Vascular mitochondrial respiratory function: the impact of advancing age

Little is known about vascular mitochondrial respiratory function and the impact of age. Therefore, skeletal muscle feed arteries were harvested from young (33 ± 7 yr, n = 10), middle-aged (54 ± 5 yr, n = 10), and old (70 ± 7 yr, n = 10) subjects, and mitochondrial respiration as well as citrate synthase (CS) activity were assessed. Complex I (CI) and complex I + II (CI+II) state 3 respiration were greater in young (CI: 10.4 ± 0.8 pmol·s−1·mg−1 and CI+II: 12.4 ± 0.8 pmol·s−1·mg−1, P \u3c 0.05) than middle-aged (CI: 7 ± 0.6 pmol·s−1·mg−1 and CI+II: 8.3 ± 0.5 pmol·s−1·mg−1) and old (CI: 7.2 ± 0.4 pmol·s−1·mg−1 and CI+II: 7.6 ± 0.5 pmol·s−1·mg−1) subjects and, as in the case of complex II (CII) state 3 respiration, were inversely correlated with age [r = −0.56 (CI), r = −0.7 (CI+II), and r = 0.4 (CII), P \u3c 0.05]. In contrast, state 4 respiration and mitochondria-specific superoxide levels were not different across groups. The respiratory control ratio was greater in young (2.2 ± 0.2, P \u3c 0.05) than middle-aged and old (1.4 ± 0.1 and 1.1 ± 0.1, respectively) subjects and inversely correlated with age (r = −0.71, P \u3c 0.05). As CS activity was inversely correlated with age (r = −0.54, P \u3c 0.05), when normalized for mitochondrial content, the age-related differences and relationships with state 3 respiration were ablated. In contrast, mitochondrion-specific state 4 respiration was now lower in young (15 ± 1.4 pmol·s−1·mg−1·U CS−1, P \u3c 0.05) than middle-aged and old (23.4 ± 3.6 and 27.9 ± 3.4 pmol·s−1·mg−1·U CS−1, respectively) subjects and correlated with age (r = 0.46, P \u3c 0.05). Similarly, superoxide/CS levels were lower in young (0.07 ± 0.01) than old (0.19 ± 0.41) subjects and correlated with age (r = 0.44, P \u3c 0.05). Therefore, with aging, vascular mitochondrial respiratory function declines, predominantly as a consequence of falling mitochondrial content. However, per mitochondrion, aging likely results in greater mitochondrion-derived oxidative stress, which may contribute to age-related vascular dysfunction

Vasodilatory and vascular mitochondrial respiratory function with advancing age: evidence of a free radically mediated link in the human vasculature

Recognizing the age-related decline in skeletal muscle feed artery (SMFA) vasodilatory function, this study examined the link between vasodilatory and mitochondrial respiratory function in the human vasculature. Twenty-four SMFAs were harvested from young (35 ± 6 yr, n = 9) and old (71 ± 9 yr, n = 15) subjects. Vasodilation in SMFAs was assessed, by pressure myography, in response to flow-induced shear stress, acetylcholine (ACh), and sodium nitroprusside (SNP) while mitochondrial respiration was measured, by respirometry, in permeabilized SMFAs. Endothelium-dependent vasodilation was significantly attenuated in the old, induced by both flow (young: 92 ± 3, old: 45 ± 4%) and ACh (young: 92 ± 3, old: 54 ± 5%), with no significant difference in endothelium-independent vasodilation. Complex I and I + II state 3 respiration was significantly lower in the old (CI young: 10.1 ± 0.8, old: 7.0 ± 0.4 pmol·s−1·mg−1; CI + II young: 12.3 ± 0.6, old: 7.6 ± 0.4 pmol·s−1·mg−1). The respiratory control ratio (RCR) was also significantly attenuated in the old (young: 2.2 ± 0.1, old: 1.1 ± 0.1). Furthermore, state 3 (CI + II) and 4 respiration, as well as RCR, were significantly correlated (r = 0.49–0.86) with endothelium-dependent, but not endothelium-independent, function. Finally, the direct intervention with mitochondrial-targeted antioxidant (MitoQ) significantly improved endothelium-dependent vasodilation in the old but not in the young. Thus, the age-related decline in vasodilatory function is linked to attenuated vascular mitochondrial respiratory function, likely by augmented free radicals

