Objective To clone amastin coding genes from different strains of Leishmania parasites. Methods Using amastin cDNA sequence as the reference, dbEST data base established by National Center Biotechnology Information (NCBI), USA, was searched by BLAST tool. A 309 bp DNA fragment of Leishmania major was found and used as the probe for the screening of a DNA library. The amastin gene of Leishmania major Abdou was cloned and sequenced. Specific primers were designed and amastin genes for Leishmania mexicana WR972, Leishmania brizeliensis and Leishmania amazonensis joseph were amplified by polymerase chain reaction. Results The amastin genes from 4 strains of Leishmania parasites were cloned and sequenced. It was found that all 4 amastin genes contained unique open reading frame of 552 bp and encoded amastin protein of 183 amino acid residues. Conclusion The amastin genes of 4 strains of Leishmania parasites were successfully cloned.

Objectives To explore the effect and advantages of seperatable nylon ring ligation under endoscope for esophageal varices hemorrhage.Methods 24 cases of esophageal varices hemorrhage accepted ligation treatment under endoscope guide by using Olympus separatable nylon ring ligation apparatus. The effect was recorded. Results The average ligation points on one case were 10 2. The emergency hemostatic rate hemorrhage from esophageal varices repture was 100%. The disappearance rate of esophageal varices was 73 7%. No severe complication was occurred during operation.Conclusions Seperatable nylon ring ligation treatment under endoscope is a new safe and effective method for esophageal varices hemorrhage. This method is simple and cheap.so it has valuable for spreading use in clinic.

objective To examine nuclear transIocation of peroxisome proliferator-activated receptor γ(PPARγ)in rats following focal cerebral ischemia/reperfusion(I/R),and to explore the significance of altered PPARγ,nuclear translocation in ischemic brain injury.Methods Healthy adult male SD rats underwent 60-min cerebral artery occlusion followed by reperfusion of 4,8,or 24 h,respectively.The cytoplasmic-to-nuclear shuttling of PPARγ was characterized by Western blot,immunohistochemical and immunofluoreseence staining.The effects of PPARγ agonist rosiglitazone (Ros) and antagonist GW9662 on I/R-induced PPARγ nuclear translocation were also examined in the present study. Furthermore,TTC staining war adopted to determine the change in cerebral infarction volume. Results (1)Western blot analysis revealed an increase of PPARγ in the nucleus and a simultaneous reduction in the cytosol following ischemia and reperfusion for 4 h(tcytosol=9.03,tmuclear=27.19,P=0.00).Prolonged the reperfusion further enhanced this I/R induced PPARγ translocation in a time-dependent manner.Using immunohistochemistry and immunofluorescence,nuclear PPAR γ positive staining increased from 48.3%in the sham control to 80.3% following ischemia and reperfusion for 24 h.(2)Western blot analysis revealed that PPARγ agonist Ros further increased I/R-induced nuclear enrichment of PPARγ,whereas PPARγ antagonist GW9662inhibited I/R-stimulated change in PPARγ.(3)When compared to the L/R group using TTC staining,Ros treatment significantly decreased the infarction volume by 48.40%(15.46±4.94 versus 29.96±3.39,t=5.93.P=0.00),whereas GW9662 increased by 58.95%(47.62±4.93 versus 29.96±3.39,t=7.23,P=0.00).Conclusions Cerebral I/R injury induces PPARγ translocation from the cytosol to the nucleus.This change may represent an intrinsic neuroprotective response against brain I/R injury.

Objective To establish the standard for quality control ofQingyan Granule. Methods The chief components of the preparation, Sophora Tonkinensis radix et rhizoma, Adenophorae radix, Lonicera japonica caulis, and Ophiopogonis radix were identified by TLC qualitatively. The contents of licorice glycosides and glycyrrhizic acid were determined by HPLC. The separation was performed on Thermo Syncronis C18 column (4.6 mm×250 mm, 5μm) with mobile phase consisted of acetonitrile with 0.05% phosphoric acid solution (A)-0.05% phosphoric acid solution (B), and gradient elution (0-8 min, 19%A;8-35 min, 19%→50%A). Detection wavelength was 237 nm, and flow rate was 1 mL/min.Results The spots in TLC were clear. There were spots with same color on the corresponding location of reference substance and reference herbal, negative control without interference. The linear range for licorice glycosides was 0.05-0.5μg (r=0.999 9). The average recovery was 99.97%, RSD=1.74% (n=9). The linear range for glycyrrhizic acid was 0.1-2μg (r=0.999 9). The average recovery was 99.74%, RSD=1.28% (n=9). Conclusion The method is simple, accurate, with high reproducibility, which can be used for quality control ofQingyan Granule.

