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.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Arrhythmias, Cardiac ; diagnosis ; genetics ; therapy ; Child ; Child, Preschool ; Electrocardiography ; Humans ; Infant ; Male ; Middle Aged ; Syndrome

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.

To compare the activity of RGD-TRAIL in different expression systems, RGD-TRAIL in both Escherichia coli (E.coli) and Pichia pastoris was constructed and expressed. In vitro activity of RGD-TRAIL from Pichia pastoris expression system was also analyzed. Genetic engineering techniques were used to construct recombinant plasmid pET30-rgd-trail and pHBM-rgd-trail. The recombinant protein RGD-TRAIL was purified with Ni ion affinity chromatography after induction. MTT assay, ELISA, scratch wound healing, transwell migration assay and Hoechst 33342 staining were performed to detect the effects of RGD-TRAIL on proliferation, binding activity, migration and apoptosis. The expression of apoptosis-associated proteins was detected by Western blotting. Recombinant protein RGD-TRAIL was successfully expressed in a form of inclusion body in E.coli, while expressed secretorily in Pichia pastoris. It possessed more potent cytotoxicity than RGD-TRAIL in E.coli by MTT assay. The RGD-TRAIL expressed by Pichia pastoris showed powerful binding affinity with cancer cells expressing α(v), DR4, DR5 and highly potent cytotoxicity through inducing apoptosis of cancer cells. Nuclear fragmentation was examined by Hoechst 33342 staining. Cleaved PARP and caspase-3 were also detected after incubation with RGD-TRAIL. Additionally, RGD-TRAIL inhibited migration significantly in A549 and HT1080 cells. The results demonstrate that Pichia pastoris expression system is more suitable for the recombinant protein RGD-TRAIL. Its binding affinity and antitumor activity might make RGD-TRAIL a promising candidate for cancer therapy.
Antineoplastic Agents ; pharmacology ; Apoptosis ; Blotting, Western ; Cell Line, Tumor ; Chromatography, Affinity ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Humans ; Oligopeptides ; biosynthesis ; pharmacology ; Pichia ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; pharmacology

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

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

Acupuncture Points ; Acupuncture Therapy ; instrumentation ; Aged ; Catgut ; Female ; Humans ; Male ; Middle Aged ; Muscle, Skeletal ; physiopathology ; Spasm ; physiopathology ; therapy ; Treatment Outcome