Objective: Peroxisomes are near-ubiquitous organelles of eukaryotic cells which perform a range of functions. The common metabolic activities of peroxisomes are the oxidation of fatty acids and generation and removal of hydrogen peroxide, but peroxisomes have also been implicated in b-oxidation of aromatic and cyclic compounds, synthesis of plasmalogens, metabolism of purines and pyrimidines, and catabolism of polyamines, D-amino acids and methanol. One of the peroxisomal matrix proteins which have already been cloned termed peroxisomal protein .Amino acid alignment analysis revealed two hydrophobic domains. The First hydrophobic domain comprises twenty amino acid residues between 12-31 residues and the second one, is located at 152-169 residues. There is a tripeptide (SKI) at carboxy terminus responsible for sorting of this protein to the matrix of peroxisome. There is a fibronectin type III (FnIII) domain between residues 31-114 in pep. In order to see the importance of above sorting signal, we performed a site-directed mutagenesis to delete SKI tripeptide, FnIII domain and two hydrophobic domains.Materials and Methods: Amplified mutant PEP cDNA were constructed downstream of EGFP cDNA under regulation of CMV promoter in pEGFP-C1 vector and were send for sequenceResults: After transfection of EGFP-PeP/DSKI cDNA, cytosolic localization of EGFP fluorescency was observed. Transfection of plasmids containing chimera of EGFP-PEP/D 31-114, EGFP-PEP/D 12-31 and EGFPPEP/ D152-169 cDNAs into P19 showed several punctuate structures presumably peroxisomes as merged with the pattern of catalase staining in those cellsConclusion: Taken together, these data strongly suggest that SKI tripeptide located at the C-terminus of protein is essential for peroxisomal targeting

Objective: The P19 murine embryonal carcinoma (EC) cell line is a valuable in vitro model cell that can be differentiated into neurons by cellular aggregation in presence of the differentiating agent retinoic acid (RA).Materials and Methods: Total RNA from P19 cells was extracted and cDNA was synthesized. Using specific primers, respective cDNAs were amplified and were analyzed by semi-quantitative RT-PCR.Results: In this project, a peroxisomal gene such as catalase has been selected and the profile of its expression has been investigated in P19 cells. Expression of peroxisomal gene like Catalase, as peroxisomal matrix protein in comparison with pluripotency markers such as Oct4 and Nanog, neural markers such as Pax6, Ngn-1, Map2 and a house keeping gene such as b-tubulin have been investigated by RT-PCR.Conclusion: Data indicated that during neural differentiation, expression of pluripotency markers have been down regulated while, expression of neural markers was significantly increased. However incase of Catalase gene expression, there was an increase in Catalase gene expression upon Retinoic acid treatment, during neural differentiation, which was observed at the final steps of neurogenesis.

Background: P19 cells are mouse embryonic carcinoma cells which contain pluripotent ability, like stem cells, to differentiate into different cell lines. There are several properties for this cell line that make it a valuable cell model for study of developmental stages.Methods: At the first step, PTS2-EGFP coding sequence which was cloned in pUcD2.hygro vector was used for transfection in to P19 cells. As the plasmid contained hygromycin (Hygro1) resistance gene, stable cells were selected using hygromycin as an antibiotic. Stable transformed cells were characterized by RT-PCR and immunostaining analyses. Findings: RT-PCR results indicated EGFP was expressed in these cells. Moreover immunocytochemical analysis of the cells confirmed the preserved pluripotency states of transfected cells.Conclusion: As P19 Cells are able to differentiate into several cell lines, using this stable cell line we will able to chase the molecular kinetics of peroxisomes.

