ABSTRACT
MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem [ES] cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma [A-172, 1321N1, U87MG] and medulloblastoma [DAOY] cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein [EGFP] or luciferase encoding genes. Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues
Subject(s)
Humans , Glioma , Serum , Cell Line , MedulloblastomaABSTRACT
The secreted aspartic proteinases [Sap2] of Candida albicans has prominent role on Candida adherence, invasion, and pathogenicity. The aim of this study was cloning, expression and characterizing of Sap2 enzyme. Also in this study for the first time, the expression system P. pasturis was used for expressing the recombinant protein. C. albicans Sap2 gene was amplified by PCR with sticky ends, EcoR1 and SacII, and it was subcloned into the T/A vector. The sequencing of this gene was done with universal primers and then the Sap2 gene was cloned into pGAPZ?A expression vector. The construct was transformed into P. pasturis yeast; the Sap2 gene integration into the yeast genome was accomplished by the homologous recombination. The expressed protein was confirmed by western blotting using monoclonal antibody against Sap2 protein. Finally, the recombinant protein was purified by Ni-NTA chromatography column, and the activity of the enzyme was confirmed. In this study, we successfully amplified C. albicans Sap2 gene and subsequently integrated into the yeast pichia pasturis genome by homologous recombination. Moreover, we were able to identify a yeast clone secreting the recombinant protein. The optimum over expression of sap2 protein was obtained after 96 h, at 30 C. Expression of Sap2 gene in P. pasturis, in comparison to bacterial expression system, leads to a high-level expression, and also need for post translation modifications, that might be required for the activity of enzyme, is obviated in the yeast system. Based on our results, the purified acid aspartyl proteinase purified from P. pasturis was capable of degrading BSA as a substrate in-vitro. The recombinant Sap2 protein had maximum activity in an acidic pH
ABSTRACT
To study the mechanisms involved in amyloid formation processes, we made a mammalian cell culture model of amylin aggregation and characterized its properties. Amyloid fibrils were extracted from CHO cells and binding affinity to Thioflavin T and Congo red was investigated. Then the apple-green birefringence of extracted fibrils was detected under polarized light to confirm the presence of aggregated protein in the extracts. To better investigate amyloid formation in CHO cells, we decided to overexpress an amyloidogenic protein in these cells. To do so, we amplified ProIAPP gene and subcloned it in EGFP-N1 expression vector. Then, CHO cells were transfected with EGFP-N1-ProIAPP and EGFP-N1 as a control. The phenotypes of around 100 transfected cells were characterized for several days after the transfection, using fluorescence microscopy. Additionally, the presence of amyloid structure in these cells was detected by Congo red staining under exposure to polarized light. Cell viability assay was performed using Trypan blue staining. Low level natural amyloid was detected in CHO cells. In addition, the ProIAPP-EGFP transfected cells exhibited aggregated phenotype in which the cells with round morphology had far more aggregates than oval ones. We found amyloid fibrils in CHO cells at low level. Overexpression of amylin protein in CHO cells caused aggregation phenotypes. These cells can be used as a model of cell culture for studying protein aggregation. Amyloidogenic properties of this protein could immensely help us to study the mechanisms that are involved in amyloid formation in mammalians including humans
Subject(s)
Cell Culture Techniques , CHO Cells , Peptides , Cell AggregationABSTRACT
Clinical application of embryonic stem[ES] cells faces difficulties regarding tissue rejection as well as ethical limitations. One solution for these issues is to reprogram somatic cells by the injection of their nucleus into an enucleated oocyte or zygote. However, technical complications and ethical considerations have impeded the therapeutic implications of this technology. An approach which is most recently developed is in vitro induction of reprogramming in adult cells. This was first achieved by using four transcription factors, including Oct4, Sox2, c-Myc and Klf4. Subsequently, many ongoing efforts were performed for enhancing this method, also for making it compatible with clinical applications. However, there is still a long road ahead. In this paper we review strategies to reprogram somatic cells to embryonic state and discuss about the recent strategy and the relevant developments