Astaxanthin (Asx) is known to be a potent quencher of singlet oxygen and an efficient scavenger of superoxide anion. Therefore, Asx would be expected to be a useful antioxidant for the prevention of oxidative stress, a causative factor in severe diseases such as ischemic reperfusion injury. However, it is still unclear whether Asx has scavenging capability against the most potent reactive oxygen species (ROS), hydroxyl radical, because the hydrophobicity of Asx prevents analysis of hydroxyl radical scavenging ability in aqueous solution. In this study, to solve this problem, liposomes containing Asx (Asx-lipo), which could be dispersed in water, were prepared, and the scavenging ability of Asx-lipo for the hydroxyl radical was examined. The liposomal formulation enabled encapsulation of a high concentration of Asx. Asx-lipo gave a dose-dependent reduction of chemiluminescence intensity induced by hydroxyl radical in aqueous solution. Hydroxyl radical scavenging of Asx was more potent than α-tocopherol. The absorbance of Asx in the liposome decreased after reduction of hydroxyl radicals, indicating the direct hydroxyl radical scavenging by Asx. Moreover, Asx-lipo prevented hydroxyl radical-induced cytotoxicity in cultured NIH-3T3 cells. In conclusion, Asx has potent scavenging capability against hydroxyl radicals in aqueous solution, and this paper is the first report regarding hydroxyl radial scavenging by Asx.
The N-terminal (1-83) fragment of the major constituent of plasma high-density lipoprotein, apolipoprotein A-I (apoA-I), strongly tends to form amyloid fibrils, leading to systemic amyloidosis. Here, using a series of deletion variants, we examined the roles of two major amyloidogenic segments (residues 14-22 and 50-58) in the aggregation and fibril formation of an amyloidogenic G26R variant of the apoA-I 1-83 fragment (apoA-I 1-83/G26R). Thioflavin T fluorescence assays and atomic force microscopy revealed that elimination of residues 14-22 completely inhibits fibril formation of apoA-I 1-83/G26R, whereas ⌬32-40 and ⌬50-58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to -sheet structures in all deletion variants except for the ⌬14-22 variant, indicating that residues 14-22 are critical for the -transition and fibril formation. Thermodynamic analysis of the kinetics of fibril formation by apoA-I 1-83/G26R indicated that both nucleation and fibril growth are enthalpically unfavorable, whereas entropically, nucleation is favorable, but fibril growth is unfavorable. Interestingly, the nucleation of the ⌬50-58 variant was entropically unfavorable, indicating that residues 50-58 entropically promote the nucleation step in fibril formation of apoA-I 1-83/G26R. Moreover, a residue-level structural investigation of apoA-I 1-83/G26R fibrils with site-specific pyrene labeling indicated that the two amyloidogenic segments are in close proximity to form an amyloid core structure, whereas the N-and C-terminal tail regions are excluded from the amyloid core. These results provide critical insights into the aggregation mechanism and fibril structure of the amyloidogenic N-terminal fragment of apoA-I. Apolipoprotein (apoA-I) 3 is the major structural and functional constituent of plasma high-density lipoprotein (HDL) This work was partly supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant JP17H03979 (to H. S.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S9.
The type III secretion apparatus (T3SA) participates in the secretion of bacterial proteins called effectors, although the detailed mechanism of effector secretion remains unclear. T3SA and flagellum were shown to branch from a common ancestor and also show structural similarity. In addition, both T3SA-dependent effector secretion and flagellar rotation were reported to require proton-motive force (PMF) for activity. From these reports, we hypothesized that T3SA, like the flagellum, would rotate via PMF and that this rotation is responsible for effector secretion. To observe T3SA rotation, we constructed a novel observation system by modifying the tip of T3SA on bacterial cell membranes with an observation probe, which allowed documentation of T3SA rotation for the first time. T3SA rotation was stopped by the addition of a protonophore that decreases PMF. Moreover, increased viscosity of the observation medium inhibited both rotation of T3SA associated with beads and effector secretion. These results suggested that effector secretion would follow the PMF-dependent rotation of T3SA and could be inhibited by preventing T3SA rotation. Moreover, the motion-track analysis of bead rotation suggested that the T3SA needle might be flexible. Consequently, we propose a "rotational secretion model" as a novel effector secretion mechanism of T3SA.
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