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ABSTRACT: Recombinant tissue-type plasminogen activator (rt-PA) has been produced in different hosts. In this research, transgenic tobacco was selected for production of human rt-PA. Transgenic plants were analyzed by polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR. The protein was extracted by Lysine Sepharose chromatography column and was further purified by HiTrap desalting column. The function of eluted protein was analyzed on zymography gel. The results showed that the 1.7-kb cDNA of tissue-type plasminogen activator (t-PA) (as well as a shortened 650-bp transcript of t-PA) has been expressed in transgenic plants. The anticipated 63-kD protein band and an additional 53-kD protein were observed in transgenic plants. Finally, zymography assay revealed that the purified rt-PA has anticipated appropriate activity comparable to a positive control drug (Alteplase). On the whole, we can say that transgenic tobacco is a good alternative host for production of t-PA.
Preparative Biochemistry & Biotechnology 04/2011; 41(2):175-86. · 0.47 Impact Factor
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ABSTRACT: Bone marrow stromal stem cells (BMSC) express two neurotrophins nerve growth factor (NGF) and brain derived growth factor (BDNF) constitutively and can be differentiated into neuronal-like cells and used to treat neural injuries and diseases. The neurotrophins are required for repair of neural tissues. However, it is not evident whether these cells supply the sufficient amounts of the functional growth factors following neuronal differentiation. This study investigates the expression of NGF, BDNF and their processing enzymes Prohormone convertases (PC) Furin, PC5 and PC6 by Real-time RT-PCR during neural differentiation of rat BMSC. The results showed that all inspected processing enzymes are expressed in the cells. The expression of NGF, BDNF and PC5 decreases following differentiation. In addition, BMSCs express Survivin, an anti-apoptotic gene; however, the differentiated cells reduce its expression similar to two neurotrophins, which could make them susceptible to apoptotic death.
Brain Research 05/2008; 1203:26-31. · 2.73 Impact Factor
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ABSTRACT: Neural-like cells derived from bone marrow stromal stem cells (BMSCs) have potential usefulness in cell therapy of degenerative or traumatic diseases of the central nervous system (CNS). The functional recovery mediated by these cells, however, depends on the secretion of neurotrophins (NTs) and their cognate receptors, as the main regulators of neural survival and death. The function of NTs is further modulated by proprotein convertase (PC) enzymes which function in converting proproteins (including proNTs) into their functional end products. Accordingly, failure in converting proprotein forms of NTs into their mature forms may lead to neuronal cell death. In the present study, we have investigated the expression profile of PCs before and during neural differentiation of rat BMSCs by RT-PCR. Our results show that major members of the PC family functioning in the constitutive secretory pathway (furin, PACE4 and PC7/LPC) are highly expressed in both undifferentiated and neurally differentiated BMSCs. In contrast, while PC1/PC3 and PC2 (specific to neural and endocrine cells) are absent in undifferentiated BMSCs, their expression is initiated upon the induction of differentiation. In conclusion, our results suggest that neurally differentiated BMSCs have acquired the functional machinery to process the precursor forms of proteins in both the constitutive and regulated pathways.
Neuroscience Letters 07/2007; 420(3):198-203. · 2.11 Impact Factor
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ABSTRACT: Bone marrow stromal cells (BMSCs) are pluripotent stem cells with self-renewal property and potential to differentiate into a variety of cell types. Identification of the genes responsible for coordination of these processes and elucidation of the mechanisms underlying these events are of fundamental importance. Nucleostemin, a novel p53 binding protein localized in the nucleoli of ESCs, is not expressed in the differentiated cells of adult tissue, suggesting a role in maintaining stem cell self-renewal. In the present study, we have evaluated the expression profile of nucleostemin in rat BMSCs before and after the induction of neural differentiation by RT-PCR and immunocytochemistry. The profile of nucleostemin expression is then compared to the key regulators of proliferation/differentiation such as: Oct-4, Nanog, Neuro D, and Cyclin D1. Our data reveal that there is no detectable expression of Oct-4 and Nanog in either non-differentiated or neurally differentiated BMSCs. In contrast, the expression of nucleostemin is relatively high in non-differentiated BMSCs, then sharply diminishes upon induction of differentiation and finally completely vanishes by 6h after initiation of differentiation. The disappearance of nucleostemin expression coincides with the appearance of Neurofilament-M and -H, MAP2, synaptophysin- and neuron-specific enolase as revealed by RT-PCR and/or immunocytochemistry. Expression of Neuro D and Cyclin D1 also diminish as differentiation proceeds but at much slower rates as compared to nucleostemin. In conclusion, our results suggest that nucleostemin, but not Oct-4 or Nanog, is expressed in BMSCs and it possibly regulates self-renewal proliferation in BMSCs.
Neuroscience Letters 01/2006; 390(2):81-6. · 2.11 Impact Factor
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ABSTRACT: Neural-like cells derived from bone marrow stromal cells (BMSC) have potential usefulness in repair of the CNS injuries or diseases. The functional recovery mediated by these cells, however, depends on secretion of specific growth factors and their designated receptors. In the present study, we have investigated the expression profile of neurotrophins NGF, BDNF and NT-3 and their high-affinity (TrkA, TrkB, TrkC) and common low-affinity (p75NTR) receptors before and during neural differentiation of rat BMSCs by RT-PCR. Results indicate that NGF and BDNF but not NT-3 are expressed in both un-differentiated as well as neurally differentiated BMSCs. In contrast, the expression of TrkA and TrkB is restricted to neurally differentiated cells, while TrkC is not expressed in these cells either before or after differentiation. Interestingly, p75NTR expression is absent in un-differentiated cells but is initiated upon the induction of neural differentiation, and then shut off in fully differentiated neuron-like cells.
Neuroscience Letters 397(1-2):149-54. · 2.11 Impact Factor
