Shear stress activation of nitric oxide synthase and increased NO levels in human red blood cells

Since the discovery of nitric oxide (NO) as a vasoactive molecule, red blood cells (RBC) have been considered to participate in NO-mediated control of the circulation. The classical role attributed to RBC was scavenging of NO, thereby impacting the local bioavailability of this important regulator of vascular tone^1^. RBC have been shown to be a source of NO, primarily via its transport bound to haemoglobin^2, 3^. Under specific conditions, haemoglobin plays an active role in converting NO derivatives (e.g., nitrite) to NO^4, 5^, with this NO originating from RBC being an effective modulator of vascular smooth muscle tone^6^. Interestingly, RBC contain a NO synthase (NOS) protein^7^, can actively synthesize NO using L-arginine as a substrate^8^, and can export NO under appropriate conditions^8, 9^. It has been previously hypothesized that RBC NOS may be activated by shear forces acting on the cell^10^, and we have recently shown that RBC NOS phosphorylation can be enhanced by subjecting RBC in suspension to shearing forces^9^: NO concentration in the suspending medium was increased subsequent to flow of RBC suspensions through five [mu]m pores^11^. We have now directly demonstrated increased RBC NOS activity and intracellular NO levels in immobilized RBC exposed to well-defined fluid shear stress. Immunostaining for serine 1177 phosphorylation and the NO-sensitive fluorescent probe diaminofluorescein were employed. Our results suggest that RBC deformation in constricted vessels may increase NO levels and favor vasodilation, thereby providing an important role for RBC in regulating the circulation

Utility investigation of automated techniques in hematopoietic progenitor cell count and viability assessment in the Good Manufacturing Practice (GMP) settingg

Aim: To compare our parameters as regards: i) cell count via two different automated cell count techniques, and ii) viability via automated trypan blue exclusion and 7-aminoactinomycin D (7-AAD) staining. Method: We used the trypan blue exclusion technique and an automated cell counter and for viability testing, and the trypan blue exclusion technique and the 7-AAD evaluation by flow cytometry. The trypan blue exclusion and the radio frequency techniques were used for automated cell counting. Flow cytometric analysis was performed by evaluating the yielded cellular products for 7-AAD uptake during the cell count of CD34+ cells. Results: The mean values for cell count were estimated as 3.44±1.22x106/ml (range, 2.48-5.71x106/ml) and 4.14±1.94x106/ml (range, 1.77-7.43x106/ml) for the trypan blue exclusion and radio frequency techniques, respectively. Additionally, the mean values for viability analyses via the automated trypan blue exclusion and 7-AAD were 93.38±6.09% (range, 79.00-98.00%) and 99.49±0.60% (range, 98.40-100.00%), respectively. Conclusions: Our study has responded to two fundamental questions: whether the results of both of the automated techniques for cell count correspond with each other, and whether the results of the automated viability assessment conform those of the 7-AAD technique during the manufacturing processes of cellular therapy products intended for clinical use. Even though we have the opportunity to use the hemocytometer in our laboratory setting, the automated trypan blue exclusion technique gives cell count results in concordance within the range of the expectations of our Quality Management System (QMS)

Neither a Nitric Oxide Donor Nor Potassium Channel Blockage Inhibit RBC Mechanical Damage Induced by a Roller Pump

