Objective To investigate the biofilm formation,the alginate biosynthetic gene ex- pression and analyze the mucA gene sequence of mucoid Pseudomonas aeruginosa PA17 and nonmu- cold Pseudomonas aeruginosa PA01.Methods The modified plate culture method was used to estab lish the biofilm model in vitro.Semi-quantitative RT-PCR was used to determine the expression level of algD in planktonic condition and during the formation of biofilm.The mucA gene of PA17 and PA01 was amplified and the products were sequenced.Results PA17 biofilm was mature at 6th day, and PA01 biofilm was mature at 3rd day.The structures of the biofilms were both like pellicle.In planktonic condition,the algD expression of PAl7 was higher than PA01;in biofilm formation,the algD expression was maximal when the biofilm was mature.There was a 166～333 deletion mutation and 342A→G in mueA gene of PA17,the mucA gene of PA01 was the same with the sequence of Genbank.Conclusions The mucA gene mutation of PA17 was a new type,which maybe the reason for the little expression difference of algD between PA17 and PA01 during the biofilm formation than it in planctonic condition and the same structure of PA17 and PA01 biofilm.

Objective To study human prostate cancer tissue specific hytk gene therapy vector using prostate specific antigen (PSA) promoter,and to determine its feasibility. Methods A prostate tissue specific hygromycin phosphotransferase thymidine kinase fusion gene(hytk)vector was established with the substitution of CMV promotor in tgCMV/hytk vector with PSA promotor.The vector was transferred into prostate carcinoma cell lines with FuGENE TM 6.Antitumor effects were observed after GCV treatment. Results PSA promoter that actively drives hytk gene was expressed only in the PSA producing prostate cell line(LNCaP).In vitro experiments demonstrated the LNCaP cells transferred by HyTK gene were killed in the presence of GCV treatment. Conclusions Hytk based vector harboring PSA promoter is a novel ideal candidate vector for prostate carcinoma gene therapy.

Objective: To study the influence of high cell incubating temperature on AMP-activated protein kinase(AMPK) activity and lipid metabolites of piglets hepatocytes in vitro.Method: Primary hepatocytes of piglets about age 55d were separated and cultured under 37 ℃(control) or 42 ℃(heat stress).The anabolic and catabolic products of [14C]-oleic acid were detected for hepatocytes and culture media at 60min,120min and 180min.There were 9 replicates per time point.Result: Heat stress activated AMPK activity and enhanced fatty acid oxidation.The production of [14C]-CO2 and [14C]-acid soluble metabolites(ASM) was higher in heat stress group than in the control.At the same time,heat stress depressed the incorporation of [14C]-oleate into phospholipids,monoglycerides,triglycerides,cholesterol and cholesteryl ester.Conclusion: Heat stress activated AMPK activity and enhanced the formation of anabolic products and depressed catabolic products in piglets hepatocytes in vitro.

Delightful achievements have been obtained in forestry genetic breeding since the application of transgenic technology in this field during the past 20 years. Field trials of some genetic modified (GM) trees have been carried out, and some GM trees have been commercialized. Meanwhile, the risks of ecological safety caused by GM trees have raised attention in the public gradually. These issues mainly include the horizontal transfer and vertical flow of foreign genes, and the potential effects on insects, soil ecosystems and virus. The current status of field trials, commercial applications and the potential ecological risks of GM trees were summarized. Then the prospects of GM trees were also presented.

AIM: To isolate peptides targeted binding and internalizing into glioblastoma cell line SWO-38. METHODS: Tumor cells were screened five rounds of whole cell screen through the Ph.D.-12 phage display library. The monoclone specific binding efficiency to the tumor cell was analyzed, and the DNA of phages were extracted, sequenced and translated to the sequences of amino acid. RESULTS: In the phage library after five rounds of screen , 10 of 13 monoclones had highly selective binding to SWO-38 cells. We found two repeated peptide sequences. CONCLUSION: Whole cell screening against tumor cells through random phage peptide library can obtain phage peptides with highly specific binding and internalizing ability. The peptides could be used as a therapy vector for tumor targeted delivery.

To explore the feasibility of stable expression of Hantavirus H8205 strain G1 segment and human IL-2 fusion gene in Vero cells, and to examine the immune protection effects on mice vaccinated with this recombinant eukaryotic expression vector containing Hantavirus G1 gene and IL-2 gene. With the help of lipofectamine, the Vero cells were transfected with pcDNA3.1/HisB-IL-2-G1 and the positive cells were selected by G418. IFAT and SDS-PAGE electrophoresis were used to determine the stable transfection and expression of recombinant protein. Each mouse was inoculated with plasmids intramuscularly (i.m.) three times, 2 boosts were given at 2-week intervals, serum anti-hantavirus antibodies were detected by ELISA and neutralizing antibodies (NAb) were detected by Plaque Reduction Neutralization Test. The fusion protein expressed in Vero cells was 78 kD, corresponding to the estimated molecular size. The neutralizing antibody titers of mice with pcDNA3.1/HisB-IL-2-G1 were 1:20-1:80. IL-2/G1 fusion gene could be transferred in Vero cells and stably express the fusion protein. Specific humeral immune responses in mice can be induced with the recombinant eukaryotic expression vector containing the fusion gene, which lays the foundation for further development of therapeutic HTNV vaccine.

