Human Red Cells Research Articles

Reduction of Prion Infectivity in Packed Red Cells by Separation of Whole Blood and Washing of the Red Cell Fraction.

Although prion diseases are rare in humans, the established link between a new variant form of CJD (vCJD) and the consumption of cattle meat contaminated by BSE have raised concerns about a possible outbreak of a large epidemic in the human population. Over the past few years, BSE has become a significant health concern in several countries, and it now seems apparent that vCJD can also be iatrogenically transmitted from human to human by blood transfusion. Exacerbating this state of affairs is the lack of a reliable test to identify individuals incubating the disease during the long and silent period from the onset of infection to the appearance of clinical symptoms. The purpose of this research study was to evaluate the effectiveness of separation of whole blood and washing of the red cell fraction for the removal of infectious scrapie prion protein (PrPSc) from red blood cell (RBC) suspensions. Samples of human, whole blood were spiked with 5 × 106 LD50 263K PrPSc. Analysis of the treated sample supernatants by Western blot revealed that approximately >88% of the PrPSc was removed with the initial plasma expression and the equivalent of 6% was detected in a saline wash (300 mL; 0.9% saline). The final sample of RBCs revealed no detectable levels of PrPSc by Western blots. Further analysis of the treated RBCs using the PMCA assay indicated detectable amounts of PrPSc only after 2 consecutive amplification rounds. Semi-quantitative analysis of PMCA amplification enabled us to estimate that the treated RBCs contained less than 1 × 104 LD50 PrPSc. This corresponded to removal levels exceeding ≥99% of spiked material in whole blood. These in vitro estimations were confirmed by in vivo infectivity studies in a hamster model of disease transmission.Results from in vivo studies displayed significant differences in the incubation periods of the spiked blood inoculated hamsters (100.1 ± 1.7) versus washed RBCs (135.8 ± 6.7). Moreover, a substantial difference in the attack rate (6/15: 40% in washed RBC, versus 13/13: 100% in spiked blood) further indicated a substantial removal of infectious prions. Comparison of this data with results of animals inoculated with different dilutions of infectious material, indicated a >99.94% reduction of infectivity. Washed, packed human red cells produced by this procedure were able to be stored in standard additive solutions (AS-3) for 42 days while still meeting all in vitro blood bank standards for acceptable red cell quality.Conclusion This data suggests that separation of plasma coupled with a simple, low volume wash of red cells may represent an efficient method to remove prions from red blood cell fractions, thus reducing possible infectivity of these products.

Herbicide 2,4‐dichlorophenoxyacetic acid (2,4‐D)‐induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.

Tank-Tread Frequency of the Red Cell Membrane: Dependence on the Viscosity of the Suspending Medium

Single human red cells were suspended in media with viscosities ranging from 12.9 to 109 mPa s and subjected to shear flow ranging from 1/s to 290/s in a rheoscope. This is a transparent cone-plate chamber adapted to a microscope. The motion of the membrane around red cells oriented in a steady-state fashion in the shear field (tank-tread motion) was videotaped. The projected length and width of the cells as well as the frequency of tank-tread motion were measured. One-thousand eight-hundred seventy-three cells of three blood donors were evaluated. The frequency increased with the mean shear rate in an almost linear fashion. The slope of this dependence increased weakly with the viscosity of the suspending medium. No correlation was found between the frequency and four morphological red cell parameters: the projected length and width of the cells as well as the ratio and the square root of the product of these quantities. The energy dissipation within the red cell membrane was estimated based on the measured parameters and compared to the energy dissipation in the undisturbed shear flow. At constant mean shear rate the rise of the energy dissipation with viscosity is slower whereas at constant viscosity the rise with the shear rate is steeper than in the undisturbed shear flow. A fit of the data collected in this work to a theoretical red cell model might allow one to determine intrinsic mechanical constants in the low deformation regime.

Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex ®-GS, 0.22 μm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax ® and Potter–Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5 h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 μM) or HI 6 (30 μM) of sarin-inhibited human muscle acetylcholinesterase (0.142 ± 0.004 min −1 and 0.166 ± 0.008 min −1, respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.

