Objective To express insulin-degrading enzyme(IDE)mutant T142A in prokaryotic cells and detect its activity.Methods According to the results of multi-sequence alignment and IDE substrate co-crystal structure,an active residue in β6-strand structure of IDE were predicted.The recombinant plasmid ppSUMO-T142A,with the site mutation of threonine 142 to alanine,was constructed by point mutation technique and expressed by E.coli prokaryotic expression system.After purification by nickel ion column affinity chromatography,ion exchange chromatography and gel filtration chromatography,the mutant T142A was obtained and determined for the activity by fluorescence method.Results IDE amino acid sequence is highly conserved among 16 species.T142 directly participates in substrate binding,interacts with substrate cleavage sites,and is close to important structures such as catalytic active sites and door-subdomains.The mutation of recombinant plasmid ppSUMO-T142A was proved to be correct by sequencing.The expressed fusion protein His-SUMO-T142A was mainly existed in soluble form in the supernatant at a concentration of 18 mg/mL,with a relative molecular mass of about 131 000;After three steps of purification,the purity of mutant T142A reached 86%.The maximum reaction rate(V_(max))of T142A catalytic degradation of fluorescent substrate V was 501.06 min~(-1) and the Michaelis constant(K_m) was 9.01μmol/L.Compared with wild-type IDE(V_(max) was 2 814.32 min~(-1),K_m was 11.93μmol/L),the activity of T142A decreased significantly.Conclusion The activity of IDE mutant T142A expressed in this study greatly decreases,while T142 is an important residue for IDE to play its enzymatic function,which provides an experimental basis for the development of new IDE activity regulatory molecules.

Cardiovascular Diseases ; Follistatin-Related Proteins ; physiology ; Humans

Objective: To study the chemical constituents of Artemisia anomala. Methods: Various chromatographic techniques were adopted to separate the constituents, and the spectroscopic analysis was used to identify their structures. Results: Seven compounds were isolated and identified as (4aS, 7S, 7aR)-methyl 7-hydroxy-7-methyl-1, 4a, 5, 6, 7, 7a-hexahydrocyclopenta [c] pyran-4- carboxylate (1), rehmaglutin D (2), (E)-6-hydroxy-2, 6-dimethylocta-2, 7-dienoic acid (3), chrysoeriol (4), luteolin (5), apigenin (6), and p-coumaric acid (7). Conclusion: Compound 1 is a new compound named artanoiridoid; Compound 2 is firstly reported in this genus; Compounds 3, 4, and 7 are separated from this plant for the first time.

The middle cerebral artery occlusion(MCAO) in rats remained a focus cerebral ischemic model that is non-incisioned,reliable,as well as the ischemic and refusion time can be controlled,which is widely used since it was created.However,because it is needed complex operations,associatied with many factors,it is difficult to establish.Many scholars have modified the operation,shape of the occlusion line,as well as the variety,narcotic drugs and so on.

Child ; Humans ; Myelodysplastic Syndromes ; classification ; diagnosis ; Pediatrics ; World Health Organization

Humans ; Larynx ; surgery ; Neurilemmoma ; surgery

Adolescent ; Child ; Female ; Fever ; etiology ; Histiocytic Necrotizing Lymphadenitis ; classification ; complications ; diagnosis ; Humans ; Lymph Nodes ; pathology ; Male

Accidents, Occupational ; Acute Disease ; Humans ; Hydrogen Sulfide ; poisoning ; Poisoning ; mortality

Blood Cells ; cytology ; Bone Marrow Cells ; cytology ; China ; Hematologic Tests ; standards ; Humans

Based on the thought of Zhishen (a kind of mind regulation), Professor SHI Xue-min, academician of the China Academy of Engineering, found the Xingnao Kaiqiao (to refresh the mind and to cause resuscitation) acupuncture method, which still plays an important role in the acupuncture treatment of wind stroke nowadays. Meanwhile, great importance is attached to the comprehensive treatment of wind stroke. Danqi Piantan capsule (see text) is developed and "wind stroke unit" is set up. In recent years, Professor SHI shifts the center of research to the treatment of hypertension, the risk factor of wind stroke. Taking Renying (ST 9) as the major acupoint, acupuncture with standard measurement and manipulations is established. And good clinical effect has been obtained as well. Therefore, this article focuses on the introduction of Professor SHI Xue-min's contribution to wind stroke treatment.
Acupuncture ; education ; Acupuncture Points ; Acupuncture Therapy ; China ; Humans ; Stroke ; therapy