Purpose To clone and express Trypanosoma Leucyl-tRNA synthetase (LeuRS) gene and to complete the purification and activity assay of LeuRS. Methods We cloned the LeuRS gene from T. Brucei genomic DNA,and cloned it into pBS-T and then into the expression vector pET21a( + ) .The expression of T. Brucei LeuRS was carried out in E. coli BL21 (DE3) RIPL host by optimizing the expression conditions. The products are purified by His-Bind affinity column and verified by Western blot . Enzymatic activity was detected by isotope labeling. Results A DNA fragment at length of 3.2 kb was amplified. Both restriction analysis and sequencing analysis proved that recombinant plasmid pET21a( + )/LeuRS was correctly constructed. The expressed product contained about 20% of total somatic soluble protein and reached a purity of 85% after purification . It was verified by the Western blot. Enzyme unit per millilitre of purified products was about 72. Conclusion Here we report the successful clone, expression and purification of LeuRS from Trypanosoma Brucei ( T. Brucei) in bacterial host. In addition, we tested its enzymatic activity by isotope labeling.This work would be helpful to the design and in vitro screening of Trypanosoma LeuRS inhibitors.

Objective To explore the value of intraoperative contrast-enhanced ultrasonography in radiofrequency ablation completeness evaluation in patients with hepatic carcinoma of differently sized nodules.Methods Fifty five patients (69 lesions) with hepatic carcinoma were included and were divided into the experimental group (20 cases,30 lesions in whom CEUS were used) and the control group (35 cases,39 lesions,conventional ultrasound was used).After RAF,the treatment effect of the two groups were evaluated by enhanced CT regularly.Differences between conventional ultrasound and CEUS were compared and their judgement on ablation residual tumor tissue was analyzed.Results After 1 to 24 months of follow-up,the total residual rate of the experimental group and the control group was 13.3％ (4/30) and 35.9％ (14/39) respectively.The significant difference was found in the residual rate between the experimental group and the control group (x2 =5.366,P ＜ 0.05).For the two groups (＞ 5 cm and ＜3-5 cm),the residual rate of the experimental group was 30.0％ and 10.0％,the residual rate of the control group was 87.5％ and 46.2％,there were statistically significant difference (respectively x2 =5.951,4.790,all P ＜ 0.05).The significant difference was found in the recurrence rate (the experimental group:20.0％ (4/30),the experimental group:75.0％ (6/8)),when the lesion was larger than 5 cm in diameter(x2 =5.445,P ＜ 0.05).Conclusions CEUS has advantage over conventional ultrasound for the detection of residual tumor tissues after radiofrequency ablation for hepatic carcinoma,it reduces after ablation tumor recurrence especially in large tumors.

AIM:To establish a HPLC fingerprint of Stellera chamaejasme L. from Inner Mongolia Region. METHODS: The RP-HPLC method was used with Akzonobel Kromasil C_ 18 (250 mm?4.6 mm, 5 ?m) the acetonitrile-0.5% phosphoric acid (gradient elution) was used as mobile phase, analytic time was 60 min, and detective wavelength was at 297 nm, the column temperature of 15℃ were adopted. RESULTS: The HPLC fingerprint of Stellera chamaejasme L. set up showed that 14 peaks were co-possessing in different sources. The results of method validation met technical standard of fingerprint, the similarities of Stellera chamaejasme L. were 0.9 to 1.0. CONCLUSION: The method is stable and reliable with a good reproducibility and provides a reference standard for the quality control of Stellera chamaejasme L. from Inner Mongolia Region.

