell, flow cytometric analysis using an antibody against the extracellular region of band 3 or Kell revealed reduction of these two proteins, suggesting a conformational change of band 3 and Kell epitopes. Taken together, we suggest that 4.1R organizes a macromolecular complex of skeletal and transmembrane proteins at the junctional node and that perturbation of this macromolecular complex not only is responsible for the well characterized membrane instability but may also remodel the red cell surface.

KINETICS OF FORMATION OF (±)-ANTI-7, 8-DIHYDROXY-9α,10α -EPOXY-7, 8, 9, 10-TETRAHYDROBENZO[A]PYRENE ADDUCTS WITH MOUSE AND HUMAN HEMOGLOBIN

Previous studies on the metabolic activation of polycyclic aromatic hydrocarbons have shown conclusively that activation to reactive electrophilic intermediates occurs via metabolism of the unsubstituted aromatic hydrocarbon to electrophilic diol-epoxide intermediates. These reactive electrophilic derivatives undergo reaction with various biological nucleophilic centers to form covalent modifications, which can be assessed by a variety of analytical techniques. While the formation of these adducts with various cellular nucleophiles has been clearly established, the relationships between relative rates of formation of various adducts in biological systems has not been significantly investigated. In the present study, we investigated the pharmacokinetics of reaction of the ultimate carcinogenic metabolite of benzo(a)pyrene, (±)-anti-7,8-dihydroxy-9α,10α -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), with both male C57BL/6 mouse red cell hemoglobin and human red cells, in vitro. Comparative pharmacokinetic studies using both mouse and human hemoglobin were carried out by incubation of the packed red cells together with solutions of (±)-anti-7,8-dihydroxy-9α,10α -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Similar rate constants for the binding of the epoxide to hemoglobin in both mouse and human were found, suggesting a similarity of amino acids that were adducted in both species. Overall binding of (±)-anti-7,8-dihydroxy-9α,10α -epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to mouse hemoglobin was found to be slightly higher than that found using human hemoglobin, suggesting the potential of a facilitation of the binding of the epoxide with mouse protein or an additional reactive amino acid to which the epoxide was bound. In parallel studies, mice were injected intraperitoneally with benzo(a)pyrene to compare the kinetics of binding of this carcinogen to hemoglobin, in vivo. Blood samples were obtained at specific intervals, and the kinetics of binding to hemoglobin characterized. These results suggested that the binding to hemoglobin in the in vivo studies was slightly lower than that compared to the in vitro assessments. However the differences in adduct levels strongly suggests that other metabolic pathways are involved in the metabolic activation of benzo(a)pyrene as well as other related carcinogenic compounds.

Reduced DIDS-sensitive chloride conductance in Ae1−/− mouse erythrocytes

The resting membrane potential of the human erythrocyte is largely determined by a constitutive Cl − conductance ∼ 100-fold greater than the resting cation conductance. The 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive electroneutral Cl − transport mediated by the human erythroid Cl −/HCO 3 − exchanger, AE1 (SLC4A1, band 3) is > 10,000-fold greater than can be accounted for by the Cl − conductance of the red cell. The molecular identities of conductive anion pathways across the red cell membrane remain poorly defined. We have examined red cell Cl − conductance in the Ae1 −/− mouse as a genetic test of the hypothesis that Ae1 mediates DIDS-sensitive Cl − conductance in mouse red cells. We report here that wildtype mouse red cell membrane potential resembles that of human red cells in the predominance of its Cl − conductance. We show with four technical approaches that the DIDS-sensitive component of erythroid Cl − conductance is reduced or absent from Ae1 −/− red cells. These results are consistent with the hypothesis that the Ae1 anion exchanger polypeptide can operate infrequently in a conductive mode. However, the fragile red cell membrane of the Ae1 −/− mouse red cell exhibits reduced abundance or loss of multiple polypeptides. Thus, loss of one or more distinct, DIDS-sensitive anion channel polypeptide(s) from the Ae1 −/− red cell membrane cannot be ruled out as an explanation for the reduced DIDS-sensitive anion conductance.

