Incorporation of nonnatural amino acids into proteins is a powerful technique in protein research. Amber suppression has been used to this end, but this strategy does not allow multiple incorporation of nonnatural amino acids into single proteins. In this article, we developed an alternative strategy for nonnatural mutagenesis by using four-base codons. The four-base codons AGGU, CGGU, CCCU, CUCU, CUAU, and GGGU were successfully decoded by the nitrophenylalanyl-tRNA containing the complementary four-base anticodons in an Escherichia coli in vitro translation system. The most efficient four-base decoding was observed for the GGGU codon, which yielded 86% of the full-length protein containing nitrophenylalanine relative to the wild-type protein. Moreover, highly efficient incorporation of two different nonnatural amino acids was achieved by using a set of two four-base codons, CGGG and GGGU. This work shows that the four-base codon strategy is more advantageous than the amber suppression strategy in efficiency and versatility.

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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Expanding translation initiation. Incorporation of unnatural carboxylic acids without α‐amino groups was achieved by using chemically acylated initiator tRNA (see figure). The results suggest that various unnatural compounds with a carboxyl group can be incorporated into the N terminus of proteins.

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A 56-year-old Japanese housewife presented with multiple erythematous lesions in association with ocular hyperemia and pain in the right upper and lower extremities, including the hands and feet. These symptoms were preceded by a sore throat with persistent fever higher than 38.5 °C for about 1 week.Dermatologic examination showed tender, dull-red, erythematous lesions, measuring 1-2 cm in diameter, located predominantly on the forehead, cheeks, auricular region, neck, forearm, hands, and feet. A biopsy specimen obtained from an erythematous lesion on the right forearm revealed prominent edema in the papillary dermis and remarkable inflammatory cell infiltration throughout the entire dermis ( Fig. 1). The infiltrate predominantly consisted of neutrophils and nuclear dust without signs of vasculitis. In routine examination, the leukocyte count was 15,000/mL (normal range, 4000 -8000 / mL) with severe neutrophilia (80%). The C-reactive protein (CRP) level was 17.65 mg /dL (normal range, < 0.5 mg /dL) and the anti-streptolysin (ASLO) level was 611 IU/mL (normal range, < 166 IU/mL). In human leukocyte antigen (HLA) testing, HLA-A2, -B39, -B35, -Cw2, and -Cw7 were positive, and HLA-B51, -B54, and -Cw1 were negative.Ocular hyperemia was caused by episcleritis forming a nodule and surrounding congestion of the superficial episcleritic vessels at the central portion of the sclera (Fig. 2). The patient suffered from pain once an hour, continuing for about 3 min, at the lateral portion of the right upper and lower extremities, as well as the right small finger. Neurologic examination demonstrated moderate or slight muscle weakness in the extremities. Hand grasping powers were 9 and 7 kg on the right and left, respectively. The patient was right-handed. Dysesthesia and paresthesia were also observed on the hands and feet. The deep tendon reflexes were preserved, however, even in the distal portion of the upper and lower limbs. In addition, essential tremor localized to the neck was recognized. Magnetic resonance imaging did not show any episodes of transient abnormal signal intensity in the central nervous system.The patient was treated with prednisolone (initial dose of 30 mg / day) and intravenous injection of cefazolin sodium (2 g / day for 5 days). Almost complete regression of the ocular and neurologic manifestations, as well as the skin lesions, was achieved in 2 weeks. Prednisolone was reduced gradually and suspended after 4 weeks. Leukocyte and neutrophil counts, CRP, and ASO returned to normal on suspension of therapy. Slight paresthesia remained in the right small finger even after stopping steroid. There was no recurrence at follow-up 6 months later.