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The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.

At moderate to fast walking speeds, the human ankle provides net positive work at high-mechanical-power output to propel the body upward and forward during the stance period. On the contrary, conventional ankle-foot prostheses exhibit a passiveelastic response during stance, and consequently, cannot provide net work. Clinical studies indicate that transtibial amputees using conventional prostheses have higher gait metabolic rates than normal. Researchers believe that the main cause for these higher rates is due to the inability of conventional prostheses to provide sufficient positive power at terminal stance in the trailing leg to limit heel strike losses of the adjacent leading leg. In this investigation, we evaluate the hypothesis that a powered ankle-foot prosthesis, capable of providing human-like ankle work and power during stance, can decrease the metabolic cost of transport (COT) compared to a conventional passive-elastic prosthesis. To test the hypothesis, a powered prosthesis is built that comprises a unidirectional spring, configured in parallel with a force-controllable actuator with series elasticity. The prosthesis is shown to deliver the high mechanical power and net positive work observed in normal human walking. The rate of oxygen consumption and carbon dioxide production is measured as a determinant of metabolic rate on three unilateral transtibial amputees walking at self-selected speeds. We find that the powered prosthesis decreases the amputee's metabolic COT on average by 14% compared to the conventional passive-elastic prostheses evaluated (Flex-Foot Ceterus R and Freedom Innovations Sierra), even though the powered system is over twofold heavier than the conventional devices. These results highlight the clinical importance of prosthetic interventions that closely mimic the mass distribution, kinetics, and kinematics of the missing limb.

The results of this study provided scientific data to support the efficient and safe use of HC to combat SARS.

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