Peroxisome proliferators-activated receptor γ (PPARγ) belongs to a nuclear receptor superfamily. Many studies have shown that PPARγ can help to improve the outcome of cerebrovascular disease. PPARγ can reduce inflammatory response, oxidative stress as wel as enhance the hematoma removal abilities of microglia and macrophages, and it plays an important protective role in intracerebral hemorrhage.

Objective The aim of this study was to describe the clinical outcomes and improvement of clivo-axial angle (CAA) during the posterior approach surgery of upper cervical anomaly instability patients.Methods All of 32 patients with symptomatic upper cervical anomaly instability were followed up from 2004 Apr.to 2014 Aug.in which 1 patient was excluded because of reoperation.There were 10 patients with OS odontiodeum,5 with odontoid deformity,4 with Klipple-Feil syndrome,4 with atlantoaxial dislocation,4 with atlantoaxial instability,1 with rheumatoid arthritis,1 with old odontoid fracture,1 with OS odontoideum and foramen magnum stenosis,1 with basilarinvagination and Klipple-Feil syndrome.They were all performed posterior reduction internal fixation and fusion,and then released spinal cord compression with the promotion of CAA.The patients were divided into two groups on the basis of decompression or not:non-decompression group 17 cases (male 8 cases,female 9 cases;age 13-65 years old,average 50.1 years old) and decompression group 14 cases (male 7 cases,female 7 cases;age 19-68 years old,average 49.6 years old).The CAA numbers were measured and compared.The clinic date was compared between preoperatively and postoperatively,which included Neck Disability Index and Nurick Classification System for Myelopathy.Results All patients were followed up,the follow-up time of non-decompression group was 0.7-10.7 years,average 2.9 years;the follow-up time of compression group was 0.3-5.5 years,average 2.9 years.Analysis of CAA,NDI and Nurick score were performed with t-test.The postoperative CAA,NDI and Nurick score improved significantly compared to preoperative ones.There was no difference between non-decompression group and decompression group.One patient felt nausea and vomited,one got soleal vein thrombosis,one had a swelling face and blurring vision and one had decompression syndrome after operation,all of these were improved using respectively therapy.No such serious complications as injury to vertebral artery,infection or internal fixation loosening occurred.Conclusion Neck ability and spinal cord function was significantly improved through increasing CAA for the upper cervical anomaly instability patients underwent posterior reduction internal fixation and fusion.

Polymerase chain reaction was employed to amplify the whole HBV CP region from the serum of patients with chronic hepatitis B virus(HBV) infection, and then the PCR products were subcloned into pGEM Teasy vectors. Clones were randomly selected to be sequenced and the selected clones were compared to look for the difference.The sequencing results suggested that each sequence of selected clones was different and there were HBV quasispecies groups in patients. There were hot deletion region and point mutation near the TATA like box of CP gene. To address whether the mutations were responsible for the transcriptional activity, the wild type(wt) and the mutants of HBV CP genes were subcloned into pcDNA3 1( ) vectors, respectively. The reverse oriented clones were digested with KpnI and XhoI, and cloned into the KpnI and XhoI sites of the chloramphenicol acetyltransferase (CAT) expressing vector (pCAT3 basic).The recombinant CAT plasmids were transfected into HepG2 cells using lipofectamine PLUS reagent, and the CAT expression which indirectly represented the transcriptional activity of HBV CP lying upstream of CAT gene was detected with a CAT ELISA kit. The restriction enzyme digesting results indicated that the recombinant CAT plasmids were successfully constructed, and the transfection tests indicated that the transcriptional activity of the mutants with deletion or substitute point mutation of TATA like box were reduced in comparison with that of CPwt. The HBV CP gene heterogeneity downregulated the transcriptional activity to some extent.

Objective: To investigate whether lipopolysaccharide induced parkin expression and mitophagy in macrophages.Methods:The murine peritoneal primary macrophages were aseptically isolated from Kunming mice and cultured in complete medium.The mitochondrial membrane potential of macrophages was detected by flow cytometry,after the cells were stimulated with 200 ng/ml LPS and labeled mitochondria with JC-1.The parkin mRNA level of macrophages was detected by RT-PCR, protein levels of parkin and autophagic related protein LC3 Ⅱ and LC3 Ⅰ were determined by Western blot.The distribution and co-localization of parkin with LC3 and mitochondria in macrophages were respectively observed by laser scanning confocal microscope, before and after the cells were treated with LPS.Results: Flow cytometry results after JC-1 staining showed that mitochondrial membrane potential in macrophages was declined after stimulation with 200 ng/ml LPS, and continuously decreased with prolonged treatment time.The mRNA levels of parkin were increased slightly within 6 h after LPS stimulation,but parkin proteins were increased significantly within 6 h after LPS stimulation.The results of parkin distribution showed that parkin was evenly distributed in the cytoplasm at normal status, but became the obvious punctate distribution after LPS stimulation in macrophages.Western blot results showed LC3 Ⅱ/LC3 Ⅰ levels were increased after LPS stimulation, indicating the appearance of macrophage autophagy.Confocal microscopy showed that there were co-localization of parkin,LC3 and mitochondrial in macrophages after LPS stimulation.Conclusion:Parkin expression is increased significantly and mediated mitochondrial autophagy in macrophages after LPS stimulation, which is involved in the clearance of damaged mitochondria,thereby playing a role in regulating macrophage inflammatory response.