A Simple Method for the Production of Anti-C3d Monoclonal Antibody

Production of monoclonal antibodies to C3d usually involves the purification of protein. Our method does not require C3 purification; it relies on attachment of C3b to mouse erythrocytes by activation of alternative pathways and further conversion in C3d. We prepared human complement-coated mouse red cells and sensitized mice of the same strain with our own schedule of immunization and applied the classical methods to obtain a mouse monoclonal antibody. We obtained a clone called BMS-11 which produces a monoclonal antibody of IgM class, to C3d with a title of 1:500000. The monoclonal antibody obtained has shown that it is suitable for use as an antiglobulin reagent.

ETIO-PATHOGENETICAL RESEARCHES IN COCCIDIOSIS OF THE LAMBS BRED IN BIG GROUPS, IN AN UNIT FROM THE WEST OF ROMANIA

The diarrheic syndrome can be attributed to various causes: biotic agents (bacteria, parasites, fungi and viruses), alimentary, inobservance of the hygiene conditions, intervention of some maternal, genetic, immunity and human factors. An important place in producing this syndrome in l ambs devolves upon the Eimeria . In this study we had in view the qualitative estim ation of the value of the biotic agents which inter vene in the beginning of the diarrheic syndrome in lambs , from the neonatal period till to the age of 3 mon ths. Observations concerning the share of the coccidian infection in producing this syndrome in lambs were performed. Clinical exam were accomplished, specifying the incidence of the diarrheic syndrome, the lo sses by mortality in lambs, also anatomo-pathological exams by necropsy, histo-pathological exam (hematoxilin- eosine method). The coproscopical exam was done, using the Blagg modified byuteu, McMaster, sedimentation by centrifugation and Baermann methods. The coccidian species were determined by micrometry, according to the morphological features cited by various authors. Fo r determining the bacterial charge in faeces sample s from lambs, selective (Istrate-Maitert) and differentiat ion (MIU, TSI, MILF, citrate Simmons) mediums were used for enterobacteria, Chapmann medium for enterococc and agar with blood for the investigation of the hemoly tic capacity. The presence of Corona and Corona viruses were investigated by electrophoresis in poliacryla mide gel, respectively the hemagglutination assay with mouse red cells. As a result of the etio-pathogenetical and diagnost ic investigations performed, we came to the conclusions: the diarrheic syndrome evolved in the lambs aged from 4 weeks to 3 months, with an incidence of 26%. The mortality occurred in 16% of the sick lambs; the clinical aspects were represented by diarrhe a with aqueous yellow-chocolate colored feces, with abunda nt mucus, anomaly, then anorexia and dehydration; anatomo-pathologically, catharal enterocolitis, wit h punctiform hemorrhages, whitish foci on the intes tinal mucous membrane, hypertrophy of the mesenteric lymph nodes were observed. As a histopathologic result, the necrotic catharal enteritis, caused by coccidian ev olution forms, and mesenteric hyperplastic lymph ad enitis were observed. The diarrheic syndrome in lambs took place by the intervention of some unor multiple biotic agents: protozoa like Eimeria (extensivity 95%, intensivity 17500 EPG); Cryptosporidium (17.82%) and Giardia (22%); and the suprainfection with bacterial germs, unpath ogen, firstly E. coli (45%) and Proteus (65%). We didn't find Rota and Corona-viruses by the usual methods. The specific structure of the Eimeria population in the lambs with diarrheic syndrome is: E. ovinoidalis (42%); E. crandallis (28%); E. faurei (10%); E. parva (17%) and E. pallida (3%).

Soluble Urokinase Plasminogen Activator Receptor Is Increased in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and Thrombosis and Inhibits Plasmin Generation In Vitro and Promotes Thrombosis in the Mouse Model.