N-carbamoylase is a part of hydantoinase operon which can transform N-carbamoylamino acid to corresponding ammo acids. The L-N-carbamoylase of Arthrobacter BT801, codied by the HyuC gene, is the rate-limiting and the only stereoselective enzyme. HyuC DNA fragment was amplified by PCR from the plasmid of pUC18-169. The target fragment was introduced into pPIC3. 5K plasmid to construct the pPIC3. 5K-hyuC expressing vector which was then transduced into Pichia pastoris GS115 cells after being linearized by BglⅡ digestion. Multi-copies insertion transformants were screened on G418 plates. The recombinant protein was proved to have biological activity of hydrolyzing N-carbamoylphenylalanine into phenylalanine through enzyme activity determination.

Objective To investigate the feasibility of spin-echo echo-planar imaging(SE-EPI) of magnetic resonance elastography (MRE) for the diagnosis of pancreatic mass and to evaluate the difference of stiffness value in different pancreatic mass. Methods In a retrospective study, MRE was performed in 20 healthy volunteer (control group) and 62 patients with surgery-proved pancreatic masses, including pancreatic cystadenoma in 5 cases and solid masses of pancreas in 57 cases (39 with malignant mass and 18 with benign mass) using 3.0 T MRE. The differences of stiffness between two groups were evaluated using non-parametric Mann-Whitney U test. ROC was used to assess the diagnostic ability of elastogram for the detection of pancreatic masses, and to evaluate MRE-determined stiffness for the differentiation of masses from healthy pancreas, and malignancy from benign solid masses. Results The total detection rate of elastogram was 91.9%(57/62),with false positive rate of 5.0%(1/20) and false negative rate of 8.1%(5/62). The median stiffness value in the control group vs patient group were 1.18 kPa (interquantile range:1.15 to 1.30) kPa and 2.43 kPa (interquantile range:1.94 to 3.64 kPa) respectively, with statistically significant differences (Z=-5.967,P<0.01). The sensitivity and specificity of stiffness (≥1.36 kPa) to differentiate pancreatic mass from healthy pancreas was 93.6%and 100.0%, respectively, and to differentiate benign and malignant solid masses (≥2.17 kPa) was 82.1%and 83.3%, respectively. Conclusion MRE is a promising method in detecting and distinguishing different pancreatic masses.

Objective To investigate the role of mitochondrial cytochrome c on hepatocyte apoptosis in non-alcoholic fatty liver disease in rabbits and its pathogenesis.Methods Forty Japanese white rabbits were randomly assigned to control group and model group.The model group was divided into three subgroups: 4-week, 6-week, and 8-week groups, with 10 rabbits in each group.The model groups were subcutaneously injected with peanut oil (1.2 mL/kg), twice a week for 4 weeks, 6 weeks or 8 weeks.The rabbits of all groups were killed at the right time.Serum samples were collected to detect the serum biochemical index levels.Liver tissue samples were taken for pathological observation using HE staining.The hepatocyte apoptosis index ( AI ) was measured by flow cytometry, and mitochondrial permeability transition pore ( MPTP) was evaluated by ultraviolet spectrophotometry.Immunohistochemistry was employed to detect the hepatic expressions of Bcl-2, Bax, CYT C and caspase-3.Western blot was performed to detect the changes of CYT C and caspase-3 protein expressions.Results The model groups showed hepatic injury and high level of TC, TG, CRP, IL-6 and TNF-αbeginning from 4 weeks.With the NAFLD process, the hepatocyte apoptosis index was significantly increased at 4-8 weeks and the MPTP was gradually increased.In the model group, hepatic Bcl-2, Bax, CYT C and caspase-3 expressions were increased steadily with the time passing.Conclusions NAFLD-induced liver damage is associated with apoptosis, and the mitochondrial cytochrome c-mediated apoptotic pathway plays a role in the occurrence of NAFLD.

OBJECTIVETo analyze the relationship between the pulse pressure (PP) and atherosclerotic renal artery stenosis (ARAS) in middle-age and eldery paients with hyperensio, so as to provide the basis for the diagnosis and treatment of ARAS.

METHODSWe tetro spectively analyzed the data of 257 patients with hypertension undergoing renal arteriography at General Hospital of PLA between 2009 and 2014. Their mean age was (64.09 +/- 7.81) years. According the results of arteriography, they were divided into two groups: ARAS group (n = 145), and non-ARAS group (n = 112). Their PP and multinomial clinical indexes with ARAS were statistically analyzed.

RESULTSSingle factor correlation and Logisitic analysis showed that age, PP, serum creatinine (SCr) levels, and new onset of hypertension after 50 years of age were strongly positively related to ARAS. The age and PP and SCr was a team of continuous variable.

CONCLUSIONPP was the first new risk factor of ARAS in middile-aged and elderly patients with hypertension. It is suggested that PP may be a early predictive indicator and a new therapeutic target for ARAS.

Angiography ; Arteriosclerosis ; physiopathology ; Blood Pressure ; Humans ; Hypertension ; Middle Aged ; Renal Artery ; physiopathology ; Renal Artery Obstruction ; Risk Factors