Red blood cells (RBC) are exposed to various levels of shear stresses when they are exposed to artificial flow environments, such as extracorporeal flow circuits and hemodialysis equipment. This mechanical trauma affects RBC and the resulting effect is determined by the magnitude of shear forces and exposure time. It has been previously demonstrated that nitric oxide (NO) donors and potassium channel blockers could prevent the sub-hemolytic damage to RBC, when they are exposed to 120 Pa shear stress in a Couette shearing system. This study aimed at testing the effectiveness of NO donor sodium nitroprussid (SNP, 10-4 M) and non-specific potassium channel blocker tetraethylammonium (TEA, 10-7 M) in preventing the mechanical damage to RBC in a simple flow system including a roller pump and a glass capillary of 0.12 cm diameter. RBC suspensions were pumped through the capillary by the roller pump at a flow rate that maintains 200 mmHg hydrostatic pressure at the entrance of the capillary. An aliquot of 10 ml of RBC suspension of 0.4 L/L hematocrit was re-circulated through the capillary for 30 minutes. Plasma hemoglobin concentrations were found to be significantly increased (~7 folds compared to control aliquot which was not pumped through the system) and neither SNP nor TEA prevented this hemolysis. Alternatively, RBC deformability assessed by laser diffraction ektacytometry was not altered after 30 min of pumping and both SNP and TEA had no effect on this parameter. The results of this study indicated that, in contrast with the findings in RBC exposed to a well-defined magnitude of shear stress in a Couette shearing system, the mechanical damage induced by a roller pump could not be prevented by NO donor or potassium channel blocker

Effect of magnesium on vascular reactivity in NOS inhibition-induced hypertension

This study investigated the effect of magnesium on the vascular reactivity of conduit and resistance arteries in a nitric oxide synthase inhibition-induced hypertension model. The aorta and third-order branches of the mesenteric artery were dissected from normotensive control and hypertensive rats, and their constriction and dilation responses in physiological saline solution containing normal (1.2 mM) or high (4.8 mM) magnesium concentrations were examined. The responses of the vessels were evaluated using potassium chloride (KCl) and phenylephrine (Phe), acetylcholine (ACh) and sodium nitroprusside. The Phe-induced constriction response of the aortic rings increased, whereas the ACh-induced dilation response decreased, in the hypertensive group compared to controls, in the presence of a normal magnesium concentration. High magnesium did not alter these responses in either group. Both the KCl- and Phe-induced constriction responses of the mesenteric arteries increased, and the ACh-induced dilation response decreased in the hypertensive group compared to controls, in the presence of a normal magnesium concentration. High magnesium significantly decreased the KCl and Phe-induced constriction and increased the ACh-induced dilation response of the mesenteric arteries in the hypertensive group, while it did not alter these responses in controls. This study suggests that high magnesium improves vascular reactivity of resistance-, but not conduit-type arteries in the nitric oxide synthase inhibition-induced hypertension model

Influence of tourniquet application on venous blood sampling for serum chemistry, hematological parameters, leukocyte activation and erythrocyte mechanical properties

Background: Venous blood sampling is usually performed using a tourniquet to help locate and define peripheral veins to achieve successful and safe venipuncture. Despite widespread usage of tourniquets for venipuncture by medical and laboratory staff, very few are aware of the effects of tourniquet application on laboratory parameters. In addition, definitive guidelines regarding when and how to use a tourniquet for blood sampling are lacking. The aim of the present study was to define the optimal sampling time after tourniquet removal to avoid adverse impact on laboratory analytes. Methods: Blood oxygen and carbon dioxide partial pressure, pH, oxyhemoglobin saturation (satO2), hematological parameters, serum electrolyte concentrations, erythrocyte, deformability and aggregation, leukocyte activation and nitrite/nitrate concentrations obtained 180 s after tourniquet release were compared with baseline values for 10 healthy subjects. Results: Blood gases, hematological parameters and serum electrolyte levels were not affected by the application and removal of a tourniquet. However, there were significant decreases in erythrocyte deformability at 90, 120, 180 s, and increases in erythrocyte aggregation at 5 and 30 s following removal of the tourniquet. A significant increase in granulocyte respiratory burst at 60 s was observed, confirming leukocyte activation due to application of the tourniquet. There were no significant alterations of blood nitrite/nitrate levels. Conclusions: Our blood sampling technique which mimicked the application and release of a tourniquet indicated unaltered values for routine blood gases, hematological testing and serum electrolyte levels. Conversely, hemorheological measurements can be affected. Therefore, it is strongly recommended that tourniquet application should be avoided during blood sampling or, if this is not possible, the procedure should be well standardized and details of the sampling method should be reported. Clin Chem Lab Med 2009;47:769–76.Peer Reviewe