Venous thrombotic events produce serious morbidity and are the most frequent cause of death in patients with paroxysmal nocturnal hemoglobinuia (PNH). Urokinase plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked protein expressed on neutrophils and endothelial cells which mediates endogenous thrombolysis through a urokinase plasminogen activator (uPA)-dependent mechanism. On the cell surface, pro-uPA binds to uPAR, promoting the conversion of pro-uPA to active uPA; uPA then cleaves plasminogen, yielding active plasmin. Fibrinolysis may play an important role in PNH where ongoing hemolysis produces procoagulant-rich red cell microvesicles (Ninomiya h et al, Br J Haematol 106, 224); that thrombotic events frequently occur in the context of a hemolytic event in PNH suggests a role of erythrocyte or platelet microvesicles in clot formation. Membrane GPI-anchored uPAR is decreased or absent on granulocytes and soluble uPAR levels are increased in patients' plasma. In the current study of 45 patients with PNH and 20 normal controls, we demonstrate that plasma suPAR concentrations correlated with the proportion of neutrophils lacking CD16, another GPI-anchored protein (R2=0.9), and that levels are 4–10-fold greater in PNH than in controls. Patients with a history of thrombosis had a higher soluble uPAR levels (in stored samples pre-dating the thrombosis) than did patients without a history of thrombosis (mean 2.2 ng/mL compared to 0.5 ng/mL; p=0.0001). In vitro suPAR was released from PNH hematopoietic cells during short-term culture in three experiments; suPAR levels were 20 fold greater than those obtained from normal CD34 cells. Activated platelets also appear to be a substantial source of suPAR: uPAR was expressed on the surface of ADP-activated CD59-positive but not of CD59-negative platelets. By ELISA specific for suPAR, we showed that substantial quantities of suPAR were released following activation of PNH but not normal platelets. Immunoblot of released material using suPAR mAb demonstrated a band at 35kDa, consistent with absence of the GPI-anchor. We hypothesized that suPAR might bind to plasma uPA, preventing its interaction with the active membrane-anchored form. In vitro we demonstrated dose-dependent decrease in the activity of single-chain urokinase following the addition of recombinant suPAR, with loss of 25% of the activity after the addition of 0.45nM suPAR. Next we injected suPAR (to achieve a intravascular concentration of 3ng/mL) and autologous mouse red cell microvesicles into three mice for three consecutive days; six additional mice were injected with uPAR or microvesicles alone and three mice injected with PBS served as controls. Mice were sacrificed after 7 days and samples obtained from liver and lung for pathological examination. All 3 mice receiving both suPAR and red cell microvesicles had numerous thrombi in the lungs and liver, while these changes were not observed in the control mouse or in mice receiving either microvesicles or suPAR alone. These data suggest that absent leukocyte and platelet membrane uPAR, high levels of suPAR, and the presence of red cell microvesicles all contribute to produce thrombosis in patients with PNH.

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates

Fluorescence emission of free protoporphyrin IX (PPIX, em.∼626 nm), zinc protoporphyrin IX (ZPP, em.∼594 nm) and fluorescent heme degradation product (FHDP, em.∼466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F365/594) correlates with the ZPP fluorescence obtained from acetone/water extraction (R2 = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R2 = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.

Positively Charged Alpha-Chains Can Stimulate K-Cl Cotransport in Transgenic Mouse Red Cells.

