Objective To isolate specific humanized anti-D-dimer scFv(single chain Fv) antibody from scFv phage libraries. Methods Isolate anti-D-dimer positive clones from Tomlinson I + J phage libraries by three rounds of panuing, then sequence monoclonal genes by bideoxy-mediated chain termination and express soluble scFv antibody; Pick out anti-D-dimer antibodies with high specificity and affinity by ELISA.Results After three rounds of selection from human scFv phage libraries Tomlinson I and J, 38 monclonal specific anti-D-dimer scFv fragments were selected. By polyclonal and monoclonal phage ELISA and gene sequencing, 20 different full-length monoclonal scFv phages were identified, the result of soluble scFv ELISA showed that 20 full-length monoclonal scFv were expressed smoothly. According to the result of soluble scFv ELISA, in 5 scFv antibodies with high value of A450 selected, 3 scFv antibody fragments showed high specific and affinity. Conclusion Antibody phage display was an effective, rapid method to isolate anti-D-dimer antibodies with high specificity and affinity.

Objective To analyze the relationship between the expression of protection of telomeres 1 (POT1) and the pathogenesis of acute myeloid leukemia (AML). Methods 62 patients with de novo AML (case group) and 10 patients with iron deficiency anemia (control group) were enrolled in this study. The quantitative real-time polymerase chain reaction (PCR) and Western blot were used to detect the expression of POT1 in AML patients. Results There were 62 de novo AML patients, including 2 cases M1, 14 cases M2, 12 cases M3, 14 cases M4, 17 cases M5, 2 cases M6 and 1 case AML without classification. According to the risk stratification, high risk group (24 cases), medium risk group (22 cases) and low risk group (16 cases) were divided. Compared with that in the controls, POT1 expression levels in patients with AML were significantly decreased both in mRNA and protein level (P< 0.05). The relative expression levels of POT1 mRNA and protein in patients with M2, M4 and M5 were significantly lower than those in the controls (P< 0.05). The expression levels of POT1 in high risk group, medium risk group and low risk group were significantly decreased than those in the controls (P<0.05). Compared with that in the controls, The relative POT1 mRNA expression was significantly decreased in M3 patients (P< 0.05), but not in protein level. POT1 protein expression was showed both in the cytoplasm and nucleus. There was no significant difference of the expression of POT1 protein between cytoplasm and nucleus (P> 0.05). Conclusions POT1 may be involved in the pathogenesis of AML. POT1 protein expresses in both cytoplasm and nucleus, and the regulatory mechanism may be related to the telomere length.

Objective The aim of the experiment is to make an intracranial aneurysm model in canine.Methods A digital tube was made based on raw magnetic resonance images of the human intracraaial carotid artery.Then 6 tubes were made in the 3 D rapid prototyping machine and coated with silicone.Finally we isolated the common carotid arteries of 6 canines and made them go through the tubes and anastomosed them end-to-side to get the aneurysm model.Six stents were implanted after one week.Results Six aneurysm models were successfully made in canines.The parent artery had similar geometrv of the human carotid siphon.All the aneurysms and parent arteries were patent in one week's follow-up.One canine died of excessive anesthesia after stentingr Two vaseular models kept patent in one month without stenosis.The other 3 had some stenosis on the bends of the vessel.Conclusions The aneurysm model in tIle experiment has high flexibility and reliability.The model provides an effective tool for research and testing neurovascular devices.It's also a useful device to train the neuroradiologists and interventional physicians.

The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.

In order to improve the accuracy for quantitating the bovine foamy virus(BFV)in vitro,we developed a baby hamster kidney cell(BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter(LTR,from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection(MOI)of BFV and the activated ratio of luciferase expression in BFVL. Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.

Objective To study the anticoagulation therapy of lower molecular weight heparin in the treatment of patients with acute pancreatitis.Methods Seventy-three patients with acute pancreatitis were divided into anticoagulation group(n=38)and control group(n=35).The serological indexes and prognosis of patients were detected.Results Anticoagulation treatment with lower molecular weight heparin significantly decreased the white blood cell count,increased the oxygen partial pressure in arterial blood,shoaened the duration of hospitalization,and reduced the aggravation rate,secondary operation rate and mortality of patients with acute pancreatitis.Conclusions Anticoagulation treatment with lower molecular weight heparin is safe,effective and can improve the prognosis of patients with acute pancreatitis.

