Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis

WOS: 000314273600006Background: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate the anti-apoptotic efficacy of Bcl-xL expressing dual promoter plasmid system as a candidate to be used for recombinant protein production and DNA vaccination approaches. For this purpose, Bcl-xL anti-apoptotic protein gene was inserted in a dual expressing vector system in frame with EGFP (enhanced green fluorescence protein) after IRES (internal ribosomal site). The plasmid has a multiple cloning site after CMV (cytomegalovirus promoter) left empty to be inserted a biopharmaceutical protein gene region or DNA vaccine antigens. Results: In order to determine the anti-apoptotic efficacy of Bcl-xL inserted dual expressing vector, BHK-21 cells were transfected both with this plasmid and empty vector as control. Apoptosis was stimulated by several apoptosis inducing agents and serum deprivation in the transfected cells for 48 hrs. Cells expressing Bcl-xL protein in frame with EGFP were determined by flow cytometry as an indicator of cell viability. Additionally, apoptosis were determined by intracellular cleaved Casp 3 staining in Bcl-xL expressing EGFP positive cells. The dual expression plasmid bearing Bcl-xL anti-apoptotic protein prolonged the cell survival rate and protected cells from apoptosis upon apoptosis induction by doxorubicin and camptothecin in which the anti-apoptotic efficacies are inhibited through over expressing of Bcl-xL. pIRES2EGFP/Bcl-xL transfected cell ratio was significantly higher compared to empty vector transfected cells (P < 0.001). In contrast, apoptotic cell ratio was significantly lower in pIRES2EGFP/Bcl-xL transfected cell population compared to empty vector transfected cells (P < 0.001). Conclusion: In conclusion, it was shown that in vitro transient expression of Bcl-xL efficiently inhibited apoptosis induced by serum deprivation, doxorubicin and camptothecin. Thus, the dual expression plasmid bearing Bcl-xL anti-apoptotic protein could be a good candidate for recombinant protein production and DNA vaccination applications.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [110O809]; Ege University, Science and Technology CenterEge University [2011BIL020]This work was partly funded by Scientific and Technological Research Council of Turkey (TUBITAK) by grant number 110O809 and Ege University, Science and Technology Center (2011BIL020)

Isolation and in vitro cultivation of human urine-derived cells: an alternative stem cell source

WOS: 000410005000021PubMed ID: 28861309Objective: For in vitro tissue engineering in urology, stem cells are commonly isolated from tissue specimens obtained during open or endoscopic surgery. Within the context of the present study our aim was to isolate cells from human urine by an alternative and safe technique rather than using the indicated method. Material and methods: After human urine samples had been collected from young and healthy donors via urethral catheterization, cells were precipitated by centrifugation and cultured. Following this isolation procedure, cells were characterized by immunocytochemical method using specific antibodies. Results: When these cells were characterized by immunocytochemical methods using specific antibodies some of them were positive for mesenchymal stem cell marker CD90 while the others were labelled with urothelial marker cytokeratin 7. When all these results were taken into consideration, urothelial cells together with stem cells were observed in the urine-derived cell population. Conclusion: According to the results obtained from this study human urine may be preferred as an alternative stem cell and urothelial cell source in that this method is and easily reproducible non-invasive method.TUBITAK-BIDEB National Scholarship Programme [2211]Secil Erden Tayhan, one of the researchers of this study, was finantially supported by TUBITAK-BIDEB 2211 National Scholarship Programme for PhD Students

Comparison of the osteogenic differentiation capacity of adipose tissue derived mesenchymal stem cells from humans and rats

WOS: 000388500800015Mesenchymal stem cells (MSCs) can be found in many types of adult tissues such as the bone marrow, adipose, placenta, liver, and periosteum. Recently, adipose tissue-derived MSCs (ADMSCs) have become one the most preferred MSC types because of their fast proliferation rate, abundance, and high plasticity for variable cell types. It is known that ADMSCs are able to differentiate into various cells, including osteoblasts, so they are quite promising for orthopedic clinical trials. The present study aimed to compare the osteogenic differentiation conditions of MSCs from human adipose tissue (hADMSC) and those of MSCs from rat adipose tissue (rADMSC). Therefore, differentiation experiments with five different media and two (human and rat) ADMSC types were performed and the mineralization responses of hADMSCs and rADMSCs were different.Ege University Scientific Research ProjectEge University [11-MUH-008]; Celal Bayar University Scientific Research Project [2011-TIP-013]; TUBITAK-BIDEB 2211 National Scholarship Program; TUBITAK-BIDEB 2210 National Scholarship ProgramThis study was financially supported in part by the Ege University Scientific Research Project (11-MUH-008) and the Celal Bayar University Scientific Research Project (2011-TIP-013). Secil ERDEN TAYHAN is supported by TUBITAK-BIDEB 2211 National Scholarship Program for PhD Students. Seyma TASDEMIR is supported by TUBITAK-BIDEB 2210 National Scholarship Program for MsC Students