Transgenic mice were generated with human α-chain anti-sickling mutations at contact sites for the HbS polymer. Some of these mice were found to have elevated K-Cl cotransport. Elevation of K-Cl cotransport in patients with homozygous HbS or SC disease increases red cell MCHC and contributes to pathology. In contrast to C57Bl mouse red cells (mRBC) and mRBC expressing only HbA that have little volume-stimulated K-Cl cotransport, we previously reported that HbC under all conditions, and HbS + γ, in the absence of mouse globins, are able to stimulate the activity of K-Cl cotransport in mRBC. These observations support the contention that HbS and HbC stimulate K-Cl cotransport activity in both mouse and human red cells and may do so via the positive charge on the human β-chain. We report here that positively charged α-chains also stimulate K-Cl cotransport in mRBC. Mice expressing α-chain mutants were generated: α49 (HbSavaria, α49S→R, +1 positive charge vs human α) with no human β-globin; α49 and βS; α49 and NY1 (that expresses human α and βS); α49–114, that expresses both α49 and α114 (HbChiapas α114P→R, +2 positive charge vs human α) with no human β-globin; α49–114 and βS; α20 (HbLeLamentin, α20 H→Q, -1 negative charge vs human α) and βS; and α20–114 (that has no average charge difference from human α) with no human β-globin. Mice were bred with α- and β-KO mice and mice expressing the NY1 transgene to produce mice expressing various levels of mutant α, human α, βS, and mouse globins. Density gradients detected increased red cell density relative to wild type mice (C57Bl) in mice expressing α-globins with a positive charge relative to human α. To isolate the effect of charged α-globin, we first examined mice with only mutated α-globins and no βS. We previously measured volume-stimulated K-Cl cotransport in C57Bl and HbAKO mice (that only express HbA) as 2.0±0.9 and 2.4±1.7 mmol/L cells x hr (FU) respectively. We found a similar value (2.4±1.1 FU) in mRBC expressing either 32% or 100% α20–114 with no βS (no charge difference from human α). mRBC expressing α49 at 44 or 100% with no βS had an average value of 12.9±3.3 FU; similarly, mRBC expressing 48% α114-49 with no βS averaged 9.4±1.2 FU. The volume-stimulated K-Cl cotransport was both chloride and okadaic acid sensitive. These results demonstrate that positively charged α-chains stimulate K-Cl cotransport while mutant α-chains without a charge difference do not. We have also examined mRBC expressing α49 or α49–114 with βS or NY1 and found that all mice exhibited increased K-Cl cotransport with the exception of mRBC from founder α49–114βS mice that express 31% mutant α and 9% βS. We conclude that a positive charge in excess of that found on human α can stimulate K-Cl cotransport and result in increased MCHC. In the presence of βS, this effect results in mouse red cells with properties similar to human SC disease and prevents the birth of mice that are fully knocked out, due to polymer formation, despite the presence of anti-sickling mutations. In constrast, mice with α20βS (−1 vs human α) were viable with exclusively human globins. These observations could only have been made by creating mouse models and imply that charge must also be considered when anti-sickling globins are proposed.

Regulation of K–Cl cotransport by protein phosphatase 1α in mouse erythrocytes

The K–Cl cotransport (KCC) is an electroneutral-gradient-driven-membrane transport system, which is involved in regulation of red cell volume. Although the regulatory cascade of KCC is largely unknown, a signaling pathway involving phosphatases and kinases has been proposed. Here, we investigated the expression and the activity of protein phosphatase 1(PP-1) isoforms in mouse red cells, focusing on two models of abnormally activated KCC: mice genetically lacking the two Src-family tyrosine kinases, Hck and Fgr, (hck-/-fgr-/-) and the SAD transgenic sickle-cell-mice. The PP-1α, PP-1γ, PP-1δ isoforms were expressed at similar levels in wild-type, hck-/-fgr-/- and SAD mouse erythrocytes and in each case were predominantly localized to cytoplasm. The PP-1α activity was significantly higher in both membrane and cytosol fractions of hck-/-fgr-/- and of SAD erythrocytes than in those of wild-type red cells, suggesting PP-1α as a target of the Hck and Fgr kinases. The PP2, a specific inhibitor of Src-family kinase, significantly increased KCC activity in wild-type mouse red cells, but failed to modify the already increased KCC activity in SAD erythrocytes. The lag-time for activation of KCC was considerably reduced in both hck-/-fgr-/- and SAD erythrocytes, suggesting that the rate limiting activation steps in both strains are freed from their tonic inhibition. Sulfhydryl reduction by dithiothreitol (DTT) lowered KCC activity only in SAD red cells, but did not affect the PP2-treated erythrocytes. These data suggest up-regulation of KCC in SAD red cells is mainly secondary to oxidative damage, which most likely reduces or removes the tonic KCC inhibition resulting from PP-1α activity controlled in turn by Src-family kinases.

