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It is important to determine a critical micelle concentration (CMC) of a surfactant in a protein formulation for stabilizing the protein at maximum by preventing it from interfacial denaturation. There are several techniques for CMC determination. Among them, surface tensiometry is the most common approach because this has a long history and much data at many research fields. However, large amount of sample solution is usually required for the measurement (e.g., more than 1 mL is necessary when a standard reservoir like a glass petri dish is used). This is one of the hurdles for protein formulators because only a small amount of protein could be used at the early-stage development. In this research, we tried to minimize the required amount of sample solution for surface tension measurement by developing appropriate probe and reservoir using a three-dimensional printer (3D printer). The advantages and capabilities of 3D printer are (1) to control the shape and size of the printed material precisely, (2) to change the figure freely, and (3) to prepare the prototype quickly. After the experiments and thereby the refinement of probe as well as reservoir, we found that CMCs of polysorbate 20, polysorbate 80, and poloxamer 188 in water and protein formulations could be precisely detected using a probe 0.5 mm in diameter and small reservoir with a pocket of 7.5 mm in diameter/0.25 mm in depth which were made by a 3D printer. Furthermore, the required sample solution per each measurement could be reduced to 80 µL, which means more than 90% reduction against a standard reservoir.

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Highly purified and organic solvent-free fullerene-C60 was dissolved, at nearly saturated concentration of 278 ppm, in squalane prepared from olive oil, which is designated as LipoFullerene (LF-SQ) and was examined for usage as a cosmetic ingredient with antioxidant ability. The aim of this study was to assess the anti-wrinkle formation efficacy of LF-SQ in subjects. A total of 23 Japanese women (group I: age 38.9 +/- 3.8, n = 11, group II; age 39.4 +/- 4.3, n = 12) were enrolled in an 8-week trial of LF-SQ blended cream in a randomized, matched pair double-blind study. The LF-SQ cream was applied twice daily on the right or left half of the face, and squalane blended cream (without fullerene-C60) was applied as the placebo on another half of the face. As clinical evaluations of wrinkle grades, visual observation and photographs, and silicone replicas of both crow's feet areas were taken at baseline (0 week) and at 4th and 8th weeks. Skin replicas were analyzed using an optical profilometry technique. The wrinkle and skin-surface roughness features were calculated and statistically analyzed. Subsequently, trans-epidermal water loss (TEWL), moisture levels of the stratum corneum, and visco-elasticity (suppleness: RO and elasticity: R7) were measured on cheeks by instrumental analysis. LF-SQ cream enhanced the skin moisture and the anti-wrinkle formation. LF-SQ cream that was applied on a face twice daily was not effective at 4th week, but significantly more effective than the placebo at 8th week (p < 0.05) without severe side effects. The roughness-area ratio showed significant improvement (p < 0.05) at 8th week with LF-SQ cream as compared to 0 week with LF-SQ cream, but no significant difference was detected between LF-SQ cream and the placebo. We suggest that LF-SQ could be used as an active ingredient for wrinkle-care cosmetics.

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Along with differentiation of mouse stromal preadipocytes OP9 into adipocytes, intracellular ROS, especially superoxide anion radicals detected by NBT reduction assay, were found to appreciably increase, mainly in cytoplasmic area, parallelling with increases in intracellular lipid-droplet accumulation, whereas undifferentiated OP9 cells kept lower levels of ROS and lipid-droplets. beta-Carotene bleaching assay showed that super-highly hydroxylated fullerene (SHH-F; C(60) (OH)(44)) exerted higher antioxidant ability than highly hydroxylated fullerene (HH-F; C(60) (OH)(32-34)) or lowly hydroxylated fullerene (LH-F; C(60) (OH)(6-12)). Differentiation-dependent lipid-droplet accumulation was suppressed by SHH-F or HH-F more efficiently than LH-F. Furthermore, SHH-F significantly repressed intracellular ROS generation accompanied by adipocyte differentiation. Thus, lipid-droplet accumulation was shown to positively correlate with ROS upon the differentiation of OP9 preadipocytes into adipocytes and SHH-F significantly suppressed intracellular ROS together with repression of intracellular lipid accumulation.

