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

Haptoglobin genotypic distribution in Iranian patients with coronary heart disease

Haptoglobin is a plasma protein with hemoglobin binding capacity. Haptoglobin has important biological functions such as binding to free hemoglobin and removes it from the circulation, thus preventing iron loss and kidney damage during intravascular hemolysis, superior antioxidant capacity, protection against free radicals. Studies on the distribution of Hp show that the gene frequencies of Hp dependent on geographical and genetic family. In the other, several authors have showed the correlation between HP types and different diseases, such as inflammation, infection, cardiovascular diseases and malignant tumors.Smoking, hypertention,diabetes mellitus and serum lipid concentrations are risk factors for developing cardiovascular diseases. In addition, Hp polymorphism has been proposed as a risk factor for developing atherosclerotic vasculare disease. In this study, the association between Haptoglobin genotypic distribution and the incidence of coronary heart disease investigated. 50 Iranian patients with coronary heart disease were randomly selected. Genomic DNA extracted from peripheral blood leukocytes. In PCRs with primers A and B, amplification products of 1757 and 3481 bp were amplified from genomic DNA containing alleles Hp1 and Hp2, respectively. In the population studied, the distribution of haptoglobin polymorphism was 36% (n = 18) for the Hp1-1 type, 62% (n =31) for the HP1-2 type, and 2% (n = 1) for the HP2-2 type.  The trend in this study showing a lower frequency of 3-vessel disease and less severe coronary artery stenosis in patients with the HP1-1 phenotype than in patients with the HP1-2 phenotypes may be indicative of a protective effect of the HP1-1 phenotype against the development of atherosclerotic coronary artery disease

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

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

Morphological Study of Fasciola Parasites Isolated from Cattle and Sheep in Golestan Province (Iran)

Background: The genus Fasciola parasite causes fascioliasis infection. Fascioliasis is widespread all around the world and it is finding in abundance in the northern provinces of Iran. Cattle and sheep are the main hosts of the Fasciola parasite and intermediate hosts are lymnaeid snails such as Galba and Fossaria. Two main species of this genus are F. hepatica and F. gigantica. One of the most important methods of diagnosing this worm is morphological method. The aim of this study is to identify Fasciola through the morphological method in Golestan province.Materials and Methods: Fasciola worms taken from infected livestock livers were washed three times with PBS and were stained with carmine alum. After staining using Valero و and Periago Periago methods, the worms were measured morphologically by و   بcalibrated میکروسکوپ microscope, واستریومیکstereomicroscope, and True Chrome II camera. SPSS version 19 was used for analysis of the data.Results: A total of 45 livers from infected sheep and cattle with Fasciola worms were taken out of 228 samples, including 84 Fasciola hepatica (36.18%), 117 Fasciola gigantica (51.31%) and 27 Fasciola  sp. (11.84%).Conclusion: This مطالعه study نشان showed داد کهthat هر the two main species of worms that isگ شاخصF.hepatica وand F. gigantica were foundژیگانتیکا inبه فراوانیabundance یافتin Golestan province. The current study was unable to identify 11.84% genus Fasciola showed as Fasciola sp

An Improved Homologous Recombination Method for Rapid Cloning of the Antibody Heavy Chain Gene and Its Comparison with the Homologous Recombination and Traditional Cloning Methods

Background: The homologous recombination (HR) is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1(+) plasmid.Results: The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing.Conclusion: The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method.