Protein phosphatase 1α is tyrosine-phosphorylated and inactivated by peroxynitrite in erythrocytes through the src family kinase fgr

Protein serine/threonine phosphorylation is a significant component of the intracellular signal that together with tyrosine phosphorylation regulates several processes, including cell-cycle progression, muscle contraction, transcription, and neuronal signaling. Cross-talk between phosphoserine/threonine- and phosphotyrosine-mediated pathways is not yet well understood. In this study we found that peroxynitrite, a physiological oxidant formed by the fast radical–radical reaction between the nitric oxide and the superoxide anion, induced tyrosine phosphorylation of the serine/threonine protein phosphatase 1α (PP1α) in human erythrocytes through activation of src family kinases. We have previously shown in mouse red cells that upregulation of the src kinase fgr phosphorylates PP1α, acting as an upstream negative regulator of PP1α, and downregulates K-Cl cotransport. Here we found that PP1α is a selective substrate of peroxynitrite-activated fgr and that tyrosine phosphorylation of PP1α corresponds to an inhibition of its enzymatic activity. Despite fgr activation and PP1α downregulation, peroxynitrite stimulated in a dose-dependent fashion the function of the K-Cl cotransporter. In an attempt to understand the mechanism of K-Cl cotransport activation, we found that the effect of peroxynitrite is completely reversed by dithriothreitol, suggesting that peroxynitrite acts as an oxidizing agent by an SH-dependent and PP1α-independent mechanism. These findings highlight a novel function of peroxynitrite in regulating the intracellular signal transduction pathways involving serine/threonine phosphorylation and the functional role of proteins that are targets of these phosphatases.

Lineage-Specific Transcriptional Regulation of GATA1 Is Dependent on Lineage-Specific Utilisation of Multiple Cis-Elements and Haematopoietic Transcription Factors.

Normal differentiation of red cells, platelets and eosinophils from a myeloid progenitor requires expression of the transcription factor GATA1. Moreover, GATA1 expression level influences lineage output; higher levels promote erythromegakaryocytic differentiation and lower levels eosinophil maturation. Conversely, repression of GATA1 expression is required for monocyte/neutrophil development. GATA1 expression is principally controlled transcriptionally. Thus, dissecting the molecular basis of transcriptional control of GATA1 expression will be one important facet in understanding how myeloid lineages are specified. To address this question we sought to identify all DNA sequences important for GATA1 expression. Previous analysis identified 3 murine (m)Gata1 cis-elements (an upstream enhancer, mHS-3.5, a haematopoietic IE promoter and elements in a GATA1 intron, mHS+3.5) conserved in sequence between human(h) and mouse. These studies also suggested additional unidentified elements were required for erythroid and eosinophil GATA1 expression. We compared sequence, mapped DNase I hypersensitive sites (HS) and determined histone H3/H4 acetylation over ~120 kb flanking the hGATA1 locus and corresponding region in mouse to pinpoint cis-elements. Remarkably, despite lying in a ~10 MB conserved syntenic segment, the chromatin structures of both GATA1 loci are strikingly different. Two previously unidentified haematopoietic cis-elements, one in each species (mHS-25 and hHS+14), are not conserved in position and sequence and have enhancer activity in erythroid cells. Chromatin immunoprecipitation studies show both mHS-25 and hHS+14 are bound in vivo in red cells by the transcription factors GATA1, SCL, LMO2, Ldb1. These findings suggest that some cis-elements regulating human and mouse GATA1 genes differ. Further analysis of in vivo transcription factor occupancy at GATA1 cis-elements in primary mouse eosinophils and red cells, megakaryocytic cells (L8057) and control fibroblasts show lineage- and cis-element-specific patterns of regulator binding (see table below). In red cells and megakaryocytes, GATA1, SCL, LMO2 and Ldb1 bind at two regulatory elements (mhHS-25 and mHS-3.5). Interestingly, the megakaryocyte transcriptional regulator Fli1 factor binds to mHS+3.5 specifically in megakaryocytes. In eosinophils, a different pattern of DNase I HS and transcription factor binding is seen. GATA1, PU.1 and C/EBPe (all regulate eosinophil gene expression) bind IE promoter and/or mHS+3.5. Collectively, these results suggest lineage-specific GATA1 expession is dependent on combinations of cis-elements and haematopoietic trans-acting factors that are unique for each lineage.DNase I Hypersensitive sites and transcription factor occupancy at mGATA1 cis-elements.mHS-26/-25*mHS-3.5mIEmHS+3.5Primary erythroid cellsHS present, GATA1, SCL, LMO2, Ldb1HS present, GATA1, SCL, LMO2, Ldb1HS present, GATA1HS present, GATA1Megakaryocytic cellsHS present, GATA1, SCL, LMO2, Ldb1HS present, GATA1, SCL, LMO2, Ldb1HS present, GATA1HS present, GATA1 and Fli1Primary eosinophilsHS absentHS present, No TF detectedHS present, GATA1 and C/EBPεHS present, GATA1, C/EBP ε and PU.1FibroblastsHS absentHS absentHS absentHS absentm: mouse, h: human, *: HS identified in this study, TF: transcription factor