A Cyanide-Based Coordination Polymer for Hydrogen Evolution Electrocatalysis

Karadas, Ferdi/0000-0001-7171-9889; Alsac, Elif Pinar/0000-0002-4255-9676WOS: 000425116300004Research on H-2 production has recently been directed to the development of cost-efficient and robust heterogeneous catalysts for hydrogen evolution reaction (HER). Given the promising catalytic activities of several cobalt-based systems and the robustness of Prussian blue analogues in harsh catalytic processes including water oxidation, a Co-Co Prussian blue analogue was investigated as a HER catalyst for the first time. Co-Co Prussian Blue modified fluorine doped tin oxide (FTO) electrode demonstrated a significant HER activity with an onset overpotential of 257 mV, a Tafel slope of 80 mV dec(-1), and a turnover frequency of 0.090 s(-1) at an overpotential of 250 mV. Comparative XPS, Infrared, and XRD studies performed on pristine and post-catalytic electrodes confirm the stability of the catalyst. Graphical Abstract [GRAPHICS] .Science and Technology Council of Turkey, TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [215Z249]; TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [1929B011500059]This work was suppoerted by the Grants from the Science and Technology Council of Turkey, TUBITAK (Project No: 215Z249). E. U. thanks TUBITAK for support (Project No: 1929B011500059)

Netherton syndrome: report of identical twins presenting with severe atopic dermatitis

We report the cases of 4-year-old identical twin sisters who presented with severe atopic dermatitis with intractable skin manifestations and multiple food allergies. Netherton syndrome (NS) (OMIM 256500) was suspected due to very high serum IgE levels, growth retardation, severe food allergies and typical hair finding (trichorrhexis invaginata). A definite diagnosis was made by genetic analysis. Our cases are unique in being the first identical twins with NS diagnosed by a novel mutation in the SPINK5 gene. NS should be considered in differential diagnosis in children who have generalized erythema with intractable eczematous lesions and elevated levels of IgE

Effect of magnesium supplementation on blood pressure and vascular reactivity in nitric oxide synthase inhibition-induced hypertension model

The aim of this study was to assess the effect of oral magnesium supplementation (Mg-supp) on blood pressure (BP) and possible mechanism in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension and/or Mg-supp were created by N-nitro-l-arginine methyl ester (25mg/kg/day by drinking water) and magnesium-oxide (0.8% by diet) for 6 weeks. Systolic BP was measured weekly by tail-cuff method. The effects of hypertension and/or Mg-supp in thoracic aorta and third branch of mesenteric artery constriction and relaxation responses were evaluated. NOS-inhibition produced a gradually developing hypertension and the magnitude of the BP was significantly attenuated after five weeks of Mg-supp. The increased phenylephrine-induced contractile and decreased acetylcholine (ACh)-induced dilation responses were found in both artery segments of hypertensive groups. Mg-supp was restored ACh-relaxation response in both arterial segments and also Phe-constriction response in thoracic aorta but not in mesenteric arteries. The contributions of NO, prostaglandins and K+ channels to the dilator response of ACh were similar in the aorta of all the groups. The contribution of the NO to the ACh-mediated relaxation response of mesenteric arteries was suppressed in hypertensive rats, whereas this was corrected by Mg-supp. The flow-mediated dilation response of mesenteric arteries in hypertensive rats failed and could not be corrected by Mg-supp. Whereas, vascular eNOS protein and magnesium levels were not changed and plasma nitrite levels were reduced in hypertensive rats. The results of this study showed that Mg-supp lowered the arterial BP in NOS-inhibition induced hypertension model by restoring the agonist-induced relaxation response of the arteries