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 investigate whether CD4~+ CD25~+ regulatory T cells (Treg) from C57BL/6J mice express alpha7 nicotinic acetylcholine receptor (α7nAChR). Methods CD4~+ CD25~+ regulatory T cells were isolated from mouse splenocytes with a CD4~+ CD25~+ regulatory T Cell isolation kit (Mihenyi Bio-tee). The purity of isolated Tregs was analyzed by flow eytometry. Expressions of α7nAChR in mouse CD4~+ CD25~+ Tregs were examined by immunofluorescence staining, Western blotting, and reverse transeription-PCR, respectively. Results It was revealed by flow cytometry that Tregs could bind alpha-bungarotoxin (α-BGT)-F/TC, a specific α7 nAChR antagonist. Moreover, a positive binding to α-Bgt was also observed on the cell surface of Treg, as viewed by fluorescent confoeal microscopy. In addition, a clear band of a7nAChR with a molecular mass of approximately 55 kD was found from Tregs by Western blotting analysis, and α7nAChR mRNA was expressed with the expected size of 199 bp from Tregs by reverse transcription-PCR. Conclusion Natural CD4~+ CD25~+ Tregs from mice express α7nAChR.

0.05).The level of interleukin-8 in the sputum showed a positive correlation with neutrophil proportion(r=0.544).Conclusion Neutrophil,IL-8 in the sputum are associated with respiratory tract inflammation in the healthy smokers,smoking is an important factor in the inflammation.

Objective To explore the application and clinical effect of bipolar hemiarthroplasty with bipolar prosthesis on unstable intertrochanteric fracture in the elderly patients.Methods The study involved 53 patients aged over 75 years with intertrochanteric fracture treated with bipolar hemiarthroplasty in our hospital from April 2003 to January 2010.According to Evans-Jensen classification,there were 24 patients with type Ⅲ fractures,eight with type Ⅳ and 11 with type Ⅴ.Osteoporosis degree was at Singh degree Ⅰ in 13 patients,degreeⅡ in 18,degree Ⅲ in 12 and degree Ⅳ in 10.Most patients underwent operation within three days after injury.Results The operation duration was(75 ±10)min,with intraoperative blood loss of(350 ± 68)ml.Three patients were lost to follow-up after discharge and one patient was died of respiratory failure caused by chronic bronchitis five months after operation.Forty-nine patients were followed up for 12-93 months(mean 38 months),which showed no joint dislocation,or loosening,subsidence or rupture of the prosthesis.According to the Harris score of hip joint,the result at final follow-up was excellent in 29 patients,good in 13 and fair in seven,with excellence rate of 86％.Conclusions Bipolar hemiarthroplasty is an effective treatment for osteoporotic and unstable intertrochanteric fractures in the elderly patients.However,as the complemented therapeutic method of proximal femoral nail antirotation(PFNA),bipolar hemiarthroplasty has severe secondary trauma and its indications must be strictly mastered.

ObjectiveTo study the histologic changes of contracture Achilles tendon under Ilizarov distraction technique.MethodsEighteen dogs were installed ilizarov external fixation frames on the right hind leg in a neutral position.Pull traction started 1 week postoperatively with speed of 1 mm/day for 3 weeks.After 15 degrees of foot drop,the fixation was still maintained for 3 weeks.Nine animals were randomly selected and put to death(Contracture group),other animals corrected for 3 weeks with the same speed and methods,then put to death(Force group),health specimens as control group.(1)Observe tendon cells arranged distribution and collagen fiber form with HE dyeing,and analysis of tendon retinal thickness with image analysis software.(2)Observe collagen Ⅰ and Ⅲ with picrosirius red dying,and analyzed both content changes with image analysis software.(3)Determination of protein contents of polysaccharide with phloroglucinol spectrophotometry.Results (1)In the contracture group,the collagenous fibre gap was not obvious,the tendon cell assumes spindle-shaped,saccate,the collagen fibrous bundle distortion,the arrangement was scattered in disorder,difficult to see the intercellular substance.Tendon retinal thickness showed obvious differences with the health side (Control group).In the force group,the tendon cells were round or oval,located in collagen bundles in a string of alignment,inflammatory cells were seen.Tendon retinal thickness showed no differences with the health side.(2)With polarized light microscopy,the collagen Ⅰ and collagen Ⅲ composition proportion in the contracture group showed obvious differences with the control group,force group had not obvious differences with health side.(3)The content of polysaccharide in the contracture group were obviously reduced,while force group returned to normal level.ConclusionThe tissue form of contracture Achilles tendon can recovered to normal with the Ilizarov technology.

