Subcloning and expression of human alpha-fetoprotein gene in Pichia pastoris

Alpha-fetoprotein protein (AFP) is naturally found in fetal serum while production and appearance after birth is indicative of presence of malignant tumors. Therefore, by measuring this protein during fetal period and after birth, it is possible to diagnose abnormalities and tumors in fetus or the newborn. The objective of this study was to produce and purify AFP protein using DNA recombinant technology to apply in diagnostic kit preparations. In this study Pichia pastoris as metylotrophic yeast was used for AFP production. After construction of recombinant plasmid, pS1-AFP, electroporation and lithium chloride techniques were used for transferring to susceptible cells. The quantity and quality of the produced protein were checked by SDS-PAGE and ELISA methods. Selection of transformed mutant strains of Muts and culturing them in glycerol media (YPG) up to OD600=6 and their transfer to methanol media (YPM) with augmentation of methanol to 1% final concentration resulted in inducing protein production in auxotrophic media lacking histidine. This protein could be useful in monoclonal antibody production and in diagnostic kit preparations.Keywords: Alpha-fetoprotein, Pichia pastoris, cloning, expressio

Bronchodilatory and B-adrenergic effects of methanolic and aqueous extracts of Althaea root on isolated tracheobronchial smooth rat muscle

Background: The smooth muscle contractions of the tracheobronchial airways are mediated through the balance of adrenergic, cholinergic and peptidergic nervous mechanisms. This research was designed to determine the bronchodilatory and B-adrenergic effects of methanolic and aqueous extracts of root Althaea on the isolated tracheobronchial smooth muscle of the rat. Materials and Methods: In this experimental study, 116 tracheobronchial sections (5 mm) from 58 healthy male Sprague-Dawley rats were dissected and divided into 23 groups. The effect of methanolic and aqueous extracts of the root Althaea was assayed at different concentrations (0.2, 0.6, 2.6, 6.6, 14.6 mg/ml) and epinephrine (5 mm) in the presence and absence of propranolol (1 mM) under one g tension based on the isometric method. This assay was recorded in an organ bath containing Krebs-Henseleit solution for tracheobronchial smooth muscle contractions using potassium chloride (KCl) (60 mM) induction. Results: Epinephrine (5 mm) alone and root methanolic and aqueous extract concentrations (0.6-14.6 mg/ml) reduced tracheobronchial smooth muscle contractions induced using KCl (60 mM) in a dose dependent manner. Propranolol inhibited the antispasmodic effect of epinephrine on tracheobronchial smooth muscle contractions, but could not reduce the antispasmodic effect of the root extract concentrations. Conclusion: The methanolic and aqueous extracts of Althaea root inhibited the tracheobronchial smooth muscle contractions of rats in a dose dependent manner, but B-adrenergic receptors do not appear to engage in this process. Understanding the mechanism of this process can be useful in the treatment of pulmonary obstructive diseases like asthma

Evaluation of Intranasal Delivery of Human Endometrial Stem Cells to the Substantia Nigra and Their Therapeutic Effects on Rotational Behavior Recovery in Mice Model of Parkinson's Disease

Background and Aim: Recent findings in cell therapy have presented new perspectives and opportunities for the treatment of neurodegenerative disease. The experimental research with intranasal (IN) administration of Stem Cells in Parkinson’s disease (PD) mouse model can work and in some cases induce major, long-lasting improvement. Adult Human endometrial derived stem cells (HEDSCs), a readily obtainable type of mesenchymal stem-like cell were used to generate dopaminergic cells and for cell therapy. In this study, we investigated the therapeutic effects of IN-delivered HEDSCs in mice model of Parkinson. Materials and Methods: In this experimental research, 35 male mouse weighting 25-30 g were divided into 5 groups. On day 120 post cell administration, the rotational behavior was measured. Immunohistochemistry staining was used to detect HEDSCs in mice brain. Findings: IN application of HEDSCs resulted in the appearance of cells in the substantia nigra (SN) and decrease in the rotational behavior of case group. Conclusion: HEDSCs are a highly inducible source of allogenic stem cells that improve Parkinson’s disease

Study of the variations in apoptotic factors in hippocampus of male rats with posttraumatic stress disorder