Objective To measure the clivus-axial angle (CAA) of Chinese,provide a normal value,and explore the correlation between CAA with cervico-medullary angle (CMA).Methods The CAA was measured on the CT of cervical spine in 225 Chinese with normal cranio-vertebral junction (CVJ).Both the CAA and the CMA were measured on the MRI of cervical spine in 117 subjects.All measurements were performed under a bone window (window level,300 Hu; window width,1 000 Hu) with an accuracy of 0.01°.A regression analysis was used for analyzing the correlation between CAA and CMA.The angle between the clivus plane and the straight line parallel to that of the posterior margin of the C2 vertebral body was defined as CAA; moreover,the angle between the strajght line parallel to the ventral side of the cervical spinal cord and the straight line parallel to that of the ventral side of the medulla oblongata was defined as CMA.Two experienced spinal surgeons performed the measurements.The CAA and CMA were measured three times,and the mean value was considered as the result.Results The CAA of 225 subjects was 133.52°-172.16° (156.93°±6.53°).The 95％ confidence interval was 145.10°-170.19° in male and 142.67°-168.47° in female.If the patients were divided into five groups according to their age,a comparison of these groups showed that there were no significant differences among the groups with respect to CAA.It showed that there was consistency between the CAAs measured with MRI and CT.The CMA for 117 patients ranged from 130.38° to 168.75° with a mean value of 154.17°±6.54°,and its 95％ confidence interval ranged from 141.35° to 166.99°.If the patients were distributed according to their gender or to their age,there were no significant differences among the groups.The relationship between CAA and CMA was linearity,and the regression coefficient was as high as 0.95 (P=0.007).Conclusion The normal CAA of Chinese is from 133.52° to 172.16°.The 95％ confidence interval goes from 145.10° to 170.19° in male,and 142.67° to 168.47° in female,close to CMA.It is valuable for measuring the compression of spinal cord,which can be accessed easily during the procedure.

Objective To investigate the prognostic factors regarding overall survival in locally advanced stage of esophageal squamous cell carcinoma (ESCC) patients after receiving concurrent chemoradiotherapy (CRT).Methods Each of 80 patients with locally advanced ESCC was treated by doxtel 135-175 mg/m2 d1 and cisplatin 40 mg/m2 d2,3 combined with radiotherapy 60 Gy.ECOG (0-1),tumor locations,tumor sizes,TNM classifications as well as the levels of CA199,SCC-Ag were detected before concurrent treatments and 4 weeks after treatment.The relationship wasanalyzed between the prognosis of disease and CA199,SCC-Ag levels.Each index was studied by Cox statistical regression analysis and P value was determined.Results All patients had completed 4 cycles' treatments successfully.The major adverse effects included neutrophilic granuloaytopenia,calvities,nausea,emesis and diarrhea.The severe effect was Ⅲ degree neutrophilic granuloaytopenia for 2 cases (2.5 ％),all the side-affect had recovered after symptomatic treatments.The median survival time were 50 months and 72.5 months in the patients who were greater than 60 year-old and less than or equal to 60 year-old,respectively (P =0.004).Regarding to 5-year overall survival,the figures were 73.3 ％,69.4 ％,41.7 ％ and 0 in the patients with cT1-2N1M0,cT3N1M0,cT4NoM0 and cT4N1M0 classification,respectively (P =0.024),they were 42.9 ％,70.0 ％,and 60.9 ％ in the patients with upper thoracic esophagus,middle thoracic esophagus,and lower thoracic esophagus,respectively (P =0.971),they were 100 ％ in the patients with ECOG 0 and 54.1％ in the patients with ECOG 1 (P =0.044),they were 86.7 ％ in the patients with CA199≤37 kU/L,and 52.0 ％ in the patients with CA199 more than 37 kU/L (P =0.008),they were 95.5 ％ in the patients with SCC-Ag≤ 1.9 μg/L,and 53.4 ％ with ＞1.9 μg/L (P =0.012).Conclusion For ESCC patients treated with CRT,the age,TNM stage,ECOG,CA199 and SCC-Ag levels are independent forecasting factors regarding their overall survival.