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Fc domain fusion can improve the therapeutic effects of relatively small biological molecules such as peptides, cytokines, and antibody fragments. Fc fusion proteins can also be used to enhance the cytotoxic effects of small bispecific antibodies (bsAbs). However, fragmentation of Fc fusion proteins, which mainly occurs around the hinge regions during production, storage, and circulation in the blood, is a major issue. In this study, we first investigated the mechanisms of fragmentation around the hinge region during storage using Fc-fused bsAbs with specificity for epidermal growth factor receptor and CD3 as a model. The fragmentation peaks generated by gel filtration analysis indicated that both contaminating proteases and dissolved active oxygen should be considered causes of fragmentation. We designed and constructed variants by introducing a point mutation into the upper hinge region, which reduced the cleavage caused by dissolved active oxygen, and shortened the hinge region to restrict access of proteases. These hinge modifications improved fragmentation resistance and did not affect the biological activity of the bsAbs in vitro. We confirmed the versatility of the hinge modifications using another Fc-fused bsAb. Our results show that hinge modifications to the Fc fusion protein, especially the introduction of a point mutation into the upper hinge region, can reduce fragmentation substantially, and these modifications can be used to improve the fragmentation resistance of other recombinant Fc fusion proteins.

Melatonin regulates diurnal locomotor rhythms in birds as well as in other vertebrates, but the molecular mechanism by which melatonin regulates locomotor activity is poorly understood. Here, we summarize new findings showing that 7α-hydroxypregnenolone, a previously undescribed avian neurosteroid, mediates melatonin action on diurnal locomotor rhythms in birds. Recently, 7α-hydroxypregnenolone was identified as a novel avian neurosteroid in Japanese quail Coturnix japonica brain. It was found that 7α-hydroxypregnenolone acutely stimulates quail locomotor activity. Subsequently, it was clarified that diurnal changes in 7α-hydroxypregnenolone synthesis occur in parallel with changes in locomotor activity in quail. Finally, it was demonstrated that melatonin depresses the synthesis of 7α-hydroxypregnenolone, thus providing a mechanism through which the nocturnal increase in melatonin regulates diurnal changes in locomotor activity. This review highlights a novel molecular mechanism controlling circadian activity of birds by the interaction of melatonin with 7α-hydroxypregnenolone, a newly discovered neurosteroid stimulating locomotion.

Superoxide dismutase 1 (SOD1) is an important antioxidative enzyme that protects skin from oxidative stress. SOD1 (-/-) mice with a genetic background of b129Sv mice showed facial skin damage after 15 weeks of age. Eyelid swelling occurred as the initial symptom and caused impairment by triggering self-scratching. The period required for wound healing in the back was markedly delayed in 20-week SOD1 (-/-) mice. Oxidative stress markers, 4-hydroxynonenal and thiobarbituric acid-reactive substances, were unexpectedly lower in SOD1 (-/-) mice at day 1 after wounding. The decay rate of electron paramagnetic resonance signal intensity of intravenously injected nitroxide radical indicated that the half-life of the signal intensity was significantly prolonged in the wounded skin of SOD1 (+/+) mice. However, while the half-life of the signal intensity in control skin was a little longer in SOD1 (-/-) mice, it did not change in wounded skin. Taken together, these data suggest that the skin of SOD1 (-/-) mice is in redox imbalance and prone to damage by wounding.