α,β-Dehydrophenylalanine choline esters, a new class of reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase

Our goal was to design, synthesize, and evaluate new cholinesterase inhibitors. Fourteen dehydroamino acids esterified to choline and to its ternary analog were synthesized by a new method that gave a yield of 84–93%. The potency of the amino acid ester derivatives was tested by measuring K i values for inhibition of human red cell acetylcholinesterase and human plasma butyrylcholinesterase. The most potent compound was a choline ester of dehydrophenylalanine where the amine group of the amino acid was derivatized with a benzoyl group containing a methoxy in the 2-position, CH 3O(C 6H 4)CONHC(CHC 6H 5)COOCH 2CH 2N +(CH 3) 3. This compound was a strong inhibitor of both human acetylcholinesterase and human butyrylcholinesterase, with K i values of 10 μM and 0.08 μM, respectively. These K i values are comparable to that of Rivastigmine. Docking of the most potent compound into the active site of human butyrylcholinesterase showed that the lowest energy model had two benzene rings oriented towards Trp 82 and Tyr 332 whereas the positively charged nitrogen group was stabilized by Trp 231. This orientation placed the ester group 3.89 Å from the active site Ser 198, a distance too far for covalent bonding, explaining why the esters are inhibitors rather than substrates. This class of anticholinesterase agents has the potential for therapeutic utility in the treatment of disorders of the cholinergic system.

RhAG protein of the Rhesus complex is a CO2channel in the human red cell membrane

We have determined CO2 permeabilities, P(CO2), of red cells of normal human blood and of blood deficient in various blood group proteins by a previously described mass spectrometric technique. While P(CO2) of normal red cells is approximately 0.15 cm/s, we find in red blood cells (RBCs) lacking the Rh protein complex (Rh(null)) a significantly reduced P(CO2) of 0.07 cm/s +/-0.02 cm/s (P<0.02). This value is similar to the value we have reported previously for RBCs lacking aquaporin-1 protein (AQP-1(null)), suggesting that each of the Rh and AQP-1 proteins is responsible for approximately 1/2 of the normal CO2 permeability of the RBC membrane. Four other blood group deficiencies tested lack diverse membrane proteins but exhibit normal CO2 permeability. The CO2 pathway constituted by Rh proteins was inhibitable at pH(e)= 7.4 by NH4Cl with an I50 of approximately 10 mM corresponding to an I50 for NH3 of approximately 0.3 mM. The pathway independent of Rh proteins, presumably that constituted by AQP-1, was not inhibitable by NH4Cl/NH3. However, both pathways were strongly inhibited by DIDS, which accounts for the marked inhibitory effect of DIDS on normal P(CO2), while in contrast another AE1 inhibitor, DiBAC, does not inhibit P(CO2), although it markedly reduces P(HCO3-). We conclude that Rh protein, presumably the Rh-associated glycoprotein RhAG, possesses a gas channel that allows passage of CO2 in addition to NH3.

Lacking deoxygenation‐linked interaction between cytoplasmic domain of band 3 and HbF from fetal red blood cells

Several of the red blood cell's metabolic and membrane functions display dependence on haemoglobin oxygenation. In adult human red cells, the increased glycolytic rate at low O2 tension results from binding of deoxygenated HbA at negatively charged, N-terminal, cytoplasmic domain of the membrane protein band 3, which liberates glycolytic enzymes from this site. This study aims to investigate the role of fetal HbF (that has lower anion-binding capacity than HbA) in fetal red cells (that are subjected to low O2 tensions), and to elucidate possible linkage (e.g. via the major red cell membrane organising centre, band 3) between the individual oxygenation-linked reactions encountered in red cells. The interaction between band 3 and Hb is analysed in terms of the effects, measured under different conditions, of a 10-mer peptide that corresponds to the N-terminus of human band 3 protein, on the oxygenation reaction of HbF and HbA, isolated from umbilical chord red cells. Contrasting with the unequivocal interaction of the peptide with HbA that with fetal HbF is weak, and annihilated in the presence of autochthonous red cell O2 affinity modulators (chloride and organic phosphates). The data indicate that HbF does not function as a transducer mediating O2 dependence of glycolysis in fetal red cells, in accordance with the different O2 and metabolic profiles compared to those in HbA-bearing adult red cells. In conjunction with the previously discovered O2 dependence of K+ transport in HbF-rich fetal cells, they moreover argue against linkage between different, physiologically relevant, O2-dependent red cell functions.