A reduced dose of ribavirin does not influence the virologic response during pegylated interferon alpha-2b and ribavirin combination therapy in patients with genotype 1 chronic hepatitis C

Background/AimsWhen combined with pegylated interferon alpha-2b (Peg-IFN α-2b) for the treatment of genotype 1 chronic hepatitis C (CHC) in Korea, the current guideline for the initial ribavirin (RBV) dose is based on body weight. However, since the mean body weight is lower for Korean patients than for patients in Western countries, current guidelines might result in Korean patients being overdosed with RBV.MethodsWe retrospectively reviewed the medical records of patients with genotype 1 CHC who were treated with Peg-IFN α-2b and RBV combination therapy. We divided the patients into groups A (≥15 mg/kg/day, n=23) and B (<15 mg/kg/day, n=26), given that the standard dose is 15 mg/kg/day. The clinical course in terms of the virologic response, adverse events, and dose modification rate was compared between the two groups after therapy completion.ResultsThe early response rates (92.0% vs. 83.3%, P=0.634) and sustained virologic response rates (82.6% vs. 73.1%, P=0.506) did not differ significantly between the two groups. During the treatment period, the RBV dose reduction rate was significantly higher in group A than in group B (60.9% vs. 23.1%, P=0.01).ConclusionsRBV dose reduction is performed frequently when patients are treated according to the current Korean guidelines. Given that lowering the RBV dose did not appear to decrease the virologic response during therapy, reducing RBV doses below the current Korean guideline may be effective for treatment, especially in low-weight patients

Strong Relationship Between Vascular Function in the Coronary and Brachial Arteries: A Clinical Coming of Age for the Updated Flow-Mediated Dilation Test?

Early detection of coronary artery dysfunction is of paramount cardiovascular clinical importance, but a noninvasive assessment is lacking. Indeed, the brachial artery flow-mediated dilation test only weakly correlated with acetylcholine-induced coronary artery function (r=0.36). However, brachial artery flow-mediated dilation methodologies have, over time, substantially improved. This study sought to determine if updates to this technique have improved the relationship with coronary artery function and the noninvasive indication of coronary artery dysfunction. Coronary artery and brachial artery function were assessed in 28 patients referred for cardiac catheterization (61±11 years). Coronary artery function was determined by the change in artery diameter with a 1.82 μg/min intracoronary acetylcholine infusion. Based on the change in vessel diameter, patients were characterized as having dysfunctional coronary arteries (\u3e5% vasoconstriction) or relatively functional coronary arteries (\u3c5% vasoconstriction). Brachial artery function was determined by flow-mediated dilation, adhering to current guidelines. The acetylcholine-induced change in vessel diameter was smaller in patients with dysfunctional compared with relatively functional coronary arteries (−11.8±4.6% versus 5.8±9.8%, P\u3c0.001). Consistent with this, brachial artery flow-mediated dilation was attenuated in patients with dysfunctional compared with relatively functional coronaries (2.9±1.9% versus 6.2±4.2%, P=0.007). Brachial artery flow-mediated dilation was strongly correlated with the acetylcholine-induced change in coronary artery diameter (r=0.77, P\u3c0.0001) and was a strong indicator of coronary artery dysfunction (receiver operator characteristic=78%). The current data support that updates to the brachial artery flow-mediated dilation technique have strengthened the relationship with coronary artery function, which may now provide a clinically meaningful indication of coronary artery dysfunction