Background: Post-traumatic stress disorder (PTSD) is a stress-related psychosomatic disorder caused by occurrence of a traumatic event and the hippocampus volume of the patients with Post-traumatic stress disorder decreased. However, the mechanisms that cause such damage are not well-understood. The aim of this study is to detect the expression of apoptosis-related Bax, Bcl-2, Caspase-3 and Insulin-like growth Factor-I proteins in the hippocampus region in the Predatory stress rats. Materials and Methods: A total of 70 male wistar rats were divided into Predatory stress groups of 1d, 2d, 3d, 7d, 14d, 30d and a normal control group (N = 10). Rats were subjected to 5 min of predatory stress and then exposed to the elevated plus-maze (EPM). Serum corticosterone and Insulin-like growth factor-1 level of Hippocampus were measured by ELISA technique. The expression of Bax, Bcl-2, and Caspase-3 were detected by western blotting. Results: Rats spent significantly more time in closed arms of the elevated plus maze (EPM) than control group after exposure to stress. Serum levels of corticosterone significantly increased at 2d-3d. The expression of hippocampal IGF-1 was significantly up-regulated at 1d-2d after stress. Both Bax and the ratio of Bax/Bcl-2 significantly peaked at Predatory stress 2d-14d. Caspase3 was significantly active among 2d-30 compared to the normal control. Conclusion: The activation of caspase-3 in the stress groups indicates that apoptosis may be one of the reasons inducing hippocampus atrophy and play roles in the pathogenesis of PTSD. Increase in hippocampus levels of IGF-1 during early PTSD might be involved in the early molecular inhibitory mechanism of apoptosis in PTSD

Study of the variations in apoptotic factors in hippocampus of male rats with posttraumatic stress disorder

BACKGROUND: Post-traumatic stress disorder (PTSD) is a stress-related psychosomatic disorder caused by occurrence of a traumatic event and the hippocampus volume of the patients with Post-traumatic stress disorder decreased. However, the mechanisms that cause such damage are not well-understood. The aim of this study is to detect the expression of apoptosis-related Bax, Bcl-2, Caspase-3 and Insulin-like growth Factor-I proteins in the hippocampus region in the Predatory stress rats.MATERIALS AND METHODS: A total of 70 male wistar rats were divided into Predatory stress groups of 1d, 2d, 3d, 7d, 14d, 30d and a normal control group (N = 10). Rats were subjected to 5 min of predatory stress and then exposed to the elevated plus-maze (EPM). Serum corticosterone and Insulin-like growth factor-1 level of Hippocampus were measured by ELISA technique. The expression of Bax, Bcl-2, and Caspase-3 were detected by western blotting.RESULTS: Rats spent significantly more time in closed arms of the elevated plus maze (EPM) than control group after exposure to stress. Serum levels of corticosterone significantly increased at 2d-3d. The expression of hippocampal IGF-1 was significantly up-regulated at 1d-2d after stress. Both Bax and the ratio of Bax/Bcl-2 significantly peaked at Predatory stress 2d-14d. Caspase3 was significantly active among 2d-30 compared to the normal control.CONCLUSION: The activation of caspase-3 in the stress groups indicates that apoptosis may be one of the reasons inducing hippocampus atrophy and play roles in the pathogenesis of PTSD. Increase in hippocampus levels of IGF-1 during early PTSD might be involved in the early molecular inhibitory mechanism of apoptosis in PTSD.</p

Full Length Research Paper - Subcloning and expression of human alpha-fetoprotein gene in Pichia pastoris

Alpha-fetoprotein protein (AFP) is naturally found in fetal serum while production and appearance after birth is indicative of presence of malignant tumors. Therefore, by measuring this protein during fetal period and after birth, it is possible to diagnose abnormalities and tumors in fetus or the newborn. The objective of this study was to produce and purify AFP protein using DNA recombinant technology to apply in diagnostic kit preparations. In this study Pichia pastoris as metylotrophic yeast was used for AFP production. After construction of recombinant plasmid, pS1-AFP, electroporation and lithium chloride techniques were used for transferring to susceptible cells. The quantity and quality of the produced protein were checked by SDS-PAGE and ELISA methods. Selection of transformed mutant strains of Muts and culturing them in glycerol media (YPG) up to OD600 =6 and their transfer to methanol media (YPM) with augmentation of methanol to 1% final concentration resulted in inducing protein production in auxotrophic media lacking histidine. This protein could be useful in monoclonal antibody production and in diagnostic kit preparations