13 research outputs found
A dysfunctional Cl inhibitor protein with a new reactive center mutation (Arg-444âLeu)
AbstractA Pl mutation (Arg-444âLeu) was identified in a dysfunctional Cl inhibitor from a patient with type 2 hereditary angioneurotic edema. The mutation was defined at the level of the protein (by sequence analysis of the Pseudomonas aeruginosa elastase-derived reactive center peptide), and the mRNA (CGCâCTC) (by sequence analysis of PCR-amplified DNA)
A pilot study on the kinetics of metabolites and microvascular cutaneous effects of nitric oxide inhalation in healthy volunteers
RATIONALE: Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked.
OBJECTIVES: We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response.
FINDINGS: We enrolled 15 healthy volunteers. Plasma nitrite levels at baseline and during NO inhalation (15 minutes at 40 ppm) were 102 (86-118) and 114 (87-129) nM, respectively. The nitrite peak occurred at 5 minutes of discontinuing NO (131 (104-170) nM). Plasma nitrate levels were not significantly different during the study. SNO-Hb molar ratio levels at baseline and during NO inhalation were 4.7E-3 (2.5E-3-5.8E-3) and 7.8E-3 (4.1E-3-13.0E-3), respectively. Levels of SNO-Hb continued to climb up to the last study time point (30 min: 10.6E-3 (5.3E-3-15.5E-3)). The response to acetylcholine iontophoresis both before and during NO inhalation was inversely associated with the SNO-Hb level (r: -0.57, p = 0.03, and r: -0.54, p = 0.04, respectively).
CONCLUSIONS: Both nitrite and SNO-Hb increase during NO inhalation. Nitrite increases first, followed by a more sustained increase in Hb-SNO. Nitrite and Hb-SNO could be a mobile reservoir of NO with potential implications on the systemic microvasculature
Function and Distribution of Apolipoprotein A1 in The Artery Wall Are Markedly Distinct From Those in Plasma
BackgroundâPrior studies show that apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high-density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux, and lecithin-cholesterol acyltransferase activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall. Methods and ResultsâA monoclonal antibody (10G1.5) was developed that equally recognizes lipid-free and HDL-associated apoA1 in both native and oxidized forms. Examination of homogenates of atherosclerotic plaqueâladen aorta showed \u3e100-fold enrichment of apoA1 compared with normal aorta (P\u3c0.001). Surprisingly, buoyant density fractionation revealed that only a minority (\u3c3% of total) of apoA1 recovered from either lesions or normal aorta resides within an HDL-like particle (1.063â€dâ€1.21). In contrast, the majority (\u3e90%) of apoA1 within aortic tissue (normal and lesions) was recovered within the lipoprotein-depleted fraction (d\u3e1.21). Moreover, both lesion and normal artery wall apoA1 are highly cross-linked (50% to 70% of total), and functional characterization of apoA1 quantitatively recovered from aorta with the use of monoclonal antibody 10G1.5 showed â80% lower cholesterol efflux activity and â90% lower lecithin-cholesterol acyltransferase activity relative to circulating apoA1. ConclusionsâThe function and distribution of apoA1 in human aorta are quite distinct from those found in plasma. The lipoprotein is markedly enriched within atherosclerotic plaque, predominantly lipid-poor, not associated with HDL, extensively oxidatively cross-linked, and functionally impaired
Function and Distribution of Apolipoprotein A1 in The Artery Wall Are Markedly Distinct From Those in Plasma
BackgroundâPrior studies show that apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high-density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux, and lecithin-cholesterol acyltransferase activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall. Methods and ResultsâA monoclonal antibody (10G1.5) was developed that equally recognizes lipid-free and HDL-associated apoA1 in both native and oxidized forms. Examination of homogenates of atherosclerotic plaqueâladen aorta showed \u3e100-fold enrichment of apoA1 compared with normal aorta (P\u3c0.001). Surprisingly, buoyant density fractionation revealed that only a minority (\u3c3% of total) of apoA1 recovered from either lesions or normal aorta resides within an HDL-like particle (1.063â€dâ€1.21). In contrast, the majority (\u3e90%) of apoA1 within aortic tissue (normal and lesions) was recovered within the lipoprotein-depleted fraction (d\u3e1.21). Moreover, both lesion and normal artery wall apoA1 are highly cross-linked (50% to 70% of total), and functional characterization of apoA1 quantitatively recovered from aorta with the use of monoclonal antibody 10G1.5 showed â80% lower cholesterol efflux activity and â90% lower lecithin-cholesterol acyltransferase activity relative to circulating apoA1. ConclusionsâThe function and distribution of apoA1 in human aorta are quite distinct from those found in plasma. The lipoprotein is markedly enriched within atherosclerotic plaque, predominantly lipid-poor, not associated with HDL, extensively oxidatively cross-linked, and functionally impaired
The FAD-Shielding Residue Phe1395 Regulates Neuronal Nitric-Oxide Synthase Catalysis by Controlling NADP+ Affinity and a Conformational Equilibrium Within the Flavoprotein Domain
Phe1395 stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe1395 was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP+ binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP+ with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP+ dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP +-bound state as compared with wild type. We conclude that Phe 1395 regulates nNOSr catalysis in two ways. It facilitates NADP + release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer
A dysfunctional Cl inhibitor protein with a new reactive center mutation (Arg-444âLeu)
Abnormal platelet aggregation in idiopathic pulmonary arterial hypertension: role of nitric oxide
Aytekin M, Aulak KS, Haserodt S, Chakravarti R, Cody J, Minai OA, Dweik RA. Abnormal platelet aggregation in idiopathic pulmonary arterial hypertension: role of nitric oxide. Am J Physiol Lung Cell Mol Physiol 302: L512-L520, 2012. First published January 13, 2012; doi:10.1152/ajplung.00289.2011.-Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease. Several processes are believed to lead to the fatal progressive pulmonary arterial narrowing seen in IPAH including vasoconstriction, cellular proliferation inflammation, vascular remodeling, abnormalities in the lung matrix, and in situ thrombosis. Nitric oxide (NO) produced by NO synthases (NOS) is a potent vasodilator and plays important roles in many other processes including platelet function. Reduced NO levels in patients with IPAH are known to contribute to the development of pulmonary hypertension and its complications. Platelet defects have been implied in IPAH, but original research supporting this hypothesis has been limited. Normal platelets are known to have NOS activity, but little is known about NOS expression and NO production by platelets in patients with IPAH. Here we characterized the phenotype of the platelets in IPAH and show a defect in their ability to be activated in vitro by thrombin receptor activating protein but not adenosine diphosphate. We also show that endothelial NOS (eNOS) levels in these platelets are reduced and demonstrate that NO is an important regulator of platelet function. Thus reduced levels of eNOS in platelets could impact their ability to regulate their own function appropriately