Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo-and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.

The development of hemolytic alloantibodies and erythrocyte autoantibodies complicates transfusion therapy in thalassemia patients. The frequency, causes, and prevention of this phenomena among 64 transfused thalassemia patients (75% Asian) were evaluated. The effect of red blood cell (RBC) phenotypic differences between donors (mostly white) and Asian recipients on the frequency of alloimmunization was determined. Additional transfusion and patient immune factors were examined. 14 (22%) of 64 patients (75% Asian) became alloimmunized. A mismatched RBC phenotype between the white population, comprising the majority of the donor pool, and that of the Asian recipients, was found for K, c, S, and Fyb antigens, which accounts for 38% of the alloantibodies among Asian patients. Patients who had a splenectomy had a higher rate of alloimmunization than patients who did not have a splenectomy (36% vs 12.8%; P = .06). Erythrocyte autoantibodies, as determined by a positive Coombs test, developed in 25% or 16 of the 64 patients, thereby causing severe hemolytic anemia in 3 of 16 patients. Of these 16, 11 antibodies were typed immunoglobulin G [IgG], and 5 were typed IgM. Autoimmunization was associated with alloimmunization and with the absence of spleen (44% and 56%, respectively). Transfused RBCs had abnormal deformability profiles, more prominent in the patients without a spleen, which possibly stimulated antibody production. Transfusion of phenotypically matched blood for the Rh and Kell (leukodepleted in 92%) systems compared to blood phenotypically matched for the standard ABO-D system (leukodepleted in 60%) proved to be effective in preventing alloimmunization (2.8% vs 33%; P = .0005). Alloimmunization and autoimmunization are common, serious complications in Asian thalassemia patients, who are affected by donor-recipient RBC antigen mismatch and immunological factors.

Recent studies suggest that a nitric oxide (NO) deficiency and elevated arginase activity may play a role in the pathogenesis of asthma. Although much attention has been directed toward measurements of exhaled NO in asthma, no studies to date have evaluated levels of plasma arginase or arginine, the substrate for NO production, in patients with asthma. This study, therefore, measured amino acid levels, arginase activity, and nitric oxide metabolites in the blood of patients with asthma, as well as NO in exhaled breath. Although levels of virtually all amino acids were reduced, patients with asthma exhibited a striking reduction in plasma arginine levels compared with normal control subjects without asthma (45 +/- 22 vs. 94 +/- 29 microM, p < 0.0001), and serum arginase activity was elevated (1.6 +/- 0.8 vs. 0.5 +/- 0.3 micromol/ml/hour, asthma vs. control, p < 0.0001). High arginase activity in patients with asthma may contribute to low circulating arginine levels, thereby limiting arginine bioavailability and creating a NO deficiency that induces hyperreactive airways. Addressing the alterations in arginine metabolism may result in new strategies for treatment of asthma.

Artemisinin is an important new antimalarial agent containing a bridged endoperoxide. The in vitro antimalarial activity of an artemisinin derivative, arteether, is antagonized by two iron chelators, pyridoxal benzoylhydrazone and 1,2-dimethyl-3-hydroxypyrid-4-one. Similarly, the acute toxicity of artemisinin in mice is antagonized by another chelator, deferoxamine-hydroxyethylstarch. A combination of artemisinin and hemin oxidizes erythrocyte membrane thiols in vitro, and this oxidation is also inhibited by an iron chelator. Thus, iron plays a role in the mechanisms of action and toxicity of artemisinin. The combination of artemisinin and hemin also decreases erythrocyte deformability. Iron probably catalyzes the generation of free radicals from artemisinin since ao-tocopherol antagonizes the thiol-oxidizing activity of artemisinin and since a spin-trapped free radical signal can be seen by electron paramagnetic resonance only when artemisinin is incubated in the presence of iron.Malaria remains an important global health problem, affecting an estimated 270 million people per year and killing 1 to 2 million people per year (47). Resistance to currently used antimalarial drugs such as chloroquine, quinine, mefloquine, and Fansidar is spreading rapidly (47). Thus, there is a great need for new antimalarial agents.The antimalarial agent artemisinin (qinghaosu) was isolated in 1972 from Artemisia annua, an ancient Chinese herbal remedy for fever (for a review, see reference 26). Several artemisinin derivatives are currently undergoing phase I and II clinical studies, including artemether, arteether, and artesunate, which are the methyl ether, ethyl ether, and succinate esters, respectively, of dihydroartemisinin. Artemisinin and its derivatives have been widely used for the therapy of malaria in China, Vietnam, Thailand, and Myanmar; over 2 million doses of artemether have been administered in China alone. Artemisinin-type antimalarial agents are particularly useful against chloroquine-resistant Plasmodium falciparum strains and cerebral malaria (47).

These data suggest that there may be a relationship between the L-arginine-nitric oxide pathway and vaso-occlusion in SCD. Low arginine levels during VOC could reflect a state of acute substrate depletion that results in a decrease in nitric oxide production.

