Antifreeze proteins (AFP) inhibit ice growth to permit the survival of polar organisms in the cold environments. The recombinant AFP from an Antarctic bacterium, Flavobacterium frigoris PS1, FfIBP (Flavobacterium frigoris icebinding protein), was produced using Pichia pastoris expression system. The optimum fermentation temperature (30 o C) and pH (5) for FfIBP production were determined using a fedbatch culture system. The maximal cell density and purified FfIBP were 112 g/L and 70 mg/L, respectively. The thermal hysteresis (TH) activity (0.85) of FfIBP obtained using a glycerol-methanol fed-batch culture system was 2-fold higher than that of the LeIBP (Leucosporidium ice-binding protein). This work allows for large-scale production of FfIBP, which could be extended to further application studies using recombinant AFPs.
Diallyl trisulfide (DATS), one of the major organosulfur components of garlic (Allium sativum), has various biological effects such as anti-microbial and anti-cancer activities. However, the molecular mechanisms of growth inhibition related to cell cycle arrest are poorly understood. In this study, we investigated the effects of DATS on cell cycle progression in U937 human leukemia cells. Treatment with DATS in U937 cells resulted in inhibition of cell viability through G2/M arrest and apoptosis. DATS-induced G2/M arrest was associated with up-regulation of cyclin B1 and cyclin-dependent kinase 1 (CDK1). DATS also significantly increased levels of phospho-histone H3, which is a mitosis-specific marker, indicating that DATS induced mitotic arrest but not G2 arrest in U937 cells. DATS treatment also generated the reactive oxygen species (ROS) in U937 cells; however, pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, significantly attenuated DATS-induced mitotic arrest and apoptosis. Taken together, our data indicate that DATS exhibits anti-cancer effects through mitotic arrest and apoptosis in a ROS-dependent manner.
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