Apoptosis Induction by Lactobacillus casei Acidic Proteins in the Colorectal Cancer Cell Line

Background: Colorectal cancer (CRC) ranks third in cancer prevalence. Lactobacillus casei, a probioticbacterium, can optimize the microbiota population of the gastrointestinal tract and prevent the growth of harmful bacteria that induce carcinogenesis. In this study, we investigated the effect of L. casei acidic proteins on apoptosis in the SW480 cell line to identify a drug protein for treating CRC.Materials and Methods: We assayed the effect of the isolated acid-resistant proteins of the Chaperoninbacterium, a metal-dependent Hydrolase, and Lysozyme on the SW480 colorectal cancer cell line apoptosispathway gene expression with a Real-Time RT-PCR.Results: All three proteins induced apoptosis in the cells treated separately with each of the proteins. Theresults showed that the up-regulation of BAX and P53 gene expression and the apoptosis pathway weresignificantly induced. Also, BCL2 expression was down-regulated, and significant anti-apoptotic was observedat p<0.0001. Moreover, the cells treated with these three proteins down-regulated the expression of MAP2K1and provoked the opposite apoptosis pathway.Conclusion: Our results found that these proteins would be a good choice for potential CRC treatment

Effect of Human HSP90 on Secondary and Tertiary Structures of Core Protein of Hepatitis C Virus and HbsAg of Hepatitis B Virus

The secondary structure of recombinant proteins can change through complex formation with other proteins. Here, we have determined the spatial structure of two proteins, including core protein of hepatitis C virus and HbsAg of hepatitis B virus, without the effect of human HSP90 as well as with the effect of this recombinant chaperone. As a result, the increase in intensity from 297.5 to 346.64 was accompanied by different folding and being non-polar protein in complex with the chaperone. HbsAg protein, combined with HSP90, showed a reduction in the maximum peak wavelength from 385 to 369.07 nm. The property of protein of being non-polar and hydrophobic, as well as having an increase in intensity from 200 to 219, indicates the protein folding. The shift from 342 to 337 nm along with blue shift indicates hydrophobic properties and the removal of protein from the water environment

Apoptotic Effects of the B Subunit of Bacterial Cytolethal Distending Toxin on the A549 Lung Cancer Cell Line

Cytolethal distending toxin (CDT) is a secreted tripartite genotoxin produced by many pathogenic gram-negative bacteria. It is composed of three subunits, CdtA, CdtB and CdtC, and CdtB-associated deoxyribonuclease (DNase) activity is essential for the CDT toxicity. In the present study, to design a novel potentially antitumor drug against lung cancer, the possible mechanisms of cdtB anticancer properties were explored in the A549 human lung adenocarcinoma cell line. A recombinant plasmid pcDNA3.1/cdtB was constructed expressing CdtB of human periodontal bacterium Aggregatibacter actinomycetemcomitans and investigated for toxic properties in A549 cells and possible mechanisms. It was observed that plasmid pcDNA3.1/cdtB caused loss of cell viability, morphologic changes and induction of apoptosis. Furthermore, measurement of caspase activity indicated involvement of an intrinsic pathway of cell apoptosis. Consequently, the recombinant plasmid pcDNA3.1/cdtB may have potential as a new class of therapeutic agent for gene therapy of lung cancer

Cloning and Expression of Luteinizing Hormone Subunits in Chinese Hamster Ovary Cell Line

Background: Luteinizing hormone (LH) was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively.Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary (CHO) eukaryotic system.Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR.  Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody.Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly

Evaluation of the Coagulant Effect of the Zanjani and Latifi Viper Venom Endemic in Iran

The venom of the viper is very important in pharmaceutical usage, such as in the process of coagulation during medical care. This study aims to evaluate the coagulant factor of Latifi and Zanjani viper venom. In the current study, after electrophoresis of proteins found in viper venom, all of the thick and strong bands of proteins were isolated and prepared for examination of coagulation characteristics, like pro-thrombin time (PTT) and active partial thromboplastic time (APTT), followed by further study by mass spectroscopy. In this way, 11 bands of proteins were recognized in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After PT and APTT tests, one common band in 26 KDa could lead to coagulation of blood plasma in less than one second. Mass spectroscopy identified this band as serine protease isoform4. The results confirmed the coagulation effect of a 26 KDa protein fraction of venom from Latifi and Zanjani vipers

The design of the constructs of cpsD and simA genes of Streptococcus iniae

 Background & Objective: Streptococcosis is one of the bacterial infections in fish, especially rainbow trout which infects brain and nervous systems of fish and is caused by S. iniae. Estimation of the impact of disease prevalence by S. iniae in fish farming in some countries is reported about 100 million dollars per year. Some of the most effective proteins in pathogenicity of these bacteria are SimA and CpsD. In order to design new and effective vaccine, in this study cloning of two genes of Streptococcus was performed into pNZ8148 vector and expressed in Escherichia coli. Materials and Methods: simA and cpsD genes were subcloned into pNZ8148 vector. Obtained constructs were transformed to expressing E. coli BL21 strain. After induction with nisin, SDS PAGE electrophoresis and Western blotting were used to confirm the procedures.Results: Using PCR with specific and universal primers, the accuracy of cloning was confirmed. Final verification of expressed protein was carried out by SDS-PAGE and western blotting. Conclusion: With regard to the obtained results, it seems that the generated gene construct in this study can be used as a vaccine against Streptococcosis in future researches

