Structural basis for high-pressure improvement in depolymerization of interfacial protein from RFRS meat batters in relation to their solubility.

High-pressure processing (HPP) can modify the construction of interfacial proteins (IPs) to improve the properties of reduced-fat and reduced-salt (RFRS) meat batters. In this study, the relationship between the construction of IPs and their solubility at fat droplet/water interface in RFRS meat batters with HPP treatments was investigated. When 200 MPa for 2 min was applied, the IPs exhibited the highest solubility due to a high concentration of absorbed myosin with the content of random coil 65.62%, but the particle diameter was in reverse. The microscopy revealed the depolymerization of IPs occurred at low pressure, while macromolecular aggregates were produced as the cross-linking of IPs to some degree at pressure ≥ 200 MPa. This phenomenon was supported by the result of SDS-PAGE and the sulfhydryl of IPs. In conclusion, the HPP induced solubility alteration of IPs was achieved by modifying their construction through adjusting the secondary structures and regulating bond interactions.

High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system.

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.