Objective Explore the conditions of the cloning,expression and purification of FMRP.Methods The plasmid pET22b(+)-FMR1,constructed by molecular cloning,was transformed into E.coli BL21(DE3) competent cells and induced to express FMRP by IPTG.Recombinant FMRP was purified by affinity chromatography,verified by Western-blot,and tested for its RNA binding ability.Results FMR1 cDNA was successfully cloned into pET22b(+) vector and expressed in E.coli BL21(DE3).A protein with Mr 79 000 was purified and confirmed to be FMRP.This protein retained the RNA binding ability of FMRP.Conclusion We successfully expressed recombinant hFMRP with high purity and activity in E.coli,which provided a reliable material to study the function of FMRP.

The effect of the complement C1q expression on total hepatic ischemia-reperfusion (I/R) injury in rats was investigated. Sixty healthy male Sprague Dawley (SD) rats weighing 180-200 g were randomly divided into 5 groups: sham-operation group (S group, n=12); group of I/R for 1 h (I/R 1 h group, n=12); group of I/R for 3 h (I/R 3 h group, n=12); group of I/R for 6 h (I/R 6 h group, n=12); group of I/R for 24 h (I/R 24 h group, n=12). The hepatic I/R model of rats was established, and liver tissues were obtained 1 h, 3 h, 6 h and 24 h after hepatic I/R, respectively. Furthermore, the tissues were stained using hematoxylin-eosin, and the liver injuries of rats were observed using a microscope. The malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in liver tissue were determined. Real-time polymerase chain reaction (PCR) and Western blotting were used to detect the expression levels of C1q mRNA and protein, respectively. As compared with the S group, the histopathological changes in I/R 1 h-24 h groups were gradually aggravated with the extension of I/R time. As compared with the S group, SOD activity and MDA content in the I/R groups were reduced and increased respectively with the extension of I/R time (P<0.01). Furthermore, the C1q expression at mRNA and protein levels in the I/R groups (especially in the I/R 3 h group) was significantly higher than that in the S group (P<0.05). It is suggested that C1q expression may play a principal role in hepatic I/R injury, particularly at the early stage of perfusion.

Adult ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; HIV Enteropathy ; complications ; metabolism ; microbiology ; pathology ; Humans ; Male ; Mycobacterium avium Complex ; isolation & purification ; Mycobacterium avium-intracellulare Infection ; complications ; metabolism ; microbiology ; pathology ; Young Adult

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.
Aminoacyltransferases ; metabolism ; Bacterial Proteins ; metabolism ; Cyclization ; Cysteine Endopeptidases ; metabolism ; Enzymes, Immobilized ; metabolism ; Kinetics ; Peptides ; metabolism ; Peptides, Cyclic ; biosynthesis ; Staphylococcus aureus ; enzymology

Shanghai University of TCM was the first Chinese medicine university that established field-grade experimental teaching center in China.And both of the Chinese medicine and Chinese herbs experimental teaching centers became national experimental teaching demonstration centers.There is general improvement in laboratories conditions,so the experimental teaching team building is the critical factor of improving experimental teaching quality.The experimental teaching team of Shanghai University of TCM consists of excellent teachers as its backbone,and lecturers and technicians from fields in traditional Chinese medicine Chinese herbs and clinical practices.The team members cooperate with each other by setting up experimental teaching research groups to improve teaching quality,which plays an important role in building experimental teaching demonstration center.

Objective To explore experimently the effect of tumor angiogenesis on rapid progression of residual tumor of liver cancer after radiofrequency ablation ( RFA). Methods A rabbit VX2 hepatoma model was established. Inoculated tumors were treated by using RFA at 55 ℃ , 70 ℃ and 85 ℃ respectively to establish the residual VX2 hepatoma model. Rabbits implanted with VX2 hepatoma but receiving no RFA treatment served as controls. The expression of vascular endothelial growth factor (VEGF)was determined in tumors to assess the relationship between VEGF and the focal tumor volume and distant metastasis. The expression of VEGF and microvessel density ( MVD) in tumor tissues was assessed by immunohistochemistry. The protein expression of VEGF was assessed by Western blot. The expression of VEGF mRNA was detected by RT-PCR. Results There were significant differences of the local tumor volume between the control group (9.91 ±0.98) cm3 and the other groups (respectively t = -17.43,-10.11, -8.79,all P<0. 05). Compared with the 70 ℃ group (17. 08 ±2. 28 ) cm3 and the 85 ℃ group (15.95 ±4.95) cm3, the focal tumor volume of 55 ℃ group was the largest (21.26 ±2.32) cm3,( respectively t = 4. 69,6. 78, all P<0. 05). Much more metastatic lesions of lung were observed in the RFA treated groups in comparison to the control group. Moreover, the lung metastasis in 55 ℃ group was the most serious among the three RFA treated groups (respectively t = -21.65, -30. 15, all P<0. 05 ).Immunohistochemical staining indicated that the expression of VEGF and MVD in the RFA treated groups was much higher than those in control group ( MVD respectively t = -13.01, -5. 46, -5. 63, all P<0. 05), ( VEGF respectively t = 8. 00,4. 92,4. 21, all P<0. 05 ). Furthermore, the expression of both VEGF protein and VEGF mRNA in 55 ℃ group was the highest among the three RFA treated groups.Conclusions The over-expression of VEGF accelerating the tumor angiogenesis may be one of the mechanisms inducing rapid progression of residual liver tumor after RFA.

Adult ; Chronic Disease ; Electromyography ; Female ; Hexanes ; poisoning ; Humans ; Male ; Occupational Diseases ; chemically induced ; diagnosis ; Poisoning ; diagnosis

Animals ; Boron ; analysis ; urine ; Ion-Selective Electrodes ; Minerals ; chemistry ; Urine ; chemistry

Objective To investigate the relationship between insulin resistance and erythrocyte in- sulin receptors in patients with gout associated with macroalbruminuria(MAU).Methods FBG,PBG,FINS, P2hINS,CH,TG,HDL,LDL-c,UA and erythrocyte insulin receptors were determined in 44 patients with MAU,62 patients with normal MAU(NMAU).Results In MAU,the levels of FINS,TG,LDL-c and HOMA-IR were(16?4)mU/L,(2.5?0.6)retool/L,(3.2?0.5)mmol/L and 3.6~1.2 respectively.While they were(13?3) mU/L,(2.3?0.8)mmol/L,(3.0?0.5)mmol/L and 3.0~0.4 in NMAU group.The levels of FINS,TG,LDL-c and HOMA-IR were significantly higher in the MAU patients than those in NMAU patients(P

Adult ; Analgesics, Opioid ; pharmacology ; Female ; Fentanyl ; pharmacology ; Humans ; In Vitro Techniques ; Interleukin-6 ; blood ; Male ; Morphine ; pharmacology ; Narcotics ; pharmacology ; Tumor Necrosis Factor-alpha ; analysis