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Background and Purpose Atherosclerosis is associated with reduced vascular hydrogen sulfide (H2S) biosynthesis. GYY4137 is a novel slow‐releasing H2S compound that may effectively mimic the time course of H2S release in vivo. However, it is not known whether GYY4137 affects atherosclerosis. Experimental Approach RAW 264.7 cells and human blood monocyte‐derived macrophages were incubated with oxidized low density lipoprotein (ox‐LDL) with/without GYY4137. ApoE−/− mice were fed a high‐fat diet for 4 weeks and administered GYY4137 for 30 days. Lipid and atherosclerotic lesions were measured by oil red O staining. Endothelium‐dependent relaxation was assessed in response to acetylcholine. Superoxide production was detected by dihydroethidium staining. Expression of mRNA and protein were evaluated by quantitative real‐time PCR and Western blot. Key Results GYY4137 inhibited ox‐LDL‐induced foam cell formation and cholesterol esterification in cultured cells. GYY4137 decreased the expression of lectin‐like ox‐LDL receptor‐1, iNOS, phosphorylated IκBα, NF‐κB, ICAM‐1, VCAM‐1 and chemokines, including CXCL2, CXCR4, CXCL10 and CCL17, but increased the scavenger protein CD36, in ox‐LDL‐treated RAW 264.7 cells. In vivo, GYY4137 decreased aortic atherosclerotic plaque formation and partially restored aortic endothelium‐dependent relaxation in apoE−/− mice. GYY4137 decreased ICAM‐1, TNF‐α and IL‐6 mRNA expression as well as superoxide (O2−) generation in aorta. In addition, GYY4137 increased aortic eNOS phosphorylation and expression of PI3K, enhanced Akt Ser473 phosphorylation and down‐regulated the expression of LOX‐1. Conclusion and Implications GYY4137 inhibits lipid accumulation induced by ox‐LDL in RAW 264.7 cells. In vivo, GYY4137 decreased vascular inflammation and oxidative stress, improved endothelial function and reduced atherosclerotic plaque formation in apoE−/− mice.
The widely used Arrhenius equation describes the kinetics of simple two-state reactions, with the implicit assumption of a single transition state with a well-defined activation energy barrier ΔE, as the rate-limiting step. However, it has become increasingly clear that the saddle point of the free-energy surface in most reactions is populated by ensembles of conformations, leading to nonexponential kinetics. Here we present a theory that generalizes the Arrhenius equation to include static disorder of conformational degrees of freedom as a function of an external perturbation to fully account for a diverse set of transition states. The effect of a perturbation on static disorder is best examined at the singlemolecule level. Here we use force-clamp spectroscopy to study the nonexponential kinetics of single ubiquitin proteins unfolding under force. We find that the measured variance in ΔE shows both force-dependent and independent components, where the forcedependent component scales with F 2 , in excellent agreement with our theory. Our study illustrates a novel adaptation of the classical Arrhenius equation that accounts for the microscopic origins of nonexponential kinetics, which are essential in understanding the rapidly growing body of single-molecule data.single-molecule force-clamp spectroscopy | protein unfolding | ubiquitin | molecular dynamics simulations I n 1889 Svante Arrhenius proposed a simple equation for the temperature dependency of the rate of a chemical reactionwhere A is a preexponential factor, k B is the Boltzmann constant, T is the absolute temperature and ΔE is the height of the activation energy barrier (1). This widely accepted description of a single barrier crossing reaction can be readily expanded to include the effect of perturbations that alter the height of the free-energy barrier. For example, when a mechanical force, F, is applied to a molecule, the free-energy barrier height is reduced by an amount equal to FΔx, where Δx represents the actual distance from the native conformation to the transition state conformation along the reaction coordinate. As pointed out by Bell (2), the corresponding Arrhenius law then becomes kðFÞ ¼ A exp½−ð ΔG−FΔx k B T Þ , where ΔG is the height of the free-energy barrier of reaction in the absence of force. This simple description of the kinetics of a reaction under force has proven useful in a wide variety of single-molecule studies such as bond rupture events (3, 4), protein unfolding (5, 6), and chemical reactions (7,8). In this work we will focus our investigation on proteins unfolding under a stretching force. However, our conclusions can be readily generalized to other reaction schemes. Assuming a negligible refolding rate, the survival probability, SðtÞ, that a protein remains folded for a time t while under a force, F, satisfies the first-order rate equation, dSðtÞ dt ¼ −kðFÞSðtÞ. The resulting survival probability is thus a single exponential. Recent generalizations of the Arrhenius description also lead to a singleexponential survival pr...
We demonstrate a combination of single molecule force spectroscopy and solvent substitution that captures the presence of solvent molecules in the transition state structure. We measure the effect of solvent substitution on the rate of unfolding of the I27 titin module, placed under a constant stretching force. From the force dependency of the unfolding rate, we determine Δ x u , the distance to the transition state. Unfolding the I27 protein in water gives a Δ x u = 2.5 Å, a distance that compares well to the size of a water molecule. Although the height of the activation energy barrier to unfolding is greatly increased in both glycerol and deuterium oxide solutions, Δ x u depends on the size of the solvent molecules. Upon replacement of water by increasing amounts of the larger glycerol molecules, Δ x u increases rapidly and plateaus at its maximum value of 4.4 Å. In contrast, replacement of water by the similarly sized deuterium oxide does not change the value of Δ x u . From these results we estimate that six to eight water molecules form part of the unfolding transition state structure of the I27 protein, and that the presence of just one glycerol molecule in the transition state is enough to lengthen Δ x u . Our results show that solvent composition is important for the mechanical function of proteins. Furthermore, given that solvent composition is actively regulated in vivo , it may represent an important modulatory pathway for the regulation of tissue elasticity and other important functions in cellular mechanics.
Bladder cancer (BC) is one of the most commonly occurring cancers, with a high recurrence rate and poor outcomes in cases of relapsed metastatic disease. Here, we analyzed the markers and significance of myeloid-derived suppressor cells (MDSCs) for BC development and progression. MDSC markers were examined in peripheral blood from 113 BC patients and 20 healthy volunteers. We identified CD11b+CD33lowHLA-DR− CD3− cells as markers of MDSCs in peripheral blood from BC patients. We also demonstrated that MDSC numbers are higher in BC patients than healthy donors, and that MDSC numbers correlate with the clinical grade, stage, and poor prognosis. In addition, serum IL-6 levels are decreased in BC patients with higher MDSC counts. IL-6 blockade increases induction of MDSCs in vitro. Low IL-6 levels inhibit activation of Stat3, resulting in the increased formation of MDSCs in BC. These results indicate that the MDSCs numbers may serve as a novel prognostic marker in BC patients, and that targeting IL-6 signaling may be a promising strategy for BC treatment.
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