The phospholipids of the human red cell are distributed asymmetrically in the bilayer of the red cell membrane. In certain pathologic states, such as sickle cell anemia, phospholipid asymmetry is altered. Although several methods can be used to measure phospholipid organization, small organizational changes have been very difficult to assess. Moreover, these methods fail to identify subpopulations of cells that have lost their normal phospholipid asymmetry. Using fluorescently labeled annexin V in flow cytometry and fluorescent microscopy, we were able to identify and quantify red cells that had lost their phospholipid asymmetry in populations as small as 1 million cells. Moreover, loss of phospholipid organization in subpopulations as small as 0.1% of the total population could be identified, and individual cells could be studied by fluorescent microscopy. An excellent correlation was found between fluorescence-activated cell sorter (FACS) analysis results using annexin V to detect red cells with phosphatidylserine (PS) on their surface and a PS-requiring prothrombinase assay using similar red cells. Cells that bound fluorescein isothiocyanate (FITC)-labeled annexin V could be isolated from the population using magnetic beads covered with an anti-FITC antibody. Evaluation of blood samples from patients with sickle cell anemia under oxygenated conditions demonstrated the presence of subpopulations of cells that had lost phospholipid asymmetry. While only a few red cells were labeled in normal control samples (0.21% +/- 0.12%, n = 8), significantly increased (P < .001) annexin V labeling was observed in samples from patients with sickle cell anemia (2.18% +/- 1.21%, n = 13). We conclude that loss of phospholipid asymmetry may occur in small subpopulations of red cells and that fluorescently labeled annexin V can be used to quantify and isolate these cells.

The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.(Blood. 2010;116(14): 2590-2599) IntroductionThe membrane skeleton is composed of a highly cross-linked network of spectrin, actin filaments, and accessory proteins that underlies the plasma membrane of differentiated cells. This network creates membrane domains by anchoring and restricting the long-range distribution of membrane proteins and plays an important role in determining cell shapes, membrane contours, and mechanical properties. 1,2 The organization of the membrane skeleton is best known in red blood cells (RBCs), where it is organized as a quasi-hexagonal network with connecting strands formed by long, flexible spectrin molecules and vertices formed by short actin filaments. 3-5 Each short actin filament forms the core of a junctional complex with 2 rod-shaped tropomyosin (TM) molecules along the filament, 2 tropomodulin 1 (Tmod1) molecules capping the pointed filament end and an ␣/␤-adducin heterodimer capping the barbed filament end. 6,7 Dematin (protein 4.9) is also associated with the short actin filaments as are ␤1-spectrin protein 4.1R complexes that extend from the sides of the filaments to form the extended spectrin-actin network. The network is connected to membrane macromolecular complexes by multiple linkages: from ␤-spectrin by ankryin to band 3, and from the junctional complex by protein 4.1, ␣/␤-adducin and dematin to band 3, glycophorin C, the glucose transporter, and other components. 8 Disruptions either of attachments of the spectrin-actin lattice to membrane macromolecular complexes ("vertical" connections), or linkages within the plane of the spectrin-actin lattice ("horizontal" connections) lead to ...

Erythrocyte glutathione depletion has been linked to hemolysis and oxidative stress. Glutamine plays an additional antioxidant role through preservation of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels, required for glutathione recycling. Decreased nitric oxide (NO) bioavailability, which occurs in the setting of increased hemolysis and oxidative stress, contributes to the pathogenesis of pulmonary hypertension (PH) in sickle cell disease (SCD). We hypothesized that altered glutathione and glutamine metabolism play a role in this process. Total glutathione (and its precursors) and glutamine were assayed in plasma and erythrocytes of 40 SCD patients and 9 healthy volunteers. Erythrocyte total glutathione and glutamine levels were significantly lower in SCD patients than in healthy volunteers. Glutamine depletion was independently associated with PH, defined as a tricuspid regurgitant jet velocity (TRV) of at least 2.5 m/s. The ratio of erythrocyte glutamine:glutamate correlated inversely to TRV (r ‫؍‬ ؊0.62, P < .001), plasma arginase concentration (r ‫؍‬ ؊0.45, P ‫؍‬ .002), and plasma-free hemoglobin level (r ‫؍‬ ؊0.41, P ‫؍‬ .01), linking erythrocyte glutamine depletion to dysregulation of the arginine-NO pathway and increased hemolytic rate. Decreased erythrocyte glutathione and glutamine levels contribute to alterations in the erythrocyte redox environment, which may compromise erythrocyte integrity, contribute to hemolysis, and play a role in the pathogenesis of PH of SCD. IntroductionThe erythrocyte redox environment may contribute to the increased oxidative stress, hemolysis, and decreased nitric oxide (NO) bioavailability observed in pulmonary hypertension (PH), a common complication of hemolytic disorders. Reduced glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is the most abundant low-molecular weight thiol 1 and the principal thiol redox buffer in erythrocytes. 2,3 The red blood cell contributes up to 10% of whole-body GSH synthesis in humans. [4][5][6] In addition to its role as a critical antioxidant, GSH possesses diverse biological functions involved in detoxification, cell proliferation and apoptosis, redox signaling, gene expression, protein glutathionylation, cytokine production, the immune response, mitochondrial function, and integrity as well as NO metabolism. 7,8 Glutathione is synthesized from glutamate, cysteine, and glycine via reactions catalyzed by 2 cytosolic enzymes, gammaglutamylcysteine ligase and GSH synthetase. The intracellular GSH concentration is the final result of a dynamic balance between the rate of GSH synthesis and the combined rate of intracellular GSH consumption and efflux. GSH is readily oxidized to glutathione disulfide (GSSG) by free radicals and reactive oxygen and nitrogen species. GSSG efflux from cells contributes to a net loss of intracellular GSH. 1 Due to its high intracellular concentrations, GSH variations in oxidation states can significantly modify the redox environment of red blood cells. Within the erythrocyte, GSH may...