فهرست مطالب

  • Volume:17 Issue:2, 2019
  • تاریخ انتشار: 1398/03/11
  • تعداد عناوین: 12
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  • Mohamad Hassan Fouani, Maryam Nikkhah *, Javad Mowla Pages 1-7
    Background
    RADA16I represents one of promising hydrogel forming peptides. Several implementations of RADA16I hydrogels have proven successful in the field of regenerative medicine and tissue engineering. However, RADA16I peptides used in various studies utilize synthetic peptides and so far, only two research articles have been published on RADA16I peptide recombinant production. Moreover, previous studies utilized non- or less routine expression and purification methods to produce RADA16I peptide recombinantly.
    Objectives
    The main goal was to produce the self-assembling peptide, RADA16I, in Escherichia coli by exploiting routine and widely used vectors and purification methods, in shake flask.
    Material and Methods
    RADA16I coding sequence was inserted in pET31b+, and the construct was transformed into E. coli. Purified fusion constructs were purified using Nickel Sepharose. RADA16I unimers were released using CNBr cleavage. CD and FTIR spectroscopy were used to study recombinant RADA16I’s confirmation. TEM was used to confirm fibril formation of recombinant RADA16I. Furthermore, MTT assay was implemented to assess cytocompatibility of recombinant RADA16I.
    Results
    The biochemical, biophysical and structural analysis proved the ability of the recombinant RADA16I to form self-assembling peptide nanofibers. Furthermore, the nanofibers exhibited no cytotoxicity and retained their cell adhesive activity.
    Conclusions
    We successfully produced RADA16I in acceptable levels and established a basis for future investigation for the production of RADA16I under fermentation conditions.
    Keywords: Fusion protein, Hydrogel, Nanofibers, RADA16I, Self-Assembling Peptide
  • Hassan Bardania, Seyed Abbas Shojaosadati *, Farzad Kobarfard, Dina Morshedi, Farhang Aliakbari, Mohammad Taher Tahoori, Elahe Roshani Pages 8-13
    Background
    Eptifibatide (Integrilin®) is a hepta-peptide drug which specifically prevents the aggregation of activated platelets. The peptide drugs are encapsulated into nanolipisomes in order to decreasing their side effects and improving their half-life and bioavailability.
    Objectives
    In this study, the in vitro cytotoxicity and hemocompatibility of RGD-modified nano-liposomes (RGD-MNL) encapsulated a highly potent antiplatelet drug (eptifibatide) was investigated.
    Material and Methods
    RGD-MNL encapsulated eptifibatide was prepared using lipid film hydration and freeze/thawing method. The morphology and size distribution (about 90 nm) of RGD-MNL were characterized using transmission electron microscopy (TEM). The in-vitro cytotoxicity of nano-liposomes was examined using the MTT, LDH release and reactive oxygen species (ROS) generation assays. The effect of RGD-MNL on red blood cells (RBC) was investigated using hemolysis and LDH release assays.
    Results
    The results revealed that RGD-MNL had no significant cytotoxic effect on HeLa and HUVEC cell lines, and also no ROS generation increase in the cells. In addition, the adverse effect of RGD-MNL on LDH release and membrane integrity of RBC was not observed.
    Conclusions
    In conclusion, the recommended RGD-MNL formulations have not any significant cytotoxicity on normal cells or RBC and have potential for protecting and enhancing the activity of antiplatelet drugs.
    Keywords: Liposomes, Cytotoxicity, Materials Testing, Eptifibatide
  • Hassan Bardania, Jamshid Raheb *, Ayyoob Arpanaei Pages 14-20
    Background
    Magnetic separation using magnetic nanoparticles can be used as a simple method to isolate desulfurizing bacteria from a biphasic oil/water system.
    Objectives
    Magnetite nanoparticles were applied to coat the surface of Rhodococcus erythropolis IGTS8 and Rhodococcus erythropolis FMF desulfurizing bacterial cells, and the viability and reusability of magnetite-coated bacteria evaluated by using various methods.
    Material and Methods
    Magnetite nanoparticles were synthesized through a reverse co-precipitation method. Glycine was added during and after the synthesis of magnetite nanoparticles to modify their surface and to stabilize the dispersion of the nanoparticles. The glycine-modified magnetite nanoparticles were immobilized on the surface of both oil-desulfurizing bacterial strains. Reusability of magnetite-coated bacterial cells was evaluated via assessing the desulfurization activity of bacteria via spectrophotometry using Gibb's assay, after the separation of bacterial cells from 96h-cultures with the application of external magnetic field. In addition, CFU and fluorescence imaging were used to investigate the viability of magnetite-coated and free bacterial cells.
    Results
    TEM micrographs showed that magnetite nanoparticles have the size approximately 5.35±1.13 nm. Reusability results showed that both magnetite-coated bacterial strains maintain their activity even after 5 × 96h-cycles. The viability results revealed glycine-modified magnetite nanoparticles did not negatively affect the viability of two bacterial strains R. erythropolis IGTS8 and R. erythropolis FMF.
    Conclusions
    In conclusion, the glycine-modified magnetite nanoparticles have great capacity for immobilization and separation of desulfurizing bacteria from suspension.
