Expression of Aspergillus nidulans phy Gene in Nicotiana benthamiana Produces Active Phytase with Broad Specificities

A full-length phytase gene (phy) of Aspergillus nidulans was amplified from the cDNA library by polymerase chain reaction (PCR), and it was introduced into a bacterial expression vector, pET-28a. The recombinant protein (rPhy-E, 56 kDa) was overexpressed in the insoluble fraction of Escherichia coli culture, purified by Ni-NTA resin under denaturing conditions and injected into rats as an immunogen. To express A. nidulans phytase in a plant, the full-length of phy was cloned into a plant expression binary vector, pPZP212. The resultant construct was tested for its transient expression by Agrobacterium-infiltration into Nicotiana benthamiana leaves. Compared with a control, the agro-infiltrated leaf tissues showed the presence of phy mRNA and its high expression level in N. benthamiana. The recombinant phytase (rPhy-P, 62 kDa) was strongly reacted with the polyclonal antibody against the nonglycosylated rPhy-E. The rPhy-P showed glycosylation, two pH optima (pH 4.5 and pH 5.5), an optimum temperature at 45~55 °C, thermostability and broad substrate specificities. After deglycosylation by peptide-N-glycosidase F (PNGase-F), the rPhy-P significantly lost the phytase activity and retained 1/9 of the original activity after 10 min of incubation at 45 °C. Therefore, the deglycosylation caused a significant reduction in enzyme thermostability. In animal experiments, oral administration of the rPhy-P at 1500 U/kg body weight/day for seven days caused a significant reduction of phosphorus excretion by 16% in rat feces. Besides, the rPhy-P did not result in any toxicological changes and clinical signs.     

Preparation and evaluation of poly(2-hydroxyethyl aspartamide)-hexadecylamine-iron oxide for MR imaging of lymph nodes

The purpose of this study was to synthesize biocompatible poly(2-hydroxyethyl aspartamide)–C₁₆-iron oxide (PHEA-C₁₆-iron oxide) nanoparticles and to evaluate their efficacy as a contrast agent for magnetic resonance imaging of lymph nodes. The PHEA-C₁₆-iron oxide nanoparticles were synthesized by coprecipitation method. The core size of the PHEA-C₁₆-iron oxide nanoparticles was about 5 to 7 nm, and the overall size of the nanoparticles was around 20, 60, and 150 nm in aqueous solution. The size of the nanoparticles was controlled by the amount of C₁₆. The 3.0-T MRI signal intensity of a rabbit lymph node was effectively reduced after intravenous administration of PHEA-C₁₆-iron oxide with the size of 20 nm. The in vitro and in vivo toxicity tests revealed the high biocompatibility of PHEA-C₁₆-iron oxide nanoparticles. Therefore, PHEA-C₁₆-iron oxide nanoparticles with 20-nm size can be potentially useful as T2-weighted MR imaging contrast agents for the detection of lymph nodes.     

Efficient preparation of DHMF and HMFA from biomass-derived HMF via a Cannizzaro reaction in ionic liquids

2,5-Dihydroxymethylfuran (DHMF) and 5-hydroxymethylfuranoic acid (HMFA) are the versatile intermediate chemicals of high industrial potential. An efficient preparation of both DHMF and HMFA is reported from biomass-derived HMF via a Cannizzaro reaction using ionic liquids as a reusable reaction solvent under the water-free conditions. The operationally simple and environment-friendly process produces both DHMF and HMFA from HMF consistently in good to high yields up to five runs with the recycled ionic liquid.     

ROS-Mediated Anti-Tumor Effect of Coptidis Rhizoma against Human Hepatocellular Carcinoma Hep3B Cells and Xenografts

Coptidis Rhizoma is the dried rhizome from the Coptis chinensis Franch. that has been shown to have a number of beneficial pharmacological properties including antioxidant, anti-inflammatory, and anti-cancer effects. However, the anti-cancer effects of Coptidis Rhizoma on hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the anti-cancer properties of Coptidis Rhizoma ethanol extract (CR) in HCC Hep3B cells and in a xenograft mouse model. Our results showed that the CR significantly inhibited cell growth and induced apoptosis in Hep3B cells through increased expression of Bcl-2 associated x-protein (Bax) and cleavage of poly-ADP ribose polymerase (PARP), reduced expression of Bcl-2, and activated caspases. CR also increased the generation of intracellular reactive oxygen species (ROS), which caused a loss of mitochondrial membrane potential (MMP, ΔΨm) and activation of the mitochondria-mediated intrinsic apoptosis pathway. Moreover, N-acetylcysteine (NAC), a ROS inhibitor, markedly blocked the effects of CR on apoptotic pathways. CR also induced the expression of light chain 3 (LC3)-I/II, a key autophagy regulator, whereas CR-mediated autophagy was significantly suppressed by NAC. In addition, pre-treatment with NAC perfectly attenuated the inhibition of cell invasion and migration of CR-stimulated Hep3B cells. Furthermore, oral administration of CR suppressed Hep3B tumor growth in xenograft mice without toxicity, alterations to body weight, or changes in hematological and biochemical profiles. Taken together, our findings suggest that CR has anti-tumor effects that result from ROS generation, and may be a potential pharmacological intervention for HCC.