Objective To investigate the relationship between tumor necrosis factor-α (TNF-α) in ischemic brain tissue and bran edema after cerebral ischemia-reperfusion in rats.Methods Eighty four male SD rats were randomly assigned to either a cerebral ischemia reperfusion group (n =44) or a sham-operation group (n =40). A model of middle cerebral artery occlusion for 120 minutes followed by reperfusion was induced in rats using the suture method. The infarct size was determined by 2, 3, 5-triphenyi terazoloride (TTC) staining at 6 h,24 h, 3 d, and 7 d respectively after the reperfusion. Dry-wet weight method was used to measure brain water content and evaluate the extent of brain edema. The enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of TNF-α in ischemic brain tissue. Results TNF-α level in ischemic brain tissue was increased at 6 h (445.8 ±91.7 pg/ml) after the reperfusion, and reached the peak at day 3 (715.5 ±121.3 pg/ml). There were significant differences compared to the sham-operation group and other time points (all P＜0.001). After that, it was decreased gradually, but it was still higher than that in the shamoperation group at day 7 (478.1 ± 145.5 pg/ml vs. 148.5 ± 101.7 pg/ml, P＜0.005). The initial change of the water content in brain tissue lagged behind the increased TNF-α. It did not increase significantly until 24 h after cerebral ischemia-reperfusion (P ＜0.001). It reached the peak at day 3 (P ＜0.001), and it was still higher than that in the control group at day 7 (P ＜0.05). The evolution of cerebral infarct volume was in accordance with the changes of TNF-α level. Conclusions TNF-α is associated with the changes of brain edema and infarct volume,and it is harmful to brain tissue.

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.
Ammonia-Lyases/genetics* ; Escherichia coli/genetics* ; Propionates ; Rhodotorula ; Tyrosine/genetics*