ABSTRACT
Purpose: The human somatropin is a single-chain polypeptide with a pivotal role in various biological processes. Although Escherichia coli is considered as a preferred host for the production of human somatropin, the high expression of this protein in E. coli results in the accumulation of protein as inclusion bodies. Periplasmic expression using signal peptides could be used to overcome the formation of inclusion bodies; still, the efficiency of each of the signal peptides in periplasmic transportation is varied and often is protein specific. The present study aimed to use in silico analysis to identify an appropriate signal peptide for the periplasmic expression of human somatropin in E. coli. Methods: A library containing 90 prokaryotic and eukaryotic signal peptides were collected from the signal peptide database, and each signal's characteristics and efficiency in connection with the target protein were analyzed by different software. The prediction of the secretory pathway and the cleavage position was determined by the signalP5 server. Physicochemical properties, including molecular weight, instability index, gravity, and aliphatic index, were investigated by ProtParam software. Results: The results of the present study showed that among all the signal peptides studied, five signal peptides ynfB, sfaS, lolA, glnH, and malE displayed high scores for periplasmic expression of human somatropin in E. coli, respectively. Conclusion: In conclusion, the results indicated that in-silico analysis could be used for the identification of suitable signal peptides for the periplasmic expression of proteins. Further laboratory studies can evaluate the accuracy of the results of in silico analysis.
ABSTRACT
BACKGROUND AND PURPOSE: Doxorubicin (DOX) as a chemotherapeutic agent has been widely used in the treatment of various types of cancer. However, DOX exerts a toxic effect on normal tissues such as the brain. Furanocoumarins reduce the risk of cardiovascular and brain diseases because of their antioxidant activities. This study has been designed, for the first time, to evaluate the effect of known furanocoumarins oxypeucedanin and isoimperatorin extracted from Prangos ferulacea (L.) Lindl on oxidative stress and apoptosis induced by DOX toward pheochromocytoma cell line (PC12). EXPERIMENTAL APPROACH: NMR and MASS spectrometers were used to characterize the isolated compounds. The protective effects of isolated compounds on DOX-induced cytotoxicity in PC12 cells were examined by MTT assay. PC12 cells were pretreated with oxypeucedanin and isoimperatorin for 2 and 21 h, respectively, subsequently exposure to DOX at IC50 concentration. Then, mitochondrial membrane potential (MMP), Bax and Bcl2 mRNA expressions, caspase-3 activation, and the generation of intracellular reactive oxygen species (ROS) were measured after 24 h. FINDINGS/RESULTS: Pretreatment with oxypeucedanin and isoimperatorin significantly decreased DOX-induced apoptosis through reduction of caspase-3 activity and ROS generation and an increase in MMP. In addition, our finding showed pretreatment with these compounds leads to regulation of Bcl-2. CONCLUSION AND IMPLICATIONS: Taken together our observation indicated that oxypeucedanin and isoimperatorin have a protective effect against apoptosis induced by DOX in PC12 cells by inhibition of ROS production.
ABSTRACT
INTRODUCTION: Rheumatoid arthritis (RA), as one of the most disabling autoimmune diseases, is a common health problem that progressively reduces the life quality of patients. Although various biologics have been introduced for RA, attempts to establish an efficient long-term therapies failed due to the heterogeneity of this disease. METHODS: In the last decade, immunomodulatory approaches such as T cell adoptive therapy have been developed for controlling autoimmunity. Regulatory T cells (Tregs), the major self-tolerance mediator, are crucial for down-regulation of aberrant immune stimulations. Hence, recruiting ex vivo Tregs emerged as a promising therapy for a variety of autoimmune diseases. RESULTS: The major bottleneck of the Treg adoptive therapy is maintaining the in vivo stability and plasticity of these fascinating cells. Recent progress in genome editing technology clustered regularly interspaced short palindromic repeats (CRISPR) in combination with CRISPR-associated (Cas) 9 system provided a new solution for this bottleneck. CONCLUSIONS: The present paper discusses RA pathogenesis and the potential application of new developments in CRISPR-mediated Treg genome editing in personalized therapy of RA.
Subject(s)
Arthritis, Rheumatoid , CRISPR-Cas Systems , Cell- and Tissue-Based Therapy/methods , T-Lymphocytes, Regulatory/immunology , Animals , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/therapy , HumansABSTRACT
Doxorubicin (DOX) is a potent, broad-spectrum chemotherapeutic drug used for treatment of several types of cancers. Despite its effectiveness, it has a wide range of toxic side effects, many of which most likely result from its inherent prooxidant activity. It has been reported that DOX has toxic effects on normal tissues, including brain tissue. In the current study, we investigated the protective effect of osthole isolated from Prangos ferulacea (L.) Lindl. on oxidative stress and apoptosis induced by DOX in PC12 as a neuronal model cell line. PC12 cells were pretreated with osthole 2 h after treatment with different concentrations of DOX. 24 h later, the cell viability, mitochondrial membrane potential (MMP), the activity of caspase-3, the expression ratio of Bax/Bcl-2, and the generation of intracellular ROS were detected. We found that pretreatment with osthole on PC12 cells significantly reduced the loss of cell viability, the activity of caspase-3, the increase in Bax/Bcl-2 ratio, and the generation of intracellular ROS induced by DOX. Moreover, pretreatment with osthole led to an increase in MMP in PC12 cells. In conclusion, our results indicated that pretreatment with nontoxic concentrations of osthole protected PC12 cells from DOX-mediated apoptosis by inhibition of ROS production.