Production Design of Efficient Recombinant Human Interleukin-4 (rhIL-4) under Specific Promoter in Escherichia coli

Recombinant DNA technology plays a vital role in improving health conditions by developing new pharmaceuticals.  Recently, some of the cytokines as other recombinant proteins could be produced using the recombinant DNA technology. The role of Recombinant IL-4 in allergy, autoimmunity, and cancer has been investigated. The present study was aimed to clone and produce the Human IL-4 under specific promoter in Escherichia coli and assessed its biological functions. The designed hIL-4 gene construct was artificially synthesized; subsequently, it was sub cloned into the pcDNA3.1 (+) vector in HindIII restriction enzyme site. Recombinant plasmid was transferred and expressed in BL21 cells. The rhIL-4 protein was evaluated by SDS-PAGE and Western blotting. It was purified by Ni-NTA affinity chromatography. The purified protein concentration and also accuracy were determined by ELISA. MTT assay was applied to evaluate the biological activity of rhIL-4 on the Erythroleukemic cell line proliferation. The rhIL-4 gene was successfully cloned and transformed into expression E. coli cells. As a result, a specific band was observed both on the SDS-PAGE and nitrocellulose membrane after Western blotting. The purified protein concentration was equal to 500 pg/ml. The MTT assay indicated that the exposed cells with rhIL-4 were proliferated in a dose dependent manner. The rhIL-4 gene under specific eukaryotic promoter was successfully cloned in the prokaryotic system and the transcription was carried out by T7 RNA polymerase. Therefore, mass production of IL-4 could be a great help in clinical trials and research studies. Additionally, prokaryotic system used in current work was less costly and less time-consuming.HIGHLIGHTSInterleukin-4 (IL-4) is a cytokine that regulates multiple biological functions.The recombinant Human IL-4 was cloned and produced under specific promoter in Escherichia coli.Through MTT assay, the exposed cells with rhIL-4 were proliferated in a dose dependent manner

Amidolytic Activity of Factor VII Expressed in Iranian Lizard Leishmania

Over many years, variable gene expression systems have been used obtaining of Factor VII protein, but each one has certain limitations. Therefore, the main goal of this study was to assess the biological activity of purified and recombinant factor VII expressed in Iranian lizard Leishmania. After transferring recombinant construct containing FVII gene to Leishmania, first, the expression of 55 kDa FVII protein in transfected cells was confirmed by analyzing cell lysate using SDS-PAGE and Western Blotting techniques. Then, FVII was purified by NI-NTA-His Tag- resin through an affinity chromatography. The chromogenic activity of human recombinant Factor VII (amidolytic activity) in supernatant and pellet fractions of Leishmania promastigotes was done using ELISA method. The amidolytic activity of rhFVII was about 0.0125 IU/ml in the concentrated cell sediment and 0 IU/ml in the supernatant in the first 60 minutes and after that time, none of the samples showed acceptable activity. HIGHLIGHTS• Factor VII has important role in the treatment of coagulation and hemophilic disease.• Recombinant FVII was expressed in Iranian Lizard Leishmania.• The amidolytic activity of rhFVII was about 0.0125 IU/ml in cell pellet.• No amildolytic activity was observed in the supernatant

Heart Diseases Associated Genes

Background: Heart diseases are complex pathophysiologic conditions involving biomarkers. Understanding the mechanisms by which a gene selectively triggers intracellular molecular responses provide insight into the complex processes implicated in heart diseases. The aim of this study was to predict heart diseases associated genes. Materials and Methods: A number of computational methods have been developed for human gene prioritization. In this study, we used Beegle and KEGG pathway databases and two online services for gene prioritization and analysis of genes related to heart disease. Results: Over 200 genes and 5 key signaling pathways related to human heart diseases were found. The processes in which gene mutations trigger a response in cells leading to cardiac conditions involve multiple pathways. Conclusion: The genes related to heart diseases could be CRP, NPPB, IL-6, ACE2 and GATA4 with high scores and the researchers should find the diagnostic biomarker between them

Design and Production of a Novel Polypeptide with Immunogenic Potentials for Immunoassay of Brucella Melitensis

Brucellosis is one of the most common diseases in humans and it has a worldwide spread. The design and production of newly synthesized proteins can be served as a goal for the rapid and accurate detection of brucellosis. To this aim, finding the antigenic epitopes is the first step to design a diagnostic method. In this study, the epitope mapping procedure was carried out by IEDB analysis resource using Flagellin and Porin amino acid sequences. The selected sequences were linked by GS linkers and cloned into pET26b vector. After confirmation of the expressed recombinant polypeptide by western blotting, it was immunologically analyzed by gel diffusion assay. The SDS PAGE and western blot analysis confirmed the 27 KDa polypeptide production and observing an arc in gel diffusion test demonstrated the precipitation of serum antibodies and the presence of specific antigen complexes. The results showed that the recombinant polypeptide produced in E. coli BL21, could interact with antibodies present in Brucella immunized sheep serum.HIGHLIGHTS•Prediction of antigenic epitopes for Brucella membrane proteins.•Production of designed polypeptide in E. coli BL21 (DE3).•Interaction of produced polypeptide with Brucella specific antibodies