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Majidzadeh K.,Tehran University of Medical Sciences | Majidzadeh K.,Cancer Genetics Research Group | Mohseni A.,Tehran University of Medical Sciences | Soleimani M.,Tehran University of Medical Sciences
Jundishapur Journal of Microbiology | Year: 2014

Background: Due to the limitations of the classical methods to detect Coxiella burnetii, direct diagnosis of the pathogen using PCR techniques is still the preferable approach. However, false negative results owing to the presence of PCR inhibitors are troublesome. Objectives: In order to identify the inhibitors during PCR assay, an internal positive control (IPC) was designed based on 16SrRNA gene of C. burnetii. Materials and Methods: In the current study, the initial and ending parts of the target gene in an external positive control plasmid (pTZ57R/T-16S) amplified using internal primers which had a BglII restriction site on the 5 ends. Both PCR products (fragments 1 and 2) were cloned into pTZ57R/T vector. Following BglII enzyme digestion, the two obtained linear plasmids were ligated. The ligation product was transformed into Escherichia coli Top10F'. Screening of the desired recombinant clone was carried out using colony PCR. Results: The size of the PCR product was equal to the sum of the first and second fragments. Sequencing confirmed the presence of the desire insert (IPC sequence) in recombinant plasmid. Consequently, the IPC fragment was longer than the target gene while both ends had similar attachments to the same primer pair. Conclusions: The results showed that direct fusion of the recombinant plasmids containing the initial and ending parts of the target gene are simple and cost-effective techniques for increasing the length of the fragment and constructing IPC. © 2014, Ahvaz Jundishapur University of Medical Sciences; Published by Kowsar Corp. Source

Kazemali MohammadReza,Pasteur Institute of Iran | Majidzadeh-A K.,Cancer Genetics Research Group | Sardari S.,Pasteur Institute of Iran | Khalaj V.,Pasteur Institute of Iran | And 5 more authors.
Enzyme and Microbial Technology | Year: 2014

Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator. © 2014 Elsevier Inc. Source

Kazemali M.R.,Pasteur Institute of Iran | Majidzadeh K.,Cancer Genetics Research Group | Sardari S.,Pasteur Institute of Iran | Saadati A.H.,Pasteur Institute of Iran | And 4 more authors.
Bioprocess and Biosystems Engineering | Year: 2016

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µset glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µset glycerol and that of methanol to be 0.05 and 0.03 h−1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained. © 2016 Springer-Verlag Berlin Heidelberg Source

Majidzadeh-A K.,Cancer Genetics Research Group | Majidzadeh-A K.,Tehran University of Medical Sciences | Gharechahi J.,Cancer Genetics Research Group
Medical Oncology | Year: 2013

Due to ease accessibility, exploring plasma for candidate cancer biomarkers is of great interest to molecular biologist and physicians. In breast cancer, the development of tamoxifen resistance (TR) is among the major causes of recurrence and mortality. Therefore, the identification of novel biomarkers that are linked to TR is of great interest and the subject of intensive research. Here, we exploited the power of two-dimensional gel electrophoresis coupled with protein identification using tandem mass spectrometry to identify plasma proteome signatures associated with TR. Comparative proteomics analysis resulted in the identification of 15 statistically significant spots, which were up-/downregulated after tamoxifen therapy. MASCOT search of the mass spectrometry generated spectral data resulted in the identification of 9 proteins. Several differentially expressed proteins such as clusterin, serum amyloid A, serpin B4, and transthyretin are already known to be involved in cancer incidence and progression. The possible involvement of these candidate proteins in conferring TR and their potential usefulness as plasma biomarkers for predicting response to tamoxifen treatment has been discussed. © 2013 Springer Science+Business Media New York. Source

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