Preparation and characterization of dialkylphosphoryl-obidoxime conjugates, potent anticholinesterase derivatives that are quickly hydrolyzed by human paraoxonase (PON1 192Q)

The potential of the most active pyridinium-4-aldoximes, such as obidoxime and trimedoxime, to reactivate phosphorylated acetylcholinesterase is not fully exploited because of inevitable formation of phosphoryloximes (POXs) with extremely high anticholinesterase activity. Hence, a topochemical equilibrium is expected at the active site, with the freshly reactivated enzyme being rapidly re-inhibited by POX produced during reactivation. In the present study, dimethylphosphoryl-, diethylphosphoryl-, and diisopropyl-obidoxime conjugates were generated and isolated in substance. Their inhibition rate of acetylcholinesterase from human red cell membranes was by a factor of 2250, 480 and 600 higher than that observed with paraoxon-methyl, paraoxon-ethyl, and diisopropyl phosphorofluoridate, respectively. All three POXs were hydrolyzed by human paraoxonase (PON1), with the alloenzyme PON1 192Q being about 50-fold more active than PON1 192R. The rate of hydrolysis, yielding obidoxime, was 1:6:0.03 for the three POXs, respectively. The rate of non-enzymic degradation, yielding obidoxime mononitrile, was similar with the three POXs and showed a high dependency on the reaction temperature (activation energy 83 kJ/mol), while enzymic hydrolysis required less energy (16 kJ/mol). To determine POX-hydrolase activity, we preferred a reaction temperature of 20 °C to reduce the noise of spontaneous degradation. A plot of POX-hydrolase versus salt-stimulated paraoxonase activity showed a highly discriminating power towards the PON1 Q192R alloenzymes, which may be based on repulsive forces of the quaternary nitrogen atoms of the protonated arginine subtype and the bisquaternary POXs. It is concluded that the pharmacogenetic PON1 Q192R polymorphism may be another contributor to the large variability of susceptible subjects seen in obidoxime-treated patients.

Receptor binding specificity of recent human H3N2 influenza viruses

BackgroundHuman influenza viruses are known to bind to sialic acid linked α2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection.ResultsViruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of α2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for α2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of α2-6 and α2-3 sialic acids, including human A549 and HeLa cells with high levels of α2-6 sialic acid, and CHO cells that have only α2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells.ConclusionAgglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan Array, and may result from increased avidity due to density of hemagglutinin and not increased affinity. Absence of α2-6 sialic acid does not protect a cell from influenza infection and the presence of high levels of α2-6-sialic acids on a cell surface does not guarantee productive replication of a virus with α2-6 receptor specificity.

A conductive pathway generated from fragments of the human red cell anion exchanger AE1

Human red cell anion exchanger AE1 (band 3) is an electroneutral Cl-HCO3- exchanger with 12-14 transmembrane spans (TMs). Previous work using Xenopus oocytes has shown that two co-expressed fragments of AE1 lacking TMs 6 and 7 are capable of forming a stilbene disulphonate-sensitive (36)Cl-influx pathway, reminiscent of intact AE1. In the present study, we create a single construct, AE1Delta(6: 7), representing the intact protein lacking TMs 6 and 7. We expressed this construct in Xenopus oocytes and evaluated it employing a combination of two-electrode voltage clamp and pH-sensitive microelectrodes. We found that, whereas AE1Delta(6: 7) has some electroneutral Cl-base exchange activity, the protein also forms a novel anion-conductive pathway that is blocked by DIDS. The mutation Lys(539)Ala at the covalent DIDS-reaction site of AE1 reduced the DIDS sensitivity, demonstrating that (1) the conductive pathway is intrinsic to AE1Delta(6: 7) and (2) the conductive pathway has some commonality with the electroneutral anion-exchange pathway. The conductance has an anion-permeability sequence: NO3- approximately I- > NO2- > Br- > Cl- > SO4(2-) approximately HCO3- approximately gluconate- approximately aspartate- approximately cyclamate-. It may also have a limited permeability to Na+ and the zwitterion taurine. Although this conductive pathway is not a usual feature of intact mammalian AE1, it shares many properties with the anion-conductive pathways intrinsic to two other Cl-HCO3- exchangers, trout AE1 and mammalian SLC26A7.

