Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris.

BACKGROUND: Proprotein convertase furin is responsible for the processing of a wide variety of precursors consisted of signal peptide, propeptide and mature peptide in mammal. Many precursors processed by furin have important physiological functions and can be recombinantly expressed in Pichia pastoris expression system for research, pharmaceutical and vaccine applications. However, it is not clear whether the furin cleavage sites between the propeptide and mature peptide can be properly processed in P. pastoris, bringing uncertainty for proper expression of the coding DNA sequences of furin precursors containing the propeptides and mature peptides. RESULTS: In this study, we evaluated the ability of P. pastoris to process furin cleavage sites and how to improve the cleavage efficiencies of furin cleavage sites in P. pastoris. The results showed that P. pastoris can process furin cleavage sites but the cleavage efficiencies are not high. Arg residue at position P1 or P4 in furin cleavage sites significantly affect cleavage efficiency in P. pastoris. Kex2 protease, but not YPS1, in P. pastoris is responsible for processing furin cleavage sites. Heterologous expression of furin or overexpression of Kex2 in P. pastoris effectively increased cleavage efficiencies of furin cleavage sites. CONCLUSIONS: Our investigation on the processing of furin cleavage sites provides important information for recombinant expression of furin precursors in P. pastoris. Furin or Kex2 overexpressing strains may be good choices for expressing precursors processed by furin in P. pastoris.

Ouabain targets the Na+/K+-ATPase α3 isoform to inhibit cancer cell proliferation and induce apoptosis.

Ouabain has been used for the treatment of heart failure and atrial fibrillation. Its potential anticancer effect has also attracted great interest. The aim of the present study was to evaluate the anticancer effect of ouabain and investigate its molecular target. The effects of ouabain on the viability of and induction of cellular death on OS-RC-2 renal cancer cells were examined using the MTT assay and acridine orange/ethidium bromide staining. The levels of Ca2+ and reactive oxygen species were determined using Fura-3-acetoxymethyl ester and dichloro-dihydro-fluorescein diacetate probes, respectively. Apoptosis was examined using annexin V-fluorescein isothiocyanate/propidium iodide staining and western blotting. The expression profile of the different Na+/K+-ATPase (NKA) isoforms in NCI-H446 small cell lung cancer cells was determined using immunocytochemistry and reverse transcription polymerase chain reaction analysis. In the present study, it was demonstrated that ouabain inhibited cancer cell proliferation and induced apoptosis while no significant difference in the expression of NKA α1 and α3 isoforms was detected following 48 h of ouabain treatment. Furthermore, expression of NKA α3 but not the α1 isoform was associated with ouabain sensitivity. The results of the present study indicated that ouabain targets the NKA α3 isoform, inhibits cancer cell proliferation and induces apoptosis.

[Exercise versus immobilization in the treatment of acute deep vein thrombosis during different clot-organized stage: an animal experiment].

OBJECTIVE: To evaluate the corresponding influence on pulmonary embolism incidence between immobilization and exercise in different stage of thrombus after acute deep vein thrombosis in rabbits. METHODS: Forty-eight New Zealand rabbits were randomly divided into three groups depending on the different organized stage of thrombus: the early, medium and later stage group.Each group was subdivided into two sub groups: the immobile and mobile subgroup. Rabbit modeling of deep vein thrombosis was made by ligating the right femoral vein. Among the early-stage group, rabbits of the immobile subgroup were fixed for 3 days, while that of the mobile subgroup were free to move for 3 days, then each was euthanized to extract the lungs for pathological examination. Among the medium-stage group, each of the immobile subgroup were fixed for 7 days, while the mobile subgroup ones were fixed for 3 days, then released free-moving for 4 days following the pathological extraction. Among the later-stage group, animals in the immobile subgroup were fixed for 14 days comparing the mobile subgroup fixed for 7 days and next free-moving for 7 days, then each was euthanized. RESULTS: Among the early-stage group, pulmonary embolism incidence (PEI) of the immobile and mobile subgroup was 4/8 vs.3/8, the pulmonary lobe embolism incidence (PLEI) was 17.5% (7/40) vs. 15.0% (6/40). Among the medium-stage group, PEI of the immobile and mobile subgroup was 3/8 vs. 2/8, PLEI was 37.5% (7/40) vs. 25.0% (10/40). Among the later-stage group, PEI of the immobile and mobile subgroup was 3/8 vs. 3/8, PLEI was 12.5% (5/40) vs. 15.0% (6/40). There was no statistical difference between immobilization subgroup and mobilization subgroup among different stage group. CONCLUSION: On the premise of given anticoagulation treatment, early ambulation do not significantly increase pulmonary embolism incidence after acute deep vein thrombosis of lower extremity in rabbits.

