Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions, increasing rigidity, fragility, calcium influx-mediated dehydration, and adhesivity of red blood cells. Increased red cell fragility results in hemolysis, which reduces nitric oxide (NO) bioavailability, and induces platelet activation and inflammation leading to adhesion of circulating blood cells. Nitric Oxide inhibits adhesion and platelet activation. Nitrite has emerged as an attractive therapeutic agent that targets delivery of NO activity to areas of hypoxia through bioactivation by deoxygenated red blood cell hemoglobin. In this study, we demonstrate anti-platelet activity of nitrite at doses achievable through dietary interventions with comparison to similar doses with other NO donating agents. Unlike other NO donating agents, nitrite activity is shown to be potentiated in the presence of red blood cells in hypoxic conditions. We also show that nitrite reduces calcium associated loss of phospholipid asymmetry that is associated with increased red cell adhesion, and that red cell deformability is also improved. We show that nitrite inhibits red cell adhesion in a microfluidic flow-channel assay after endothelial cell activation. In further investigations, we show that leukocyte and platelet adhesion is blunted in nitrite-fed wild type mice compared to control after either lipopolysaccharide- or hemolysis-induced inflammation. Moreover, we demonstrate that nitrite treatment results in a reduction in adhesion of circulating blood cells and reduced red blood cell hemolysis in humanized transgenic sickle cell mice subjected to local hypoxia. These data suggest that nitrite is an effective anti-platelet and anti-adhesion agent that is activated by red blood cells, with enhanced potency under physiological hypoxia and in venous blood that may be useful therapeutically.
DNA methylation, which requires the universal methyl donor Sadenosyl-L-methionine (SAM), plays a pivotal role in eukaryotic gene regulation and when dysregulated, can result in severe alterations in cellular function. An emerging approach to further understand DNA methylation utilizes azide-and alkynefunctionalized N-mustard SAM analogues as biochemical tools to probe sites of DNA methylation. While the successful utility of these substituted analogues has been demonstrated with prokaryotic DNA methyltransferases, their utility with physio-logically-relevant eukaryotic DNA methyltransferase 1 (DNMT1) is examined for the first time here. A fluorescence-based magnetic bead assay was validated in initial experiments to measure the extent of DNA modification by the N-mustard analogues using Spiroplasma methylase, M.SssI, a prokaryotic model of DNMT1. Subsequent analysis with DNMT1 revealed limited utility of the analogues, as added azide-and alkynefunctionality appears to directly impact binding to DNMT1.[a] Dr.
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