Purpose: The Present study was designed to investigate the OT effects on differentiation of P19 carcinoma cell line into cardiomyocytes.Materials and Methods: P19 carcinoma cell line were cultivated in hanging drops for 2 days to form aggregates termed embryoid bodies (EBs) and in suspension for 5 days. The EBs was treated with oxytocin hormone and DMSO. The EBs were then plated onto gelatin- coated tissue culture dishes, and monitored microscopically daily for the presence of contracting areas, as well as the distribution of the timing of onset of spontaneous beating and beating frequency per minute was evaluated for each EBs. Cells from the spontaneously contracting areas within EBs were stained with anti  a- actinin, anti-desmin, and anti-cardiac troponin I in 21- day- old EBS.Results: Spontaneously contracting areas appeared in most of the EBs of OT and DMSO groups in 7+3 and 7+5 day, respectively. Immunohistological analysis showed that the harvested cardiomyocytes were stained positively with all of and bodies and displayed many features characteristic of native cardiomyocytes. Conclusion: These results indicate that oxytocin hormone can induce differentiation of P19 carcinoma cells to cardimoyocytes, but the exact mechanism of this process is unclear. Thus, the OT-OTR system can play an important role in cardiogenesis even in vitro.

Mouse P19 embryonic carcinoma (EC) cells are pluripotent and can be differentiated into a population consisting largely of neurons and glia cells using a concentration of 5×10-7 M of retinoic acid (RA). Thus, P19 EC cells are a good model system to study events occurring during the critical phase of neuronal differentiation in vitro. Honey bee venom (BV) from consists of mellitin, phospholipase A2, apamin and several other bioactive substances. Studies have shown that mellitin and phospholipase A2, two major components of BV, play an important role in differentiation of neurons and enhance neurite outgrowth in PC12 cell line. The purpose of this experiment was to examine effects of bee venom and RA on the induction of neuronal differentiation in P19 cell line. The cells were seeded at a density of 3×104 cells/ml in a-MEM containing 2.5% FBS and 7.5% calf serum, and incubated at 37oC in humidified atmosphere containing 5% CO2. Primary results obtained from morphological examination showed that six days after treatment with 5×10-7 M RA, P19 cells produced processes and gradually obtained neuronal phenotype until day 10 of culture and then all cells died on day11. P19 cells treated with 1, 3 mg/ml bee venom produced processes on day 6 and neurons appeared. Then they exceeded to total size until day 10 and produced elongated processes but all cells died on day 11. Using bee venom and RA together had the same but more pronounced differentiating results. It can be concluded that applying bee venom with RA have an additive effect on cell differentiation and proliferation.

Objective: Peroxisomal matrix protein is mainly expressed in heart, skeletal muscle, and brain tissues. To study the expression of peroxisomal protein (PEP) during neurogenesis, we here employed P19 cells as an in vitro model of neural differentiation.Materials and Methods: Expression pattern of PEP was investigated under distinct steps of differentiation by real time PCR. Time course study of the PEP expression revealed that expression increased prominently during aggregate formation in P19 cells. The expression level of endogenous genes such as MAP-2, PEP, catalase and PEX3 as peroxisomal markers, compared withβ-tubulin as housekeeping gene were monitored during different stages of neural differentiation.Results: The results were semi-quantified and revealed the highest relative expression of PEP in cell aggregates when treated by RA. In contrast, in the absence of RA, the relative expression of PEP was at the lowest level in aggregates and gradually increased in later stages. Unlike expression of PEP and MAP2 which are stage specific dependent, the expression levels of the other two peroxisomal markers, catalase and PEX3, were not stage dependent.Conclusion: The results of this study showed that elevated level of PEP expression was dependent on RA. A further insight in PEP gene expression, quantitative real time PCR results showed significant difference in PEP expression in presence and absence of RA.