Expression of HbC and HbS, but not HbA, results in activation of K-Cl cotransport activity in transgenic mouse red cells

AbstractElevation of K-Cl cotransport in patients with homozygous hemoglobin (Hb) S or HbC increases red cell mean corpuscular hemoglobin concentration (MCHC) and contributes significantly to pathology. Elucidation of the origin of elevated K-Cl cotransport in red cells with mutant hemoglobins has been confounded by the concomitant presence of reticulocytes with high K-Cl cotransport. In red cells of control mice (C57BL), transgenic mice that express only human HbA, and transgenic mice that express both mouse globins and human HbS, volume stimulation is weak and insensitive to NO3- and dihydroindenyl-oxy-alkanoic acid (DIOA). DIOA and NO3- are inhibitors in all other mammalian red cells. In contrast, in knock-out mice expressing exclusively human hemoglobin HbC or HbS+γ, replacement of isotonic Cl- media by hypotonic Cl- resulted in strong volume stimulation and sensitivity to DIOA, okadaic acid, and NO3-. In summary, we find that HbC, under all conditions, and HbS+γ, in the absence of mouse globins, have significant quantitative and qualitative effects on K-Cl cotransport in mouse red cells and activate mouse K-Cl. We conclude that human globins are able to stimulate the activity and/or regulation of K-Cl cotransport in mouse red cells. These observations support the contention that HbS and HbC stimulate K-Cl cotransport in human red cells.

Screening for carbohydrate-binding proteins in extracts of Uruguayan plants.

The presence of carbohydrate-binding proteins, namely lectins, -galactosidases and amylases, was determined in aqueous extracts of plants collected in Uruguay. Twenty-six extracts were prepared from 15 Uruguayan plants belonging to 12 Phanerogam families. Among them, 18 extracts caused hemagglutination (HAG) that was inhibited by mono- and disaccharides in 13 cases, indicating the presence of lectins. The other 8 extracts did not cause any HAG with the four systems used to detect HAG activity (rabbit and mouse red cells, trypsin-treated rabbit and mouse red cells). For the extracts prepared from Solanum commersonii, HAG activity and HAG inhibition were similar for those prepared from tubers, leaves and fruits, with the chitocompounds being responsible for all the inhibitions. Purification of the S. commersonii tuber lectin was carried out by affinity chromatography on asialofetuin-Sepharose, and SDS-PAGE under reducing conditions gave a single band of Mr of approximately 80 kDa. The monomer N-acetylglucosamine did not inhibit HAG induced by the purified lectin, but chitobiose inhibited HAG at 24 mM and chitotriose inhibited it at 1 mM. -Galactosidase activity was detected in leaves and stems of Cayaponia martiana, and in seeds from Datura ferox. Only traces of amylase activity were detected in some of the extracts analyzed. The present screening increases knowledge about the occurrence of carbohydrate-binding proteins present in regional plants.

