Formation of meso, N-diphenylprotoporphyrin IX by an aerobic reaction of phenylhydrazine with oxyhemoglobins. (1/16)

Administration of phenylhydrazine to rabbits resulted in the denaturation of hemoglobins in erythrocytes, causing the formation of intracellular precipitates known as Heinz bodies, severe hemolytic anemia, and reticulocytosis. To elucidate the molecular mechanism of the destabilization, we allowed human oxyhemoglobins to react aerobically with phenylhydrazine. After treatment with acetic acid/HCl and H2SO4/methanol, the chloroform extract contained blue-green pigments of major products accompanied by different minor products. Each product was isolated by column chromatography. By fast-atom-bombardment mass spectrometry (FAB-MS) and proton nuclear magnetic resonance (1H-NMR) spectrometry, dimethyl esters of N-phenylprotoporphyrin IX and meso, N-diphenylprotoporphyrin IX were determined. Other major products also were determined to be dimethyl esters of triphenyl-and tetraphenyl-substituted protoporphyrins by FAB-MS. The formation of meso, N-diphenylprotoporphyrin indicated that the addition of a phenyl radical to the meso-carbon atom of the protoporphyrin ring occurred. Triphenyl and tetraphenyl adducts also indicated the formation of phenyl radicals in the aerobic reaction of phenylhydrazine with oxyhemoglobins. From these results, we suggest that the formation of phenyl radicals and the replacement of heme with phenyl-substituted protoporphyrins cause the destabilization of hemoglobins to induce Heinz bodies and hemolytic anemia with phenylhydrazine.  (+info)

Inappropriately low reticulocytosis in severe malarial anemia correlates with suppression in the development of late erythroid precursors. (2/16)

Inappropriately low reticulocytosis may exacerbate malarial anemia, but the under-lying mechanism is not clear. In this study, naive and infected mice were treated with recombinant murine erythropoietin (EPO), and the upstream events of erythropoiesis affected by blood-stage Plasmodium chabaudi AS were investigated. Malaria infection, with or without EPO treatment, led to a suboptimal increase in TER119(+) erythroblasts compared with EPO-treated naive mice. Furthermore, a lower percentage of TER119(+) erythroblasts in infected mice were undergoing terminal differentiation to become mature hemoglobin-producing erythroblasts. The impaired maturation of erythroblasts during infection was associated with a shift in the transferrin receptor (CD71) expression from the TER119(+) population to B220(+) population. Moreover, the suboptimal increase in TER119(+) erythroblasts during infection coincided with a blunted proliferative response by splenocytes to EPO stimulation in vitro, although a high frequency of these splenocytes expressed EPO receptor (EPOR). Taken together, these data suggest that during malaria, EPO-induced proliferation of early EPOR-positive erythroid progenitors is suppressed, which may lead to a suboptimal generation of TER119(+) erythroblasts. The shift in CD71 expression may result in impaired terminal maturation of these erythroblasts. Thus, inadequate reticulocytosis during malaria is associated with suppressed proliferation, differentiation, and maturation of erythroid precursors.  (+info)

Modulation of the course and outcome of blood-stage malaria by erythropoietin-induced reticulocytosis. (3/16)

Severe anemia is a major life-threatening complication of malaria. The roles of erythropoietin (Epo) and erythropoiesis during blood-stage malaria were investigated. By treating Plasmodium chabaudi AS-infected C57BL/6 (B6) mice, which are resistant to malaria, with polyclonal anti-human Epo neutralizing antibody, we demonstrated that Epo-induced reticulocytosis was important for alleviating malarial anemia and for host survival. By inducing erythropoiesis in A/J mice, which are susceptible to malaria, and in B6 mice at various periods during infection, by use of exogenous recombinant murine Epo, untimely onset of reticulocytosis was shown to augment multiplication of parasites and result in lethal infection. However, timely inducement of reticulocytosis with Epo treatment alleviated malarial anemia and increased survival. Our data reveal the important role of Epo-induced reticulocytosis in modulating the course and outcome of blood-stage malaria. However, the mechanisms underlying the increased mortality associated with untimely treatment with Epo and the increased protection associated with timely treatment with Epo remain to be investigated.  (+info)

Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton. (4/16)

Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases regulate actin structures and have multiple overlapping as well as distinct roles in hematopoietic cells; therefore, we studied their role in red blood cells (RBCs). Conditional gene targeting with a loxP-flanked Rac1 gene allowed Crerecombinase-induced deletion of Rac1 on a Rac2 null genetic background. The Rac1(-/-);Rac2(-/-) mice developed microcytic anemia with a hemoglobin drop of about 20% and significant anisocytosis and poikilocytosis. Reticulocytes increased more than 2-fold. Rac1(-/-);Rac2(-/-) RBCs stained with rhodamine-phalloidin demonstrated F-actin meshwork gaps and aggregates under confocal microscopy. Transmission electron microscopy of the cytoskeleton demonstrated junctional aggregates and pronounced irregularity of the hexagonal spectrin scaffold. Ektacytometry confirmed that these cytoskeletal changes in Rac1(-/-);Rac2(-/-) erythrocytes were associated with significantly decreased cellular deformability. The composition of the cytoskeletal proteins was altered with an increased actin-to-spectrin ratio and increased phosphorylation (Ser724) of adducin, an F-actin capping protein. Actin and phosphorylated adducin of Rac1(-/-);Rac2(-/-) erythrocytes were more easily extractable by Triton X-100, indicating weaker association to the cytoskeleton. Thus, deficiency of Rac1 and Rac2 GTPases in mice alters actin assembly in RBCs and causes microcytic anemia with reticulocytosis, implicating Rac GTPases as dynamic regulators of the erythrocyte cytoskeleton organization.  (+info)

Enhanced suicidal death of erythrocytes from gene-targeted mice lacking the Cl-/HCO(3)(-) exchanger AE1. (5/16)

Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl(-) removal, and energy depletion activate Ca(2+)-permeable cation channels with Ca(2+)-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1(-/-) mice) than in their wild-type littermates (AE1(+/+) mice) despite increased percentages of reticulocytes (AE1(-/-): 49%, AE1(+/+): 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1(-/-)erythrocytes/reticulocytes ( approximately 10%) than in AE1(+/+) erythrocytes ( approximately 1%). Osmotic shock (addition of 400 mM sucrose), Cl(-) removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1(-/-) erythrocytes/reticulocytes than in AE1(+/+) erythrocytes. The increase of annexin binding following exposure to the Ca(2+) ionophore ionomycin (1 muM) was, however, similar in AE1(-/-) and in AE1(+/+) erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca(2+) permeability in AE1(-/-) erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1(-/-) mice than in AE1(+/+) mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca(2+) entry and subsequent scrambling of cell membrane phospholipids.  (+info)

Membrane remodeling during reticulocyte maturation. (6/16)

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Role for parasite genetic diversity in differential host responses to Trypanosoma brucei infection. (7/16)

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Restoration of high-density lipoprotein levels by cholesteryl ester transfer protein expression in scavenger receptor class B type I (SR-BI) knockout mice does not normalize pathologies associated with SR-BI deficiency. (8/16)

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