Objective: P19 cells are a line of pluripotent embryonal carcinoma stem cells able to grow continuously in serum-supplemented media and can be induced to differentiate along either mesodermal or ectodermal lineage. The differentiation of these cells can be controlled by nontoxic drugs. When treated with deprenyl, P19 cultures differentiate into cell types similar to those derived from neuroectoderm. The antiparkinsonian effect of deprenyl was reported by several investigators. On the other hand, there is a growing interest in the potential use of stem cell therapy in Parkinson’s disease. One of the major advantages to using P19 cells in the study of neuronal function and development is that these cells are amenable to genetic manipulation. Foreign DNA can be introduced into these cells using a standard method involving calcium phosphate precipitation. In this investigation, deprenyl was used to induce neuronal differentiation in undifferentiated pluripotent P19 embryonal carcinoma cells. We used calcium phosphate precipitation method to transfect these cells with pML8 plasmid, a vector encoding eGFP and puromycin resistance gene under the control of the murine Pgk-1 promoter.Materials and Methods: The cells were cultured using a-MEM medium that supplemented with 15% fetal bovine serum (FBS). In the treatment group, deprenyl was used to induce embryoid body (EB) differentiation to neuronal lineage. Initially, the viability test was used to select the range of nontoxic doses before transfection. The optimal inducing dose was obtained using different concentrations of deprenyl (10-6–10-11 M). The viability of untransfected P19 cells during the experiment was determined by the trypan blue dye exclusion method. The peak response was at 10-8 M, which was used for further investigation. Morphologic and immunofluorescence techniques were used to evaluate the differentiation of the P19 cells, Cresyl violet for the morphologic study, anti-synaptophysin and anti-betatubulin III antibodies for characterizing the neuronal phenotype of the cells.Results: The results show that deprenyl can induce neuronal phenotype associated with neuronal marker expression in successfully GFP-transfected P19 carcinoma cells. It induced the differentiation of the cells into neuron-like cells in a concentration-dependent manner.Conclusion: This study suggests the potential use of combined deprenyl and stem cell therapy to improve deficits in neurodegenerative diseases.

Objective: So far many research groups are exploring cells to replace destroyed insulin-producing cells in the diabetic patients. One attractive approach is the generation of functional b cells from stem cells.  Embryonal carcinoma (EC) stem cells are developmentaly pluripotent cells, can differentiate into all cell types under the appropriate conditions. The present study was carried out to examine the effects of mouse pancreas extract (MPE) on production of IPCs from undifferentiated P19 EC cells, using a simple nonselective inductive culture system rather than spontaneous differentiation.Materials and Methods: The cells were grown using low-attachment dishes to induce embryoid body (EB) formation. The resulted EBs cultured in a medium containing 3% fetal bovine serum, supplemented by concentration of 50, 100, 200 and 300 mg/mL MPE.Results: The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters resemble those of pancreatic islets. P19-derived IPCs were immunoreactive to proinsulin+insulin and insulin receptor beta. Furthermore, expression of the genes related to pancreatic b cell development and function such as, pancreatic and duodenal homeobox 1 (pdx-1), insulin 1 (Ins1) and insulin 2 (Ins2) was observed. When stimulated with glucose, these cells synthesized and secreted insulin in a glucose-regulated manner.Conclusion: The data presented in this study showed that it is possible to generate IPCs from undifferentiated EC cells with the characteristics of pancreatic β cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool to study pancreatic development and function.

Introduction: RNA interference (RNAi) is a phenomenon of gene silencing that uses double-stranded RNA (dsRNA), specifically inhibits gene expression by degrading mRNA efficiently. The mediators of degradation are 21- to 23-nt small interfering RNAs (siRNA). The use of siRNAs as inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells.Aim: To investigate the efficiency of siRNAs to eGFP gene silencing in P19 embryonal carcinoma (EC) stem cell.Materials & Methods: Here, we used a vector-based siRNA expression system, pSUPER that can induce RNAi in mammalian cells (P19 line of murine embryonal carcinoma stem cell). The vector containing a small hairpin RNA (shRNA) to target exogenous reporter gene, enhanced green fluorescent protein (eGFP). The expression of eGFP in the cells was detected by using fluorescent microscopy and flow cytometry.Results: Using eGFP as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector can specifically inhibit gene expression. Ttransfection of the plasmid into P19 cells significantly decreased the number of eGFP-expressing cells and overall eGFP fluorescence. The RNA interfering effect was successfully observed in both transient and stable transfected cells. Conclusion: The results indicate that use of hairpin siRNA expressing vectors for RNAi is a promising method to inhibition of gene expression in mammalian cells.