0.05),but it was associated with the depth of invasive cervical and lymph node metastases(P

Objective: In T_(739) inbred line mice,bladder cancer model was established via tumor source transplant.In order to get insights of the biological effects of implanting ~(125)I seed to treat bladder cancer,the development and the pathological modality of tumor tissues were observed through the experiment of implanting ~(125) I seed.The experiment was to provide theoretical gist and practical guidance for clinical(utilization) of ~(125)I seed for brachytherapy of bladder cancer. Methods: 16 T_(739) inbred line mice were divided into two groups(A:experimental group,n=8;B:control group,n=8) in random;bladder cancer model was established via tumor source transplant.When the diameter of the transplanting tumor was about 0.7 cm,~(125)I seeds were implanted at the tumor of experimental group while the empty seed was penetrated into the control group at the same position as the experimental group and the experimental mice were executed after 24 days.The longest vertical diameters(a) and(b) of tumor body were observed every 3 days and the volume of tumor body(V=ab~(2)/2) was calculated.Combining with the weight of mouse(W) and the weight of tumor body after execution of mice(w),the growth curve of tumor was protracted and the colony redouble time and tumor suppression rate were calculated.After fixation of formaldehyde,the pathological modality of tumor tissues were observed with HE staining. Results: ① The transplantation was 100% successful and there was no natural fade-away phenomenon.②After implantation of seed,the weight of group A mice increased by(4.93?0.60)g,and group B increased by(13.60?1.15)g(P

Objective To explore the protective effcets of Schisandra chinensis extract (SCE) in paraquat (PQ)-induced pulmonary fibrosis in mice ,and its intrinsic molecular mechanisms thereof. Methods A total of 108 mice were randomly allocated into six groups (n=18):control group, model group, low concentration of SCE group (200 mg/kg), medium concentration of SCE group (400 mg/kg), high concentration of SCE group (800 mg/kg) and vitamin C group (100 mg/kg). Except control group, mice were given by intragastric administration with PQ (100 mg/kg) and administered with SCE and Vitamin C once per 24 h after PQ modeling. Mice were sacrificed at 7, 14 and 21 d after modeling. Six mice were executed at different time points. The degree of lung tissue inflammation and fibrosis were observed by HE staining and Masson staining. The mRNA and protein expression levels of transforming growth (TGF)-β1, interleukin (IL)-6 and IL-17 in lung tissue were determined by RT-PCR and ELISA respectively. Results (1) Compared with control group, the lung tissue of model group showed a large number of inflammatory cell infiltration, space congestion, and its inflammation scores increased at 7 and 14 days after modeling (P<0.05). At the same time, compared with model group and vitamin C group, inflammation scores were significantly decreased in medium concentration of SCE group and high concentration of SCE group (P<0.05). (2) Compared with control group, collagen fibers and the degree of fibrosis were significantly increased in model group ,while pulmonary fibrosis were decreased in medium concentration of SCE group and high concentration of SCE group at 14 and 21 days after modeling (P<0.05). (3) With the extension of modeling time, both mRNA and protein expressions of TGF-β1 were obviously elevated, IL-6 decreased and IL-17 reduced after the first increase in PQ group. Compared with PQ group, levels of three cytokines mRNA and protein expression in medium concentration of SCE group and high concentration of SCE group changed as follows:IL-6 level was markedly decreased at 7 and 14 days after modeling;TGF-β1 level was markedly increased at 14 and 21 days after modeling. However, IL-17 level was markedly decrease at three time points(P<0.05). Conclusion SCE can relieve PQ-induced lung inflammation and fibrosis by suppressing TGF-β1, IL-6, and IL-17 expressions.

Objective To investigate the effect of sepsis on vecuronium-induced inhibition of acetylcholine release in neuromuscular junction in rats.Methods Thirty-six adult male SPF SpragueDawley rats,aged 2-3 months,weighing 200-220 g,were randomly divided into 3 groups (n=12 each) using a random number table:control group (group C),sham operation group (group S) and sepsis group (group Sep).Sepsis was induced by cecum ligation and puncture (CLP) in rats anesthetized with intraperitoneal chloral hydrate 350 mg/kg.At 12 h after CLP,the sciatic nerve-pretibial muscle was prepared.Vecuronium was added to the culture medium with the final concentration of 0.08 μg/ml,and the sciatic nerve-pretibial muscle was incubated for 15 min.Before and after administration,evoked endplate potentials (EPPs) and miniature endplate potentiais (MEPPs) were recorded by using intracellular microelectrode.EPP/MEPP ratio was calculated.Results Compared to C and S groups,EPPs,MEPPs and EPP/MEPP ratio were significantly increased before and after administration in group Sep.EPPs,MEPPs and EPP/MEPP ratio were significantly lower after administration than before administration in the three groups.Conclusion Sepsis can promote acetylcholine release in neuromuscular junction,thus weakening vecuronium-induced inhibition of acetylcholine release in neuromuscular junction in rats.

Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Membrane ; metabolism ; Cell Nucleus ; metabolism ; Cytoplasm ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Signal Transduction ; TCF Transcription Factors ; metabolism ; Transcription Factor 7-Like 2 Protein ; Wnt Proteins ; physiology ; beta Catenin ; metabolism

Objective To analyze the clinical characteristics of autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and AIH/PSC or/PBC overlap syndrome in order to further understand the autoimmune liver diseases (AILD). Methods One hundred and nine patients with AILDs confirmed pathologically were collected between Jan. 2004 and June 2006. Of 109 patients, AIH was found in 27 eases, PBC in 67 cases, PSC in 4 eases, AIH-PSC overlap syndrome in 1 case and AIH-PBC overlap syndrome in 10 cases. The clinical and laboratory data of all patients were assessed retrospectively. Results The AILD was predominantly found in middle-aged women (73.3% ,80/109), and the main clinical manifestations were jaundice, malaise, anorexia and pruritus. The age distribution of patients with AIH showed a single peak at 50 years. Elevated serum gamma globulin and IgG were found in patients with AIH, of whom 62.9% (17/27) were positive for anti-nuclear antibody (ANA) and 3 were positive for liver-kidney microsomes type 1 antibody. The main histological changes in severe AIH cases included interface hepatitis (77.7 %) and bridging necrosis. Most of the PBC patients were presented with elevated serum alkaline phosphatase, glutamyl transpeptidase and IgM. Fifty patients (74.6%) were positive for anti-mitochondrial antibody (AMA) and AMA-M2. The pathological examination showed that 28. 3% of the cases were in Ⅰ or Ⅱ stage and 71.7% in Ⅲ or Ⅳ stage in patients with PBC who received liver biopsy. The pathologic change of reduction or even disappearing of bile ducts was found in 62. 6% patients with PBC. The clinical and pathological manifestations in patients with AIH-PBC overlap syndrome had both characteristics of PBC and AIH. Three out of 10 patients with AIH-PBC overlap syndrome were positive for ANA and AMA/AMA-M2. Conclusion Since AILD is not rare in Chinese, its diagnosis should be based on the clinical presentation, biochemical, immunological and histologic changes.

Phenylalany--tRNA synthetase is a key enzyme for protein synthesis in Trypanosoma. Its validation as an inhibition. target will enable the development of a new generation of anti-Trypanosoma drugs. However, little is known about the isolation of the Trypanosoma Phenylalanyl-tRNA synthetase. Here we report the cloning, expression, purification, and activity assay of Phenylalanyl-tRNA synthetase from Trypanosoma brucei in Escherichia coli host. We co-cloned the alpha-subunit and beta-subunit of Phenylalanyl-tRNA synthetase from Trypanosoma brucei genomic DNA into the co-expression vector pCOLADuet. We successfully expressed the Trypanosoma brucei Phenylalanyl-tRNA synthetase in E. coli host, purified the whole enzyme by Ni-Hind affinity column and verified it by Western blotting. In addition, we tested its enzymatic activity by isotope labeling. The whole work laid a solid foundation for in vitro the screening and optimization of Trypanosoma brucei phenylalanyl-tRNA synthetase inhibitors.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Phenylalanine-tRNA Ligase ; biosynthesis ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Trypanosoma brucei brucei ; enzymology ; genetics