RESUMO
Grape seed proanthocyanidin (GSPA) consists of catechin, epicatechin and epicatechin gallate, which are strong antioxidants that are beneficial to health and may attenuate or prevent Alzheimer's disease (AD). In the present study, the effects of GSPA on pheochromocytoma (PC12) cell viability were determined using cell counting kit-8 and lactate dehydrogenase (LDH) assays, whereas apoptosis and mitochondrial membrane potential (Ψm) were measured via flow cytometry analysis. The effect of GSPA administration on the behavior and memory of amyloid precursor protein (APP)/presenilin-1 (PS-1) double transgenic mice was assessed using a Morris water maze. APP Aß peptides and tau hyperphosphorylation were examined by western blotting; whereas the expression levels of PS-1 were evaluated by reverse transcription-quantitative polymerase chain reaction and compared with pathological sections stained with hematoxylin-eosin and Congo red. Data from the in vitro experiments demonstrated that GSPA significantly alleviated Aß25-35 cytotoxicity and LDH leakage ratio, inhibited apoptosis and increased Ψm. The findings from the in vivo experiments showed a significant enhancement in cognition and spatial memory ability, an improvement in the pathology of APP and tau protein and a decrease in PS-1 mRNA expression levels. Therefore, the results of the present study indicated that GSPA may be a novel therapeutic strategy for the treatment of AD or may, at the very least, improve the quality of life of patients with AD.
RESUMO
The yeast Pichia pastoris GS115 is a widely used microbial cell factory for the production of heterologous protein. In order to reveal the impacts of high heterologous protein expression on the central metabolism of Pichia pastoris GS115 using glucose as sole carbon source, we engineered a high ß-galactosidase expression strain P. pastoris G1HL and a low expression control strain P. pastoris GHL through controlling the initiation strength of constitutive promoter pGAP. The carbon flux distributions in these two strains were quantified via (13)C metabolic flux analysis. Compared to the control strain, G1HL showed a lower growth rate, a higher flux through glycolysis pathway, a higher flux through pentose phosphate pathway, and a lower flux through by-products secretion pathway. The metabolic flux redistribution in G1HL was thought to compensate the increased redox cofactors and energy demands caused by the high protein expression. Although the fluxes through Krebs cycle in two engineered strains were almost the same, they were significantly lower than those in wild strain. The enhanced expression of ß-galactosidase by glutamate supplementation demonstrated the potential of P. pastoris GS115 to catabolize more carbon through the Krebs cycle for even higher protein expression. In conclusion, our work indicates that P. pastoris GS115 can readjusts the central metabolism for higher heterologous protein expression and provides strategies for strain development or process optimization for enhancing production of heterologous protein.
