Objective To obtain the sequence of recombinant human basic fibroblast growth factor (rhbFGF) with endonuclease sites of BamHI and Pst Ⅰ by PCR based gene assembly and construct the recombining vector of rhbFGF gene by TA cloning technique.Methods The rhbFGF gene sequence which was designed for lactococcus with endonuclease sites of BamHI and Pst Ⅰ was divided into 22 oligonucleotides by DNASTAR 6.0 (bFGFl-bFGF22).The 22 oligonucleotides were spliced by PCR based gene assembly to get the rhbFGF eDNA with endonuclease sites of BamHI and Pst Ⅰ.The PCR product was inserted into the PMD18-T VECTOR.The recombining vector were converted to the competent E.coli TOP10.The clones generated from LAB were analyzed by miniprep isolation from LAB host.They were identified by the restriction enzyme cuuing and sequencing.Results The rhbFGFcDNA synthesized by PCR based gene assembly with endonuclease sites of BamHI and Pst Ⅰ was verified by 2％ agarose electrophoresis.The recombining vector of rhbFGF gene by TA cloning technique was identified by enzyme digestion and gene sequencing.Conclusions The TA cloning vector of recombining hbFGF with endonuclease sites of BamHI and Pst Ⅰ was constructed successfully.

Objective To analyze the correlation between serum antinucleosome antibody (AnuA) and renal pathological characteristic,disease activity as well as some laboratory tests in patients with lupus nephritis (LN).Methods Serum AnuA levels were detected by enzyme-linked immunosorbant assay in 40 patients with LN (observation group) and 40 healthy people (control group).Renal biopsy was examined in all LN patients.The relationships between serum AnuA level and systemic lupus erythematosus disease activity index (SLEDAI),renal pathohistology,laboratory parameters were analyzed.Results The serum AnuA level in observation group before treatment was significantly higher than that in control group [ ( 110.23 ± 80.48) kU/L vs. ( 10.45 ± 8.20) kU/L,P ＜ 0.05 ].Four cases of renal biopsies were class Ⅱ,8 cases were class m,23 cases were class Ⅳ,and 5 cases were class V.Serum AnuA level had significant difference between each class by Kruskal-Wallis rank sum test (P ＜ 0.05),and serum AnuA level of class Ⅳ was the highest (P ＜ 0.05).Serum AnuA level had positive correlation with SLEDAI,urine protein quantitation and anti-double strands DNA antibody (r =0.462,0.521,0.394,P ＜0.05),negative correlation with complement C3 and C4 levels (r =-0.403,-0.489,P ＜ 0.05 ).Serum AnuA level after treatment [ (32.45 ± 18.31) kU/L] was significantly decreased than that before treatment [(110.23 ± 80.48) kU/L](P＜0.05).Conclusions Serum AnuA level is not only a good index of LN activity,but also reflect renal involvement.That serial measurement of serum AnuA level may provide better clinical strategies for the therapy.

The present study was designed to investigate the effect of gossypol on the reabsorption and excretion of ~(42)K in rats and guinea pigs by using autoradiographic technique, and selected the well known tubulo-toxic agent, gentamicin as a positive control for a comparative study to evaluate whether gossypol exerts nephrotoxic effect. Our results confirmed that gentamicin could induce significant decrease or inhibit ~(42)K reabsorption and cause structural damage of renal tubules. Gossypol could also affect the reabsorption function of proximal tubule, but did not appear to act as a tubulotoxic agent comparable with gentamicin to cause injury of the renal tubules.

Objective To investigate the expressions of CD133 and p53 in colorectal cancer and their clin-ical significances. Methods The expressions of CD133 and p53 in 74 colorectal cancer patients were detected by the immunohistochemistry method. The relationships of CD133 and p53 with the clinicopathological parameters and prognosis were analyzed. Results The positive expression rates of CD133 and p53 in colorectal cancer tissues were 33.8%and 55.4%,respectively. The expression levels of CD133 and p53 were not related to age,sex,tumor location and histological type ,and but were significantly related to the histological differentiation ,TNM stage and distant metastasis(P<0.05,respectively). The Spearman correlation analysis showed that there was no correlation between the levels of CD133 and p53. Conclusions The high expressions of CD133 and p53 in colorectal cancer tissues were closely related to the histological differentiation and TNM stage. CD133 and p53 could be used as important biomarkers for the evaluation of malignant biological behavior,and the diagnosis and prognosis of colorectal cancer.

Objective To determine the cellular localization and expression pattern of AR and FSHR in adult rat testis must be helpful to understand the action site and mechanisms that T and FSH regulate spermatogenesis. Methods We applied in situ Hybridization to detect the expression of AR and FSHR on adult testis, in which Dig labeled cRNA probe was used to carry out the experiment on frozen sections; at the same time, following the technique of transillumination assisted microdissection we separated seminiferous epithelium into four stages(Ⅱ Ⅵ,Ⅶ Ⅷ,Ⅸ Ⅻ and ⅩⅢ Ⅰ), extracted total RNA and carried out dot hybridization, using ? 32 P labeled cDNA probe, in order to test qualitatively and quantitatively the location of AR and FSHR mRNA and their expression pattern in adult rat testis. Results Our results showed that the positive signal of AR mRNA was located in Sertoli cells and Leydig cells. The signal in Sertoli cells began to appear in Ⅱ Ⅵ stages, strongest in Ⅶ Ⅷ stages and weakest in Ⅸ Ⅰ stages ( P