[A prospective randomized control clinical trial about clopidogrel combined with warfarin versus clopidogrel alone in the prevention of restenosis after femoral-popliteal artery angioplasty].

OBJECTIVE: Using two antithrombotic treatment (clopidogrel vs. clopidogrel combined warfarin) strategies after femoral-popliteal artery angioplasty prospectively, to evaluate which strategy is more effective for the restenosis prevention. METHODS: Totally 50 patients referred for endovascular treatment (including the percutaneous transluminal angioplasty (PTA) and stent implantation) of the superficial femoral artery and popliteal artery from January 2008 to May 2009 were randomly divided into clopidogrel group (group A, 25 cases, 30 limbs) and clopidogrel plus warfarin group (group B, 25 cases, 33 limbs) before operation. Clinical outcomes and restenosis rate of the target lesions were evaluated at 3, 6 and 12 months after operation. RESULTS: Totally 88 patients were screened for participation in the study, 56 patients were included after the follow-up of 12 months. At 3 months, the rates of restenosis were 16.7% in group A and 18.2% in group B (χ² = 0.025, P = 0.874). At 6 months, the accumulated restenosis rates were 36.7% in group A and 36.4% in group B (χ² = 0.001, P = 0.98). At 12 months, the accumulated restenosis rates were 53.3% in group A and 42.4% in group B (χ² = 0.75, P = 0.387). Analysis for the critical limb ischemia sub-group showed that follow-up of 12 months, the accumulated restenosis rate was 8/10 in group A and 6/12 in group B (χ² = 1.023, P = 0.312). CONCLUSION: The clopidogrel alone treatment for PTA or PTA plus stent implantation of femoral popliteal artery has no statistically significant difference in comparison with the clopidogrel combined warfarin treatment in terms of the cumulative vascular restenosis rate at 3, 6, 12 months postoperatively.

[Conditional inhibition of hemozoin formation by chloroquine in vitro].

OBJECTIVE: To study the characteristics of inhibition on hemozoin formation by chloroquine under in vitro condition. METHODS: Under different concentrations (0.5-2 mol/L) of sodium acetate (NaAc) and at the pH range of 4.0-5.0, chloroquine was tested for inhibition of beta-hematin (hemozion) formation by using the HPIA (heme polymerization inhibitory activity) assay. The morphology of beta-hematin crystals was determined by light microscopy. Ultraviolet spectrophotometry was employed to measure beta-hematin content, and the size of beta-hematin crystal was analyzed by X-ray diffraction (XRD). RESULTS: Chloroquine exhibited varied effect on beta-hematin formation, depending on pH value and Na+ concentration. When the NaAc concentration increased from 0.5 mol/L (pH 4.2) to 2 mol/L (pH 4.8), the chloroquine inhibitory effect also increased. Results suggested that there exists a threshold pH, below which the beta-hematin formation escalates and chloroquine inhibition declines, and at or above which chloroquine exerts a stronger inhibitory effect on beta-hematin formation. With the increase of pH from 4.4 to 4.8, the crystallinity and the size of crystal changed from 6.93% and 357 angstrom to 6.32% and 264 angstrom, respectively. When pH reached to 5, no more beta-hematin formed. Chloroquine could reduce the crystallinity and crystal size of beta-hematin at same pH value. Morphology analysis on the samples was consistent with the above results. CONCLUSION: Chloroquine inhibits hemozoin formation only when the pH value is at or above threshold pH.