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.

China ; Clinical Laboratory Techniques ; Humans ; Myelodysplastic Syndromes ; diagnosis

OBJECTIVEWe aim to explore the central auditory nervous system (CANS) functioning in children with nonsyndromic cleft palates by analyzing the auditory evoked potentials and event-related potentials (ERP).

METHODSA total of 34 children with nonsyndromic cleft palates were recruited as subjects, and 27 normally developed children were selected as the normal controls. Auditory brainstem response (ABR), middle latency response (MLR), and mismatch negativity (MMN) of ERP were selected as indices to observe the function of CANS in children in both groups.

RESULTSAstatistically significant difference between the groups was obtainedin the MMN recording (F = 227.69, P < 0.01), whereas no significant group differences were obtained in the ABR and MLR results (P > 0.05). Children with nonsyndromic cleft palates showed diminished MMN responses compared with the normal controls, whereas ABR and MLR were within the normal range.

CONCLUSIONChildren with nonsyndromic cleft palates are at risk of central auditory discrimination dysfunction. The significant abnormal event-related potentials recorded in children with cleft palates suggest that the dysfunction of CANS maybe located at the cortical level and normal function of CANS was located at the brain stem and sub-cortical level.

Aortic Diseases ; Aortic Valve ; Aortic Valve Stenosis ; Calcinosis ; Humans ; Hyperlipoproteinemia Type II

OBJECTIVE： To discuss the application prospect of pharmacogenomics METHODS： To introduce the concept of pharmacogenomics and analyse its application at prospect RESULTS ＆ CONCLUSION： Pharmacogenomics is wide in the prospect of application and has a vast reservoir of economic potential， it will exert an influence on pharmacy in the 21st century

Objective To investigate the effect of propofol on hypoxic pulmonary vasoconstriction(HPV)and assess the underlying mechanism in rats.Methods Twenty male SD rats weighing 300-400 g were.anesthetizedwith intraperitoneal phenobarbital.Heart and lungs were removed after thoracotomy.Pulmonary.arterial rings 4 mmin length and 1.0-1.4 mm in diameter were prepared and suspended in Earl solution maintained at 37℃ with a pHof 7.40 and aerated with 20% O_2-5% CO_2-25% N_2.The rings were stimulated with phenylephrine(PE)10~(-6)mol?L~(-1) with different preloads(300,500,700,900,1 100 mg).The isometric tension of the arterial rings wasmeasured.The optimal preload was determined to be 900 mg which allowed best contractility.Hypoxia was inducedby aerating the solution with 95% N_2-5% CO_2 and the flow rate was adjusted to maintain PO_2 of the solution at40-20 mm Hg and pH at 7.40.HPV of the rings were recorded.Then propofol was added to achieve a finalconcentration of 1,3,10,30,100 ?mol?L~(-1) and HPV was again induced and the changes in HPV wererecorded.In addition the effects of propofol(10,30 ?mol?L~(-1))on vasoconstriction produced by KCI and PE werealso measured.Results The lower doses of propofol(10 ?mol?L~(-1))significantly inhibited HPV(P

?Corneal collagen cross-linking ( CXL ) could increase the mechanical strength, biological stability and halt ectasia progression due to covalent bond formed by photochemical reaction between ultraviolet - A and emulsion of riboflavin between collagen fibers in corneal stroma. Corneal melting is an autoimmune related noninfectious corneal ulcer. The mechanism of corneal melting, major treatment, the basic fundamental of ultraviolet- A riboflavin induced CXL and the clinical researches status and experiment in CXL were summarized in the study.