Objective Survivin is overexpressed in gastric cancer. However it not expressed in normal gastric mucosa. The expression of survivin is tightly related to the prognosis of gastric cancer.By gene reconstruction we generated pcDNA3 survivin mutant(Cys84Ala) plasmid, and observed its effect on the gastric carcinoma cell lines. Methods The survivin mRNA and protein expression levels were determined by reverse transcription polymerase chain reaction(RT PCR) analysis,Western blot and immunohistochemical staining respectively . Flowcytometry and acridine orange staning were employed to detect apoptosis. Results Overexpression of survivin mRNA and protein were detected in the gastric cancer cell lines. Inhibition of survivin by survivin mutant cDNA induced apoptosis,activated caspase 3 activity,cleaved PARP and promoted cytochrome C releasing in gastric cancer cells,and effectively sensitized gastric cancer cells to chemotherapeutic agents. Conclusion Inhibition of survivin may induce apoptosis in gastic cancer and sensitize gastric cancer cells to chemotherapeutic agents.Survivin targeted therapeutic protocol may potentially benefit gastric cancer therapy.

Objective To investigate the possible signal pathway of the apoptosis in gastric cancer cell lines(SGC 7901 and MKN 45)induced by arsenic trioxide. Methods TUNEL method was used to observe the influence of calcium antagonist, inhibitors of protein kinase C (PKC) and protein tyrosine kinase(PTK) on the apoptosis of gastric cancer cells induced by arsenic trioxide. Levels of cAMP, PKC and PTK were detected before and after the treatment with arsenic trioxide. Results Both PKC and PTK inhibitors could induce apoptosis of gastric cancer cell lines, also both of them had a cooperative action with arsenic trioxide in inducing apoptosis of gastric cancer cells, while calcium antagonist had no any effect on the apoptosis of gastric cancer cell lines. PKC and PTK levels decreased but cAMP level increased during the apoptosis of gastric cancer cells induced by arsenic trioxide ( P

Objective To investigate the clinical value for treatment of diabetic foot with PTA and PTA combined cinepazide maleate.Methods In 24 cases of diabetic associated vascular disease of lower limb,12 cases were treated with PTA and other 12 cases were treated with PTA combined einepazide maleate,We analysed and compared clinical effects before and after the procedure,together with 3 months follow up.Results In patients treated with PTA,the clinical symptom scores of posttreatment and follow-up decreased;ABI and TcPO_2 increased significantly.The clinical symptom score and ABI of follow-up remained,stable,but TcPO_2 decreased significantly.Control angiography showed improvement in degree of vascular stenosis and peripheral staining of 11 patients after treatment.The vascular patency remained in 12 patients and the peripheral staining decreased in 7 patients on follow-up.In patients treated with VIA combined cinepazide maleate,the clinical symptom score,ABI and TcPO_2 after treatment and on follow-up showed no signifcant changes compared with those in patients treated by PTA.F,Control angiography showed that the degree of vascular stenosis and peripheral staining were improved in 12 patients after treatment.The vascular pateney was maintained and peripheral staining was improved on follow-up.Before and after treatment,there were no significant differences in clinical symptom score.ABI and TcPO_2 between patients treated with PTA and PTA combined cinepazide maleate,however,there were significant differences in clinical symptom score and TcPO_2 on follow-up.Conclusion PTA can significantly improve clinical symptom of diabetic foot and the application of cinepazide maleate is a benefitial and necessary supplement.PTA combined cinepazide maleate can be taken as one of the conventional treatment plans for diabetic foot.(J Intervent Radiol,2007,16:811-815)

Bel1, a transactivator of prototype foamy virus (PFV), plays pivotal roles in the replication of PFV. Previous studies have shown that Bel1 bears a nuclear localization signal (NLS), but its amino acid sequence remains unclear and the corresponding importins have not been identified. In this report, we inserted various fragments of Bel1 into an EGFP-GST fusion protein and investigated their subcellular localization by fluorescence microscopy. We found that the 215PRQKRPR221 fragment could direct nuclear localization, which accords with the consensus sequence K(K/R)X(K/R) of monopartite NLS. Point mutation experiments revealed that K218, R219, and R221 are essential for the nuclear localization of Bel1. The results of the GST-pulldown showed that the Bel1 fragment with residues 215-223, which bears the NLS, interacts with KPNA1, KPNA6, and KPNA7. This result suggests that KPNA1, KPNA6, and KPNA7 maybe involved in Bel1 nuclear translocation.
Cell Line ; Cell Nucleus ; genetics ; metabolism ; virology ; Humans ; Nuclear Localization Signals ; genetics ; metabolism ; Protein Binding ; Protein Transport ; Retroviridae Infections ; genetics ; metabolism ; virology ; Retroviridae Proteins ; chemistry ; genetics ; metabolism ; Spumavirus ; chemistry ; genetics ; physiology ; Trans-Activators ; chemistry ; genetics ; metabolism ; alpha Karyopherins ; genetics ; metabolism