Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons.

Applications of the peroxidase activity of cytochrome P450 enzymes in synthetic chemistry remain largely unexplored. We present herein a protein engineering strategy to increase cytochrome P450BM3 peroxidase activity for the direct nitration of aromatic compounds and terminal aryl-substituted olefins in the presence of a dual-functional small molecule (DFSM). Site-directed mutations of key active-site residues allowed the efficient regulation of steric effects to limit substrate access and, thus, a significant decrease in monooxygenation activity and increase in peroxidase activity. Nitration of several phenol and aniline compounds also yielded ortho- and para-nitration products with moderate-to-high total turnover numbers. Besides direct aromatic nitration by P450 variants using nitrite as a nitrating agent, we also demonstrated the use of the DFSM-facilitated P450 peroxidase system for the nitration of the vinyl group of styrene and its derivatives.

Enabling highly (R)-enantioselective epoxidation of styrene by engineering unique non-natural P450 peroxygenases.

Unlike the excellent (S)-enantioselective epoxidation of styrene performed by natural styrene monooxygenases (ee > 99%), the (R)-enantioselective epoxidation of styrene has not yet achieved a comparable efficiency using natural or engineered oxidative enzymes. This report describes the H2O2-dependent (R)-enantioselective epoxidation of unfunctionalized styrene and its derivatives by site-mutated variants of a unique non-natural P450BM3 peroxygenase, working in tandem with a dual-functional small molecule (DFSM). The observed (R)-enantiomeric excess (ee) of styrene epoxidation is up to 99% with a turnover number (TON) of 918 by the best enantioselective mutant F87A/T268I/L181Q, while the best active mutant F87A/T268I/V78A/A184L (with 98% ee) gave a catalytic TON of 4350, representing the best activity of a P450 peroxygenase towards styrene epoxidation to date. Following this approach, a set of styrene derivatives, such as o-, m-, p-chlorostyrenes and fluorostyrenes, could also be epoxidized with modest to very good TONs (362-3480) and high (R)-enantioselectivities (95-99% ee). The semi-preparative scale synthesis of (R)-styrene oxide performed at 0 °C with high conversion, maintaining enantioselectivity, and moderate isolated yields, further suggests the potential application of the current P450 enzymatic system in styrene epoxidation. This study indicates that the synergistic use of protein engineering and an exogenous DFSM constitutes an efficient strategy to control the enantioselectivity of styrene epoxidation, thus substantially expanding the chemical scope of P450 enzymes as useful bio-oxidative catalysts.

Targeting of Rho kinase ameliorates impairment of diabetic endothelial function in intrarenal artery.

Endothelial dysfunction in kidney vasculature is the initial and key element for nephropathy in diabetes mellitus. Accumulating evidence suggests the protective role of Rho kinase inhibitors in endothelial dysfunction via modulating eNOS activity and NO production. However, the role of Rho kinase in diabetes-related endothelial dysfunction in kidney vasculature and the relevant mechanisms remain unknown. We assessed whether pharmacological inhibition of Rho kinase attenuates endothelial dysfunction in intrarenal arteries from type 1 diabetic rats. Fasudil, a Rho kinase inhibitor effectively decreased the phosphorylated level of MYPT1 without affecting the expression of ROCKs in the kidney. Fasudil treatment showed no improvement in diabetes-related abnormality in metabolic indices, but it significantly ameliorated endothelial dysfunction in intrarenal arteries and lessened the mesangial matrix expansion in the kidney cortex. Mechanistically, superoxide production in the intrarenal artery and NOX4 member of NADPH oxidase in the renal cortex that contribute to diabetic nephropathy were also prevented by the Rho kinase inhibitor. In conclusion, the present results indicate that Rho kinase is involved in endothelial dysfunction in type 1 diabetes via enhancement of oxidative stress and provides new evidence for Rho kinase inhibitors as potential therapeutic agents for the treatment of diabetic nephropathy.

Effect of treadmill training with body weight support on gait and gross motor function in children with spastic cerebral palsy.

OBJECTIVE: To examine the effect of treadmill training with body weight support (TBWS) on gait and gross motor function in children with spastic cerebral palsy (CP). DESIGN: Eight children with spastic CP participated in the study. Their temporal-distance gait parameters, Gross Motor Function Measure, muscle tone, and selective motor control were assessed three times: two times under their regular therapeutic treatment (condition A), and one time after receiving the TBWS treatment in addition to their regular therapeutic treatments (condition B). There were two treatment schedules, AAB and ABA. Except for the first one (taken at study entry), the assessments were always taken after 12 wks of treatment. The children were equally divided into two groups and randomly assigned to the two schedules. The two groups were matched according to category of the Gross Motor Function Classification System. RESULTS: The TBWS treatment significantly improved the children's gait (increases in stride length and decreases in double-limb support percentage of gait cycle) and their Gross Motor Function Measure (dimension D and E scores as well as the total score). No significant improvements on muscle tone or selective motor control were noted. CONCLUSIONS: The TBWS treatment improved some gait parameters and gross motor functions in children with spastic CP.

[Protective effects of puerarin on radiation injury of experimental rats].

OBJECTIVE: To observe the protective effects of puerarin on radiation injury of experimental rats and to discuss the possible mechanism of its radiation protection. METHODS: Wistar rats were divided randomly into 4 groups with 8 rats in each group: physiological saline non-radiation (SN) group, puerarin non-radiation (PN) group, physiological saline radiation (SR) group, puerarin radiation (PR) group. The source of radiation was cobalt-60 gamma rays, and the rats were exposed to radiation (1.2 Gy/min) at a dose of 18 Gy. Following irradiation of the rats, puerarin was injected intravenously at each dose of 30 mg/kg in 6 consecutive days (24 h interval). Samples were collected and assayed one week later. RESULTS: Puerarin delayed effectively the declines of the quantity of red blood cells and white blood cells in circulation due to ionizing irradiation, improved the index of thymus and spleen in ionizing irradiation rats, increased the activity of superoxide dismutase (SOD) and decreased the level of malondialdehyde (MDA) in the rat myocardial tissue. CONCLUSION: Puerarin has a marked protective effect on ionizing irradiation injury of experimental rats, and its protective mechanism is probably based on oxidation-resistance.