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Incorporation of non-natural amino acids into proteins in response to amber or four-base codons is a useful technology for protein research. In the case of the amber codon, however, release factor 1 can competitively decode the same codon, and consequently inhibit the incorporation of non-natural amino acids. To improve amber codon-mediated incorporation, we carried out a comprehensive screening of amber suppressor tRNAs derived from all tRNAs encoded in the genomes of Escherichia coli K12 and Mycoplasma capricolum. The amber suppressor tRNAs were synthesized from synthetic genes, aminoacylated with a fluorescent non-natural amino acid, and added to an E. coli cell-free translation system. Fluorescent SDS-PAGE analysis indicated that Trp tRNAs showed high suppressor activity in both organisms. Further mutagenesis and screening revealed that M. capricolum Trp(1) tRNA with G1C72A73 mutation is the most suitable for efficient and specific incorporation of non-natural amino acids into proteins in response to the amber codon.

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An amber suppression method has been used for incorporation of nonnatural amino acids into proteins. However, the incorporation efficiency of nonnatural amino acids through an amber codon has been low, which restricts the application of the proteins containing nonnatural amino acids. In this study, we screened a wide variety of amber suppressor tRNAs to discover tRNAs capable to incorporate nonnatural amino acids with high efficiency. To this purpose, synthetic amber suppressor tRNAs of E. coli and Mycoplasma capricolum were screened for the incorporation of a fluorescently labeled nonnatural amino acid in an E. coli cell-free translation system. tRNAs that showed high capability for the incorporation were then mutated not to be aminoacylated by any of endogenous aminoacyl-tRNA synthetases of E. coli and to enhance the incorporation capability. As a result of these investigations, we successfully obtained several amber suppressor tRNAs with high ability for the incorporation of nonnatural amino acids.

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Position-specific incorporation of non-natural amino acids into proteins is a useful technique in protein engineering. In this study, we established a novel selection system to obtain tRNAs that show high decoding activity, from a tRNA library in a cell-free translation system to improve the efficiency of incorporation of non-natural amino acids into proteins. In this system, a puromycin-tRNA conjugate, in which the 3'-terminal A unit was replaced by puromycin, was used. The puromycin-tRNA conjugate was fused to a C-terminus of streptavidin through the puromycin moiety in the ribosome. The streptavidin-puromycin-tRNA fusion molecule was collected and brought to the next round after amplification of the tRNA sequence. We applied this system to select efficient frameshift suppressor tRNAs from a tRNA library with a randomly mutated anticodon loop derived from yeast tRNA CCCG Phe. After three rounds of the selection, we obtained novel frameshift suppressor tRNAs which had high decoding activity and good orthogonality against endogenous aminoacyl-tRNA synthetases. These results demonstrate that the in vitro selection system developed here is useful to obtain highly active tRNAs for the incorporation of non-natural amino acid from a tRNA library.

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Various four-base codons have been shown to work for the introduction of non-natural amino acids into proteins in an Escherichia coli cell-free translation system. Here, a four-base codon-mediated non-natural mutagenesis was applied to a eukaryotic rabbit reticulocyte cell-free translation system. Mutated streptavidin mRNAs containing four-base codons were prepared and added to a rabbit reticulocyte lysate in the presence of tRNAs that were aminoacylated with a non-natural amino acid and had the corresponding four-base anticodons. A Western blot analysis of translation products indicated that the four-base codons CGGU, CGCU, CCCU, CUCU, CUAU, and GGGU were efficiently decoded by the aminoacyl-tRNAs having the corresponding four-base anticodons. In contrast, the four-base codons AGGU, AGAU, CGAU, UUGU, UCGU, and ACGU were not decoded. The stop codon-derived four-base codons UAGU, UAAU, and UGAU were found to be inefficient, whereas the amber codon UAG and opal codon UGA were efficient for the incorporation of non-natural amino acids. The application of the expanded genetic code in a eukaryotic cell-free system opens the possibility of a four-base codon-mediated incorporation of non-natural amino acids into proteins in living eukaryotic cells.

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