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 compare the clinical efficacy of once daily regimen with Glipizide GITS (Glucotrol XL) and multiple dose regimen with Gliclazide (Diamicron) in the treatment of Chinese patients with type 2 diabetes mellitus. Methods One hundred and fifty one Chinese type 2 diabetic patients meeting the 1985 WHO diagnostic criteria were recruited from 4 clinical centers. The patients were assigned to Glipizide GITS (78 cases) or Gliclazide (73 cases) treatment group. Results The trial consisted of 1 week washout period followed by 6 week dosage regulation period and 6 week dosage maintenance period. Comparing to the baseline, HbA 1c , fasting plasma glucose (FPG) and 24 hour glucose profile levels in two groups were significantly reduced after 3 month treatment. In Glipizide GITS group, the mean reductions of blood HbA 1c and FPG level were 0.55% and 2.05 mmol/L respectively (P

AIM:To establish a method of determining the content of chlorogenic acid,paracetmol,vitamin C and chlorphenamine maleate in Vitamin C Yinqiao Tablets. METHODS: The separation was performed in the C_(18) colunm with the mobile phase of methanol-acetonitril-0.02% phosphate acid(5∶10∶85).The flow rate was(1.0 mL/min) with the wavelength at 219 nm and 310 nm,the temperature of column was 35 ℃. RESULTS: The calibration curves were linear in the ranges of 0.03-0.13 ?g for chlorogenic acid,0.94-4.68 ?g for paracetmol,0.49-2.44 ?g for Vitamin C,0.01-0.05 ?g for chlorphenamine maleate,the average recoveries were not less than 98%,respectively. CONCLUSION: The method is simple,rapid and with satisfactory results.It is suitable for quality control of Vitamin C Yinqiao Tablets.

Objective To sum up the clinical experience of operative management of tibia plateau fracture in senile patients. Methods 35 cases of senile tibia plateau fracture were treated with different ways of internal fixation, including screw, cannulated compression screw, L shaped plate, and T shaped plate. Results All the 35 cases were followed up and the time ranged from 6 months to 2 years. X ray films showed that the anatomical reduction was reached and the fractures healed in 33 cases. The final results were rated according to Merchant criteria: excellent in 22 cases, good in 9 cases, fair in 2 cases and poor in 2 cases. The excellent and good rates were 85.7%. Conclusions Operation on senile patients with tibia plateau fracture should be performed early so as to reconstruct the articular surface. Rigid fixation and bone transplantation are impartment means to avoid plateau collapse, and early functional exercise can reduce postoperative complications.

AIM:To establish the method of determining notoginsenoside R_1,ginsenoside Rg_1 and Rb_1 in Xinning Tablet(Radix et Rhizoma Salvae Miltiorrhizae,Radix et Rhizoma notoginseng,Flos Carthami,Rhizoma Chuanxiong,ect).METHODS:HPLC-ELSD was used to determine notoginsenoside R_1,ginsenoside Rg_1 and Rb_1 in Xinning Tablet.The separatrion was performed on C_ 18 colunm with acetonitrile and water being used as a gradient program at 35 ℃.The elution program was(0-5 min,20%-25% acetonitrile;5-20 min,25%-45% acetonitrile),drift tube temperature was at 70 ℃,gas flow rate of 2.0 L/min.RESULTS:3 saponins were separated well.Average recoveries were 102.32% for notoginsenoside R_1 100.73% for ginsenoside Rg_1;101.40% for ginsenoside Rb_1,respectively.CONCLUSION:The method is simple and rapid and with satisfactory results and is suitable for quality control of Xinning Tablet.

Objective To compare the efficacy and tolerability of nateglinide with those of acarbose in Chinese type 2 diabetes mellitus (T2DM) patients.Methods This multi-center,randomized,double-blind,parallel-arm study compared the efficacy and tolerability of nateglinide( 120 mg,3/d,n = 119) and those of acarbose( 100 mg,3/d,n = 118) during a 12-week treatment in T2DM patients uncontrolled by diet with glycosylated haemoglobin (HbA1c) 6.5% - 11.0% .Results Monotherapy with nateglinide (120 mg,3/d)or acarbose (100 mg,3/d)decreased HbA1c to a similar extent during 12-week treatment.The mean change from baseline to end-point in HbAlc was ( -0.90±0.98)% and ( -0.83±0.81 )% in patients receiving nateglinide and acarbose,respectively,with no significant difference between the two groups (P>0.05).The decrease in fasting plasma glucose (FPG)was similar between nateglinide and acarbose (P > 0.05).The mean change in 2-hour postprandial plasma glucose ( PG2h ) was ( - 1.45 ± 2.74) mmol/L and ( -2.20±2.21 ) mmol/L in patients receiving nateglinide and acarbose(P =0.0017).Body weight was significantly decreased in both groups at the end-point ( P < 0.05 ),although the decrease was more with acarbese than nateglinide [( -0.66±1.79)kg vs (-2.06±2.00) kg,P=0.0000].And the proportion of patients experiencing any presumed drug related adverse events was not significantly different between the two groups.Conclusions Nateglinide ( 120 mg,3/d) is effective and well tolerated in T2DM patients uncontrolled by diet,demonstrating similar HbA1c reductions as compared with acarbose (100 mg,3/d).

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

Objective To study the effect of basic fibroblast growth factor(bFGF) on neuron-like differentiation of superparamagnetic iron oxide nanoparticles (SPIONs)-labeled amniotic membrane-derived mesenchymal stem cell.Methods Cells were cultured from enzymatic-digested amniotic membrane tissue.After that,the following steps were taken:(1) Mesenchymal stem cells derived from amniotic membrane were identified by using cell morphology,MTT method and flow cytometry.(2)SPIONs were used to label amniotic membrane-derived mesenchymal stem.(3)bFGF was imported to induce the neuron-like differentiation of SPIONs-labeled amniotic membrane-derived mesenchymal stem cell.Results (1) Primary cultures of P3,amniotic membrane-derived mesenchymal stem cell were fibroblast-like and expression of surface molecules CD29,CD44,CD90 and CD105 was detected,while expression of CD31,CD34,CD45 and CD106 was negative.(2) SPIONs of no more than 14.0 μg/ml are safe to label amniotic membrane-derived mesenchymal stem cells.Cell activity is more than 80％ and expression of surface molecules CD29,CD44,CD90 and CD105 is positive.(3)RT-PCR and immunocytochemistry analysis showed that 10.0 ng/ml bFGF induced neuron-like differentiation of amniotic membrane-derived mesenchymal stem cell (14 μg/ml SPIONs-labeled).Conclusions Enzymatic digestion and cell adherent culture method can be used to isolate mesenchymal stem cells from amniotic membrane.SPIONs of no more than 14.0 μg/ml are safe to label amniotic membrance-derived mesenchymal stem cells and have no effect on the cell activity.Neuron-like differentiation of amniotic membrane-derived mesenchymal stem cell can be induced with 10.0 ng/ml bFGF.

Objective To evaluate the clinical efficacy and safety of sibutramine (Reductile )in Chinese obese patients. Methods Obese adults (BMI 27～45 kg/m 2) in six research centers received sibutamine 10 mg or placebo one tablet each day with a controlled-energy diet for 24 weeks by randomized, double-blinded, placebo-controlled study. Results For intent-to-treat analysis, 125 sibutramine-treated subjects and 126 placebo-treated subjects were evaluated. After 24 weeks, sibutramine-treated patients lost more weight (6.52?3.95)kg than placebo-treated patients (3.18?3.59)kg(P