Origins of the discipline ‘neonatal haematology’, 1925-75

Origins of the discipline ‘neonatal haematology’, 1925-75

Single-Particle Tracking and Laser Optical Tweezers Studies of the Dynamics of Individual Protein Molecules in Membranes of Intact Human and Mouse Red Cells

Single-Particle Tracking and Laser Optical Tweezers Studies of the Dynamics of Individual Protein Molecules in Membranes of Intact Human and Mouse Red Cells

K:Cl Cotransport Activity Is Inhibited by HCO−3 in Knockout Mouse Red Cells Expressing Human HbC

ABSTRACTK:Cl cotransport (KCl) was examined in transgenic mice expressing exclusively human hemoglobin C. In contrast to previous studies in early transgenic mice expressing human α and βS and residual mouse globins, we found significant volume and pH stimulation and sensitivity to . Exposure to physiological levels of also blocked a significant fraction of KCl cotransport.

K+(Na+)/H+ Exchange in Human Erythrocytes Activated under Low Ionic Strength Conditions

The K–Cl cotransporters (KCCs) of mouse erythrocytes exhibit higher basal activity than those of human erythrocytes, but are similarly activated by cell swelling, by hypertonic urea, and by staurosporine. However, the dramatic stimulation of human erythroid KCCs by N-ethylmaleimide (NEM) is obscured in mouse erythrocytes by a prominent NEM-stimulated K+ efflux that lacks Cl−-dependence. The NEM-sensitivity of Cl−-independent K+ efflux of mouse erythrocytes is lower than that of KCC. The genetically engineered absence of the K–Cl cotransporters KCC3 and KCC1 from mouse erythrocytes does not modify Cl−-independent K+ efflux. Mouse erythrocytes genetically devoid of the Gardos channel KCNN4 show increased NEM-sensitivity of both Cl−-independent K+ efflux and K–Cl cotransport. The increased NEM-sensitivity and stimulation magnitude of Cl−-independent K+ efflux in mouse erythrocytes expressing transgenic hypersickling human hemoglobin SAD (HbSAD) are independent of the presence of KCC3 and KCC1, but absence of KCNN4 reduces the stimulatory effect of HbSAD. NEM-stimulated Cl−-independent K+ efflux of mouse red cells is insensitive to ouabain and bumetanide, but partially inhibited by chloroquine, barium, and amiloride. The NEM-stimulated activity is modestly reduced at pH 6.0 but not significantly altered at pH 8.0, and is abolished at 0 °C. Although the molecular identity of this little-studied K+ efflux pathway of mouse erythrocytes remains unknown, its potential role in the pathophysiology of sickle red cell dehydration will be important for the extrapolation of studies in mouse models of sickle cell disease to our understanding of humans with sickle cell anemia.

The mouse Kell blood group gene (Kel): cDNA sequence, genomic organization, expression, and enzymatic function.

The human Kell blood group system is important in transfusion medicine, since Kell is a polymorphic protein and some of its antigens can cause severe reactions if mismatched blood is transfused, while maternal alloimmunization may lead to fetal and neonatal anemia. In humans, Kell is an Mr 93,000 type II membrane glycoprotein with endothelin-3-converting enzyme activity that is linked by a single disulfide bond to another protein, XK, that spans the membrane ten times. An absence of XK leads to clinical symptoms termed the McLeod syndrome. We determined the cDNA sequence of the mouse Kell homologue, the organization of the gene, expression of the protein and its enzymatic function on red cells. Comparison of human and mouse Kell cDNA showed 80% nucleotide and 74% amino acid sequence identity. Notable differences are that the mouse Kell protein has eight probable N-linked carbohydrate side chains, compared to five for human Kell, and that the mouse homologue has one more extracellular cysteine than human Kell protein. The mouse Kell gene (Kel), like its human counterpart, is similarly organized into 19 exons. Kel was located to proximal Chromosome 6. Northern blot analysis showed high expression in spleen and weaker levels in testis and heart. Western blot analysis of red cell membrane proteins demonstrated that mouse Kell glycoprotein has an apparent Mr of 110,000 and, on removal of N-linked sugars, 80,000. As in human red cells, Kell is disulfide-linked to XK and mouse red cells have endothelin-3-converting enzyme activity.