Expression,purification and identification of I278T-mutant human cystathionine βsynthase / 国际检验医学杂志

Objective To investigate the expression and purification I278T-mutant human cystathionineβsynthase(CBS) in E . coli .Methods Site-directed mutagenesis by overlap extension using the polymerase chain reaction (PCR) was employed to construct mutant plasmids pGEX4T-1-CBS(I278T) ,which was induced and expressed in a medium containing 3% ethanol ,purified by affinity chromatography to obtain mutated CBS (I278T) protein .The activity ,UV-visible absorption spectroscopy ,protein particle size and Zeta potential of the purified protein were measured .Results Plasmid pGEX4T-1-CBS(I278T) was successfully constructed .The yield ,the specific activity and activity recovery of purified mutant CBS (I278T ) protein were 2 .3 mg/L ,21 .4 U/mg and 22 .6% .S-adenosylmethionine(AdoMet) with final concentration of 1 mmol/L showed no activation toward mutant CBS (I278T) protein .Ac-cording to UV-visible absorption spectroscopy analysis ,purified mutant CBS(I278T) had characteristic absorption peaks at 429 nm and 550 nm for heme-binding proteins .Protein average particle size was 7 .5 -10 .1 nm ,mainly in the form of tetramers ,and Zeta potential was - 16 .3 mV .Conclusion The methods of expression ,purification and identification of I278T-mutant human cystathionineβsynthase in E .coli were successfully established .

Effect of Vitamin E Supplementation on Intestinal Barrier Function in Rats Exposed to High Altitude Hypoxia Environment

The study was conducted to investigate the role of vitamin E in the high altitude hypoxia-induced damage to the intestinal barrier in rats. Sprague-Dawley rats were divided into control (Control), high altitude hypoxia (HH), and high altitude hypoxia+vitamin E (250 mg/kg BW*d) (HV) groups. After the third day, the HH and HV groups were placed in a hypobaric chamber at a stimulated elevation of 7000 m for 5 days. The rats in the HV group were given vitamin E by gavage daily for 8 days. The other rats were given equal volume saline. The results showed that high altitude hypoxia caused the enlargement of heart, liver, lung and kidney, and intestinal villi damage. Supplementation with vitamin E significantly alleviated hypoxia-caused damage to the main organs including intestine, increased the serum superoxide dismutase (SOD) (p< 0.05), diamino oxidase (DAO) (p< 0.01) levels, and decreased the serum levels of interleukin-2 (IL-2) (p< 0.01), interleukin-4 (IL-4) (p<0.001), interferon-gamma (IFN-gamma) (p<0.01) and malondialdehyde (MDA) (p<0.001), and decreased the serum erythropoietin (EPO) activity (p<0.05). Administration of vitamin E significantly increased the S-IgA (p<0.001) in ileum and significantly improved the expression levels of occludin and IkappaBalpha, and decreased the expression levels of hypoxia-inducible factor 1 alpha and 2 alpha (HIF-1alpha and HIF-2alpha), Toll-like receptors (TLR4), P-IkappaBalpha and nuclear factor-kappaB p65(NF-kappaB P65) in ileum compared to the HH group. This study suggested that vitamin E protectis from intestinal injury caused by high altitude hypoxia environment. These effects may be related to the HIF and TLR4/NF-kappaB signaling pathway.

Study on the Quality Standard of Xiaoer Shengxue Granules / 中国中医药信息杂志

Objective To establish a quality standard for Xiaoer Shengxue granules. Method SHU-Dihuang, yam, Chinese data in Xiaoer Shengxue granules were identified by TLC. Ferrous sulfate in the granule was determined by UV with wavelength at 509 nm. Results SHU-Dihuang, yam, Chinese data could be detected by TLC. Ferrous sulfate showed a good linear relationship at the range of 0.50～2.50 mg/mL, r=0.999 9. The average recovery was 101.3% and RSD was 1.48% (n=5). Conclusion The method is available with a good reproducibility and can be used for the quality control of Xiaoer Shengxue granule.