Objective To investigate the efficacy and tolerability of dose-dense chemotherapy with cisplatin plus 5-fluorouracil( PF regimen)in distant metastatic nasopharyngeal carcinoma( NPC)patients. Methods From April 1,2008 to April 30,2014,168 patients were assigned to traditional group(n = 83) and dose-dense group(n = 85)using digital random table in 1 : 1 ratio. All patients received PF regimen,and once every 28 days in traditional group and once every 14 days in dose-dense group. The primary endpoint was progression-free survival(PFS)and the secondary endpoint was overall survival(OS),toxicity and response rate. Results The median PFS,median OS,1,2,3-year survival rate,complete response rate and objective response rate were significantly improved in dose-dense group which were 13. 3 months,20. 2 months,80. 2% , 36. 0% ,16. 1% ,16. 5% ,84. 7% ,and those in control group were 10. 0 months,16. 1 months,59. 6% , 10. 1% ,0,3. 6% ,54. 2% respectively(χ2 = 24. 47,P = 0. 000;χ2 = 16. 65,P = 0. 000;χ2 = 8. 41,P =0. 004;χ2 = 16. 96,P = 0. 000;χ2 = 14. 91,P = 0. 000;χ2 = 7. 63,P = 0. 006;χ2 = 18. 47,P = 0. 000). The rates of grade 3-4 adverse events in dose-dense and traditional group were 38. 8% and 6. 0%(χ2 = 25. 81, P = 0. 000). Conclusion Dose-dense chemotherapy of PF is more efficient and acceptable toxic than the tra-ditional one. It can be a new treatment option for patients with distant metastases of NPC.

Escherichia coli AFP111 is a spontaneous mutant with mutations in the glucose specific phosphotransferase system (ptsG) in NZN111 (delta pflAB deltaldhA). In AFP111, conversion of xylose to succinic acid generates 1.67 molecule of ATP per xylose. However, the strain needs 2.67 molecule ATP for xylose metabolism. Therefore, AFP111 cannot use xylose due to insufficient ATP under anaerobic condition. Through an atmospheric and room temperature plasma (ARTP) jet, we got a mutant strain named DC111 that could use xylose under anaerobic condition in M9 medium to produce succinic acid. After 72 h, DC111 consumed 10.52 g/L xylose to produce 6.46 g/L succinic acid, and the yield was 0.78 mol/mol. Furthermore, the reaction catalyzed by the ATP-generating PEP-carboxykinase (PCK) was enhanced. The specific activity of PCK was 19.33-fold higher in DC111 than that in AFP111, which made the strain have enough ATP to converse xylose to succinic acid.
Atmosphere ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Industrial Microbiology ; Metabolic Engineering ; Mutation ; Plasma Gases ; pharmacology ; Succinic Acid ; metabolism ; Temperature ; Xylose ; metabolism

During the anaerobic fermentation by Escherichia coli AFP111 for succinic acid production, the viable cell concentration and productivity were decreased with the raising of succinic acid concentration. In order to restore cellular succinic acid productivity and prolong fermentation time, we collected strains and refreshed medium for repetitive succinic acid production. However, productivity is lower than that in the anaerobic fermentation before reusing strains. To enhance the productivity, strains were aerobically cultivated for 3 h in pure water before anaerobic fermentation. The activities of key enzymes were enhanced for better performance in producing succinic acid at anaerobic stage. After three rounds of repetitive fermentations, succinic acid concentration and yield reached to 56.50 g/L and 90% respectively. The succinic acid productivity was 0.81 g/(L x h), which was 13% higher than the repetitive fermentations without aerobic activation of the strains.
Aerobiosis ; Anaerobiosis ; Culture Media ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; Glucose ; metabolism ; Industrial Microbiology ; Succinic Acid ; metabolism

Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate was accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. Results in 3 L fermentor showed that OD600 is 4.64 and BA016 consumed 35.00 g/L glucose and produced 25.09 g/L succinate after 112 h under anaerobic conditions. Overexpression of pncB and pyc in BA016, the accumulation of pyruvic acid was further decreased, and the formation of succinic acid was further increased.
Anaerobiosis ; Escherichia coli ; enzymology ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; Glucose ; metabolism ; Industrial Microbiology ; Lactococcus lactis ; enzymology ; NAD ; metabolism ; Pentosyltransferases ; biosynthesis ; genetics ; Pyruvate Carboxylase ; biosynthesis ; genetics ; Succinic Acid ; metabolism

Objective:To explore the feasibility and stability of using oleic acid to establish acute respiratory distress syndrome(ARDS) model in Beagle dogs.Methods:A total of 25 Beagle dogs were injected oleic acid with a speed of 0.25?0.03ml/Kg through jugular vein after being anesthetized.The gas ventilation index,oxygen metabolism,histopathological changes,chest-ray scan and stability of each index after establishing the ARDS situation were continuously monitored and evaluated.Results:All 25 dogs were reached the diagnosis criteria of ARDS in 4.16?0.92 hours after injection of oleic acid,The arterial PaCO2 of the animals was 27.98?8.25mmHg,PaO2 was 65.40?11.48mmHg,and PaO2/FiO2 was 182.3?29.6.After mechanical ventilation treatment,PaO2/FiO2≤200.Pulmonary mesenchyme,and alveolus edema,bleeding,shrinkage and transparent formation were seen under microscope.There was darkness or coarse shadow in the lung by the X-ray scan.Conclusions:Through injection of oleic acid via jugular vein,a Beagle dog ARDS model may be quickly established with stable changes of hemodynamics,pulmonary mechanics and histology.