Master of Science

thesisIn the first study, skeletal muscle feed arteries (SMFAs) were harvested from young, middle aged, and old subjects, and mitochondrial respiration as well as citrate synthase (CS) activity were assessed. Complex I (CI) and complex I+II (CI+II), state 3 respiration were greater in the young compared to both middle aged and old and, in addition to CII state 3, were inversely correlated with age. In contrast, state 4 respiration and mitochondria-specific superoxide levels were not different across groups. The respiratory control ratio (RCR) was greater in the young compared to the middle aged and old and inversely correlated with age. As CS activity was inversely correlated with age, when normalized for mitochondrial content, the age-related differences and relationships with state 3 were ablated. In contrast, mitochondrion-specific state 4 was now lower in the young than the middle aged and old and correlated with age. Similarly, superoxide/CS levels were lower in the young than the old and correlated with age. Therefore, with aging vascular mitochondrial respiratory function declines, predominantly as a consequence of falling mitochondrial content. However, per mitochondrion, aging likely results in greater mitochondrial-derived oxidative stress, which may contribute to age-related vascular dysfunction. The second study sought to examine the link between vascular and mitochondrial respiratory function in the vasculature with aging. SMFAs were harvested from young and old subjects. Using pressure myography, vasodilation in SMFAs was assessed in iv response to flow-induced shear stress, acetylcholine (ACh), and sodium nitroprusside (SNP) and mitochondrial and respiration were measured, by respirometry, in permeabilized smooth muscle fibers. Endothelium-dependent vasodilation was significantly attenuated in the old, induced by flow or ACh, while endotheliumindependent vasodilation was not altered by age. CI+II, state 3 respiration was significantly lower in the old. Although state 4 respiration and mitochondrial-specific free radical production, assessed by electron paramagnetic resonance spectroscopy, were not different between groups, both tended to be higher in the old. RCR was also significantly attenuated in the old. State 3 and 4 respiration as well as RCR exhibited significant correlations with endothelium dependent but not endothelium-independent capacity. Free radical levels were also significantly correlated with advancing age. Therefore, the agerelated decline in vasodilatory capacity in humans is related to a concomitant attenuation in mitochondrial respiratory capacity and may be a consequence of augmented free radical production

Grid selection strategy for high-resolution cryo-EM

Cryo-electron microscopy (cryo-EM) is a revolutionary technique to study the three-dimensional structure of macromolecules and theirs complexes at molecular resolution. The first step in preparing samples for cryo-EM is to select and optimize the right grid for the specimen. This screening process needs consideration in many aspects including concentration and stability of the specimen, compatibility with grid material and optimum ice thickness across the grid. Importantly, the best signal-to-noise ratio (SNR) for a micrograph is closely related to vitrifying the grid sample with the optimum imaging condition. Here we describe overall strategies for grid selection and optimization by understanding the properties of grids and a variety of techniques for grid treatment for high-resolution electron micrographs. This review also describes the utilization of various specimen supports including amorphous carbon, graphene and functionalized support films.N

Cryo-EM structures of GroEL:ES2 with RuBisCO visualize molecular contacts of encapsulated substrates in a double-cage chaperonin

The GroEL/GroES chaperonin system assists the folding of many proteins, through conformational transitions driven by ATP hydrolysis. Although structural information about bullet-shaped GroEL:ES1 complexes has been extensively reported, the substrate interactions of another functional complex, the foot-ball-shaped GroEL:ES2, remain elusive. Here, we report single-particle cryo-EM structures of reconstituted wild-type GroEL:ES2 complexes with a chemically denatured substrate, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). Our structures demonstrate that native-like folded RuBisCO density is captured at the lower part of the GroEL chamber and that GroEL&apos;s bulky hydrophobic residues Phe281, Tyr360, and Phe44 contribute to direct contact with RuBisCO density. In addition, our analysis found that GroEL:ES2 can be occupied by two substrates simultaneously, one in each chamber. Together, these observations provide insights to the football-shaped GroEL:ES2 complex as a functional state to assist the substrate folding with visualization.Y

Integrated cryoEM imaging center at Seoul National University, Korea

The Center for Macromolecular and Cell Imaging (CMCI) is a core facility equipped with multiple cryo-capable electron microscopes including a 200 kV Thermofisher Glacios, 200 kV JEOL JEM2100Plus and 120 kV FEI TECNAI with direct electron detector technologies. The center also has auxiliary equipment for optimal specimen preparation, such as for protein purification, tissue culture, grid preparation and vitrification. The center offers to the scientific community the best practice of cutting-edge cryogenic electron microscopy (cryoEM) technology for specimen optimization, highresolution data collection and structure determination. The center also develops the strategy of an integrated cryoEM imaging platform to visualize molecular architectures from purified proteins to cells.N