Prion protein and the red cell

This review focuses on transfusion-transmission of variant Creutzfeldt-Jakob disease by red cell preparations. Recently, three cases of probable transmission of variant Creutzfeldt-Jakob infectivity by transfusion of red cell preparations have been described in humans. Experiments on transmissible spongiform encephalopathies affecting rodents have led to the conclusion that infectivity in red cell preparations is not bound to the red cells themselves but contained within the suspending medium from which it can be removed by filtration. Red cell preparations are the main transfusion product provided by blood services. If experiments demonstrating significant removal of rodent transmissible spongiform encephalopathy infections by filtration of red cell preparations are applicable to variant Creutzfeldt-Jakob in humans then a method for rendering human red cell preparations safe for transfusion is provided.

Pyruvate kinase deficiency: The genotype-phenotype association

Red cell pyruvate kinase (PK) deficiency is the most frequent enzyme abnormality of glycolysis causing chronic non-spherocytic haemolytic anaemia. The disease is transmitted as an autosomal recessive trait, clinical symptoms usually occurring in compound heterozygotes for two mutant alleles and in homozygotes. The severity of haemolysis is highly variable, ranging from very mild or fully compensated forms to life-threatening neonatal anaemia necessitating exchange transfusions. Erythrocyte PK is synthesised under the control of the PK-LR gene located on chromosome 1. One hundred eighty different mutations in PK-LR gene, mostly missense, have been so far reported associated to PK deficiency. First attempts to delineate the genotype-phenotype association were mainly based on the analysis of the enzyme's three-dimensional structure and the observation of the few homozygous patients. More recently, the comparison of the recombinant mutants of human red cell PK with the wild-type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined. However, the clinical manifestations of red cell enzyme defects are not merely dependent on the molecular properties of the mutant protein but rather reflect the complex interactions of additional factors, including genetic background, concomitant functional polymorphisms of other enzymes, posttranslational or epigenetic modifications, ineffective erythropoiesis and differences in splenic function.

Bovine hemoglobin (glutamer‐250, Hemopure)‐specific immunoglobulin G antibody cross‐reacts with human hemoglobin but does not lyse red blood cells in vitro

Bovine hemoglobin (Hb)-based oxygen carrier (HbOC-201; Hb glutamer-250, Hemopure, Biopure Corp.) is a blood replacement and augmentation drug that increases oxygen-carrying capacity of circulating blood in patients with anemia and acute blood loss. The objective of this study was to assess the biologic significance (cross-reactivity, hemolysis) of humoral immune responses in humans receiving repetitive HbOC-201 administrations. Serum samples containing immunoglobulin G (IgG) anti-HbOC-201 (n = 146) or no antibody (n = 16) were collected from subjects receiving HbOC-201 in clinical studies. IgG anti-HbOC-201 levels were quantified and the extent of cross-reactivity to human hemoglobin (HuHb) was assessed in direct-binding and competitive-inhibition immunoassays. Serum samples containing the highest levels of IgG anti-HbOC-201 were studied in a complement-mediated hemolysis assay for their ability to lyse human red cells (RBCs). The IgG anti-HbOC-201 levels in the antibody-positive serum samples ranged from 0.7 to 86.8 mug per mL. Of the 146 IgG anti-HbOC-201-positive serum samples, 88.4 percent contained IgG antibodies whose binding to solid-phase HbOC-201 was competitively inhibited by incubation with soluble HuHb (11.6% [<20% inhibition]; 63% [20%-80% inhibition]; and 25.4% [>81% inhibition]). Direct-binding analysis to solid-phase HuHb confirmed that 74 percent contained IgG antibodies reactive with HuHb. Dichotomous competitive inhibition and direct-binding IgG anti-HuHb data correlated significantly (r(2) = 0.77, p < 0.001). Serum samples with the highest levels of IgG anti-HuHb, as identified from clinical studies, did not lyse human RBCs in the presence of exogenous complement or induce the direct sensitization of RBCs with human IgG or complement. These analyses indicate that HbOC-201 administration elicits IgG antibodies in humans that react with bovine and HuHb, but do not cause hemolysis in vitro.

Scrambling of Phospholipids Activates Red Cell Membrane Cholesterol

Cholesterol is predicted to associate more strongly with the outer than the inner leaflet of plasma membrane bilayers based on the relative in vitro affinities of their phospholipids. Complex formation with the high-affinity species (especially saturated sphingomyelins) is said to reduce the chemical activity (escape potential or fugacity) of the sterol. We therefore tested the hypothesis that scrambling the sidedness of plasma membrane phospholipids of intact cells will increase the chemical activity of outer surface cholesterol. Upon activating the plasma membrane scramblase in intact human red cells by introducing ionomycin to raise cytoplasmic Ca++, phosphatidylserine became exposed and, concomitantly, the chemical activity of exofacial cholesterol was increased. (This was gauged by its susceptibility to cholesterol oxidase and its rate of transfer to cyclodextrin.) Similar behavior was observed in human fibroblasts. Two other treatments known to activate cell surface cholesterol (namely, exposure to glutaraldehyde and to low-ionic-strength buffer) also brought phosphatidylserine to the cell surface but by a Ca++-independent mechanism. Given that phospholipid scrambling is important in blood coagulation and apoptosis, the concomitant activation of cell surface cholesterol could contribute to these and other pathophysiological signaling processes.