Production of recombinant human dipeptidyl peptidase IV from Sf9 cells in microbial fermenters

WOS: 000368559100014The human dipeptidyl peptidase IV (hDPPIV/CD26) is expressed as an immune response in some cancer cells as well as intestine and incretin metabolism, and deficiency of the enzyme leads to metabolic disorders. In the present study, recombinant hDPPIV/CD26 genes were expressed in baculovirus-insect cell systems in a 5-L stirred-tank fermenter. Because of the shear sensitivity of the insect cell line, production from insect cells should be performed in new-generation type bioreactors, which are commonly more expensive than microbial fermenters. To optimize the process, hydrodynamic parameters and oxygen consumption of Sf9 cells at 1.5 L and 3 L were monitored, and a certain amount of serum was added to the production medium to decrease shear and stabilize the growth of insect cells that normally do not need serum addition. In this study, dimensionless numbers and some hydrodynamic parameters were calculated in 1.5 L, and predictions were made for 3 L fermenter volumes. Agitation rates of 60 rpm were determined to protect insect cells against damaging shear stress. Regarding the agitation rate, oxygen mass transfer coefficient (k(L)a) was 0.0129 min(-1) for 1.5 L and was kept constant for 3 L (0.0133 min(-1)). The maximum enzyme activity from microbial fermenters was 2.37-fold higher than activity from T-flask in our previous work. The infection efficiency of transfected cells was 78%-81% in the 1.5-L and 3-L fermenters

Comparison of in vitro cytotoxicity and genotoxicity of MMA-based polymeric materials and various metallic materials

WOS: 000285565200011Aim: To determine the in vitro cytotoxicity and genotoxicity of some polymeric and metallic implant materials used as base materials in dentistry, based on ISO (International Organization for Standardization) and OECD (Organization for Economic Co-Operation and Development) test protocols. Materials and methods: Three different acrylate-based polymeric materials were tested for their in vitro cytotoxicity and genotoxicity (polymethylmethacrylate microspheres [PMMA], a solid cement prepared by mixing PMMA with its monomer methylmethacrylate [PMMA+MMA], a solid cement prepared by mixing PMMA, MMA, and hydroxyapatite [PMMA+MMA+HA], as wells as 4 different metallic materials (titanium [Ti grade 41, nickel alloy 625 [Ni-625], stainless steel alloy 304L [SS-304L], and stainless steel alloy 321 [SS-321]). Cytotoxic effects of the materials were determined using L929 mouse fibroblasts by MTT assay. Cell attachment properties related to the biocompatibility of the materials were analyzed using a scanning electron microscope (SEM). Genotoxicity of the materials was determined with human peripheral lymphocytes via micronucleus assay. Results: The highest compatibility was exhibited by Ti grade 4, followed by Ni-625, SS-304L and, SS-321. Among the polymeric materials, PMMA+MMA+HA had the highest biocompatibility, followed by PMMA+MMA and PMMA. Conclusion: The biocompatibility of the metallic materials was higher than that of the polymeric materials. Ti, the most inert metal, exhibited the highest biocompatibility. The addition of HA reduced the cytotoxic and mutagenic effects of MMA monomer and leachable ingredients.Ege University, Directorate of Administrative and Financial Affairs, Izmir, TurkeyEge University [05/MUH/40]We thank Profs. Daryush Ila and Satilmis Budak of the Center for Irradiation of Materials, Department of Physics, Alabama University, for providing the metallic materials. This study was partially funded by Ege University, Directorate of Administrative and Financial Affairs (Grant No: 05/MUH/40), Izmir, Turkey