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.

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 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.

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 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.

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

The effect of vascular endothelial growth factor (VEGF) overexpression on matrix metalloproteinase-2 (MMP-2) in nasopharyngeal carcinoma (NPC) cells in vitro and the possible mechanism involved were investigated, and the correlation between the expression of VEGF and MMP-2 in NPC evaluated. The NPC cells were transfected with PAd-trackVEGF165 plasmid. The expression levels of VEGF and MMP-2 mRNA and protein in NPC cells were detected by semi-quantitative RT-PCR and Western blot respectively. It was found that the expression of VEGF and MMP-2 mRNA and protein was significantly increased in NPC cells after transfection of VEGF165. It was concluded that the expression of VEGF was correlated to the in vitro invasion of NPC cells, and the induction of MMP-2 by VEGF was a key process of NPC cell invasion.

PurposeTo investigate the role of diffusion weighted imaging (DWI) in the detection of focal liver lesions (FLL).Materials and Methods T2WI, dynamic contrast enhancement (DCE) and DWI (b=100 s/mm2 and 600 s/mm2 respectively) were performed in 205 patients with 310 FLLs. All images were read by two reviewers to determine the detection of FLLs and score the confidence. The consistency of the results given by the two reviewers was evaluated. The confidence scores between different sequences and the detection rate of different sequences were also compared.Results The consistency of the two reviewers was excellent or good in T2WI, high b value DWI, low b value DWI, and DCE (Kappa=0.71, 0.85, 0.82 and 0.64,P<0.05). As to the confidence score of lesion detection, high b value DWI and low b value DWI were both significantly higher than T2WI (P<0.01), but both were lower than DCE (P<0.01). DWI yielded higher score and detection rate for detecting FLLs than T2WI (86.5%vs. 70.3%,P<0.01), but lower than DCE (91.6%,P<0.05). For small malignant lesions (≤2 cm in diameter), DWI showed similar detection rate with DCE (P>0.05), but higher than T2WI (P<0.01). The combination of DWI and DCE detected more small malignant lesions than DWI or DCE alone (P<0.01).Conclusion DWI can detect more FLLs than T2WI, and can help DCE detect small malignant FLLs. Therefore DWI is suggested to be included in the routine protocol of liver MRI examination.