Objective To study the effect of the magnetic field-treated water on metabolism of monoaminergic nerve transmitters in mice. Methods Twenty healthy aged Kunming mice were divided into a control group and an experimental group randomly. The mice in the control group were given tap-water and those in the experimental group magnetic field-treated water for 30 days. The content of monoaminergic nerve transmitters in brain of mice in the two groups were determined. Results A significant difference was found in the content of monoaminergic nerve transmitters between the control and the experimental groups. The contents of NE, DA, 5-HT and 5-HIAA increased significantly(P

Objective To study the in vivo expression patterns of caspase-3 in normally aged primate brain. Methods Fresh brain tissue was taken from 4-year-old and 20-year-old female rhesus monkeys,4 for each age group. In vivo distribution of caspase-3-positive cells and its protein levels in different brain regions were investigated by immunohistochemical and immunoblot means,respectively. Results In the frontal cortex,most neurons lacked detectable caspase-3 immunoreactivity,whereas low to moderate caspase-3 immunostaining was found mainly in pyramidal cells in CA1,CA3 and CA4 subdivisions of hippocampus. And in the cerebellum,a small number of Purkinje cells were strongly stained in their cytosol and dendrites,while their nuclei lacked staining. Age-related changes in caspase-3 expression pattern were not found. Immunostaining of motor cortex in aged monkeys localized strong caspase-3 immunoreactivity to a limited number of large pyramidal cells in layer V. Immunoblotting revealed the 32 000 caspase-3 progenitor in all three brain regions. There were no differences in caspase-3 expression levels as a function of either brain region or age of animals. Conclusions These results suggest that caspase-3 is constitutively expressed in matured primate brain and there is no significant age-related change in its expression.

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

ObjectiveTo evaluate the effects caused by different doses of N-nitrosodiethylamine (DENA) on establishment of a multistep carcinogenesis model of liver cirrhosis in SD rats.MethodsFifty male SD rats were randomly divided into control group (n =5 ) and experimental group (n =45 ).Experimental group consisted of 3subgroups(high,median and low dosegroup ),which were intragastrically induced DENA once a week for 10 weeks,by using 70,50,35 mg/kg DENA solution,respectively.MRI was performed for inspecting pathological changes of rat livers in the following weeks.The mortality rates of different groups were calculated.The differences of the incidence of dysplastic nodule (DN)found in three groups were analyzed with Pearson Chi-square test.ResultsThe multistep carcinogenesis development from regenerative nodule (RN) to DN to hepatocellular carcinoma (HCC) was showed in the SD rat model.Two rats in median dose group and 4 rats in high dose group died.The incidence of DNs found in 3 different groups was 20.7％ ( 6/29 ),30.6％ ( 19/62 ),14.3％ (9/63),respectively,which showed no significant difference among three groups(x2 =4.901,P ＞ 0.05).However,significant difference existed between median and high dose group( x2 =4.811,P ＜ 0.05 ).Combined with the MRI presentation,the intragastric dose of 50 mg/kg could be more appropriate among three different doses.ConclusionsThe median dose DENA could induce an ideal multistep carcinogenesis model of HCC which is suitable for investigating DN on MRI.

Objective To establish and evaluate the CaV1?1?R528H gene knock?in mouse model of thyrotoxic hy?pokalemic periodic paralysis. Methods Thirty?six 8?week?old male CaV1?1?R528H gene knock?in mice and thirty?six 8?week?old wild?type male C57BL/6J mice were used in this study. Using three?factor two?level 2 × 2 × 2 factorial design ( the three factors including mutation, thyroxine and insulin, and two levels were with or without) , the mice were divided into 8 groups. The thyroxine groups were intraperitoneally injected with levothyroxine in a dose of 350 μg/kg once per day for 12 consecutive days to produce thyrotoxicosis. The insulin groups were intraperitoneally injected with short?acting insulin in a dose of 0?8 U/kg after the last administration of levothyroxine, and the potassium levels of different groups were meas?ured and recorded before (0 min) and after insulin injection (30 min, 60 min). Results (1) Compared with the control group, the following phenomena including irritability, dull coat, increased diet and water intake, and slow body weight gain, were observed in the thyrotoxic mice. Thyroid function tests showed that the levels of T3 and T4 in the thyrotoxic mice were significantly higher than those in the corresponding control mice (P<0?05), and the TSH level was significantly low?er than that of the corresponding control mice (P<0?05 ). (2) After administration of insulin or thyroxine alone, the po?tassium levels in the mutant and wild?type mice were not significantly different. However, after combined administration of thyroxine and insulin, the potassium levels in the mutant group were significantly lower than those in the wild?type mice at 30 min and 60 min ( P<0?05 for both). (3) The main effects and interactions:Mutation factor or thyroxine factor alone did not influence on the potassium level, only insulin showed hypokalemic effect (P<0?05). There were interactions be?tween thyroxine and mutation, and between insulin and mutation (P<0?05), but no interaction between thyroxine and in?sulin. Conclusions (1) A thyrotoxicosis state in mice is successfully developed in this study. (2) An CaV1?1?R528H gene knock?in mouse model of thyrotoxic hypokalemic periodic paralysis is successfully established.