The effects of pyrimethamine and sulphadoxine mixture on plasma haemoglobin/methaemoglobin ratio of Hbaa erythrocytes

Fifty(50) blood samples of human red blood cell genotype HbAA were assayed in-vitro to determine the effect of increasing combined concentrations of pyrime- thamine and sulphadoxine (ratio 20:1w/w) solution on plasma levels of hemoglobin (Fe 2+)/methaemoglobin (Fe 3+) ratio. The concentrations used were 0.2, 0.4, 0.6, and 0.8%. The mean percentage plasma hemoglobin level of the control sample was 97.86 ± 0.74%, while the test samples at 0.2, 0.4, 0.6 and 0.8% concentrations showed hemoglobin levels of 96.52 ± 0.75, 96.72 ± 0.64, 97.42 ± 0.61 and 96.07 ± 1.49% respectively. The mean percentage methaemoglobin level of the control sample was 2.14 ± 0.74%, while the test samples at 0.2, 0.4, 0.6 and 0.8% concentration gave plasma methaemoglobin levels of 3.48 ± 0.75, 3.28 ± 0.64, 2.55 ± 0.61 and 3.93 ± 1.49% respectively. These result showed a concentration dependent variability with the percentage of pyrimethamine/sulphadoxine mixture that was added to the test samples. Statistical analysis showed a significant difference between the mean values of the control and test samples (p IJONAS Vol. 3 (1) 2007: pp. 116-121

Differential Effect of HOE642 on Two Separate Monovalent Cation Transporters in the Human Red Cell Membrane

Residual K⁺ fluxes in red blood cells can be stimulated in conditions of low ionic strength. Previous studies have identified both the non-selective, voltage-dependent cation (NSVDC) channel and the K⁺ (Na⁺)/H⁺ exchanger as candidate pathways mediating this effect, although it is possible that these pathways represent different modes of operation of a single system. In the present study the effects of HOE642, recently characterised as an inhibitor of the K⁺(Na⁺)/H⁺ exchanger, on NSVDC has been determined to clarify this question. Radioisotope flux measurements and conductance determinations showed that HOE642 exerted differential effects on the NSVDC channel and the K⁺(Na⁺)/H⁺ exchanger, confirming that the salt loss observed in low ionic strength solutions represents contributions from at least two independent ion transport pathways. The findings are discussed in the context of red blood cell apoptosis (eryptosis) and disease.

Calculation of transmission coefficients for some permeant molecules in human red cell and resting axolemma squid axon membranes

Quantum mechanical calculations of transmission coefficients for some permeant molecules across the human red cell and resting axolemma squid axon membranes are carried out. The calculations depend on (i) the molecular weight of the molecule and (ii) the depth and width of the potential well of the membrane. In most cases good agreement between calculated and experimental values is found.

Diagnosing Hemolytic Disease of the Fetus — Time to Put the Needles Away?

The RhD (Rh0[D]) antigen is considered the most immunogenic of the antigens found on the surface of the human red cell. In 1941, Levine, Katzin, and Burham1 showed that antibodies to the RhD antigen in pregnant women caused hemolysis and anemia in their offspring. Originally called erythroblastosis fetalis secondary to the finding of a large number of immature red cells in the neonatal circulation, today this condition is known as hemolytic disease of the fetus and newborn.When RhD-positive fetal cells enter the maternal circulation as a result of pregnancy-related events such as miscarriage or delivery, anti-D antibody . . .

Reversible Post-translational Modification of Proteins by Nitrated Fatty Acids in Vivo

Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking.

Of mice and men: the voracious spleen

Comment on Buffet et al, page [3745][1] Over the years, physiological studies have provided many insights into the function of the spleen, and now, in this issue of Blood , Buffet and colleagues describe significant new mechanistic insights into clearance of parasitized human red cells by the