    Keywords: Rhodococcus erythropolis, Nanoparticles, Equipment Reuse
  • Li, Ying Wu, Jun, Jie Xu, Pan Xu, Bin Yong, Hong Feng * Pages 21-29
    Background
    Enantiopure epoxides are important intermediates in the synthesis of high-value chiral chemicals. Epoxide hydrolases have been exploited in biocatalysis for kinetic resolution of racemic epoxides to produce enantiopure epoxides and vicinal diols. It is necessary to obtain sufficient stable epoxide hydrolases with high enantioselectivity to meet the requirements of industry.
    Objectives
    Enhancement of soluble expression and biochemical characterization of epoxide hydrolases from Bacillus pumilus and B. subtilis.
    Material and Methods
    Homologous genes encoding epoxide hydrolases from B. pumilus and B. subtilis were cloned and expressed in Escherichia coli. The recombinant epoxide hydrolases were characterized biochemically.
    Results
    Low temperature induction of expression and aC-terminal-fused His-tag enhanced soluble expression of the epoxide hydrolases from the two Bacillus species in E. coli. These epoxide hydrolases could hydrolyze various epoxide substrates, with stereoselectivity toward some epoxides such as styrene oxide and glycidyl tosylate.
    Conclusions
    The position of the His-tag and the induction temperature were found to play a vital role in soluble expression of these two epoxide hydrolases in E. coli. In view of their catalytic properties, the epoxide hydrolases from Bacillus have potential for application in kinetic resolution of some epoxides to prepare enantiopure epoxides and vicinal diols.
    Keywords: Bacillus, Epoxide hydrolase, Hydrolysis
  • Abubakar Muhammad, Bokhari Syed Ali Imran, Vernoux Jean, Paul, Ishtiaq Ali Muhammad, Rani Faryal, Desmasures Nathalie, Imran Muhammad * Pages 30-37
    Background
    Alkaline proteases is the important group of enzymes having numerous industrial applications including dairy food formulations.
    Objectives
    The current study deals with the purification and characterization of an alkaline serine protease produced by Geotrichum candidum QAUGC01, isolated from indigenous fermented milk product, Dahi.
    Material and Methods
    In total twelve G. candidum strains were screened for their proteolytic activity by using standard protease assay. The protease production from G. candidum QAUGC01 was optimized by varying physio-chemical conditions. The protease was purified by using two-step
    method
    ammonium sulfate precipitation and gel filtration chromatography. Protease was further characterized by studying various parameter like temperature, pH, modulators, metal ions and organic solvent. A thermodynamic study was also carried out to explore the half-life of protease.
    Results
    The G. candidum grew profusely at 25 °C and at an initial pH of 4.0 for 72 h of incubation producing 26.21 U/mlmaximum extracellular protease. Protease revealed that Vmax and Km was 26.25 U.ml-1.min-1 and 0.05 mg.mL-1, respectively using casein as substrate. The enzyme was stable at a temperature range (25-45 ºC) and pH (8-9). Residual enzyme activity was strongly inhibited in the presence of PMSF (7.5%). The protease could hydrolyze proteinaceous substrates, casein (98%) and BSA (95%). The thermodynamic studies explored that the half-life of the enzyme that was 106.62 min, 38.72 min and 15.71 min at 50, 60 and 70 ºC, respectively.
    Conclusions
    Purified protease from G. candidum GCQAU01 is an ideal candidate for industrial application.
    Keywords: Geotrichum candidum, Alkaline Serine Protease, Kinetics, Thermodynamics
  • Jian Dong, Kunqiang Hong, Cuiying Zhang, Ye, Fu Chen, Dong Shengsheng, Xiao Li, Dong, Guang Xiao * Pages 38-45
    Background
    Enhancing the industrial yeast strains ethyl acetate yield through a precise and seamless genetic manipulation strategy without any extraneous DNA sequences is an essential requisite and significant demand.
    Objectives
    For increasing the ethyl acetate yield of industrial brewer’s yeast strain, all the ATF1 alleles were overexpressed through “self-cloning” integration strategy.
    Material and Methods
    Escherichia coli strain DH5α was utilized for plasmid construction. ATF1 alleles were overexpressed through a precise and seamless insertion of the PGK1 promoter in industrial brewer’s yeast strain S6. In addition, growth rates, ATF1 mRNA levels, AATase activity, the fermentation performance of the engineered strains, and gas chromatography (GC) analysis was conducted.
    Results
    The two engineered strains (S6-P-12 and S6-P-30) overexpressed all ATF1 alleles but unaffected normal growth. The ATF1 mRNA levels of the S6-P-12 and S6-P-30 were all 4-fold higher than that of S6. The AATase (Alcohol acetyl transferases, encoded by ATF1 gene) activity of the two engineered strains was all 3-fold higher than that of the parent strain. In the beer fermentation at 10 ℃, the concentrations of ethyl acetate produced by the engineered strains S6-P-12 and S6-P-30 was increased to 23.98 and 24.00 mg L-1, respectively, about 20.44% and 20.54% higher than that of S6.
    Conclusions
    These results verify that the ethyl acetate yield could be enhanced by the overexpressed of ATF1 in the polyploid industrial brewer’s yeast strains via “self-cloning” integration strategy. The present study provides a reference for target gene modification in the diploid or polyploid industrial yeast strains.
    Keywords: Acetate Ester, ATF1, Polyploidy, PGK1
  • Mina Beigmohammadi, Ali Movafeghi, Ali Sharafi *, Samineh Jafari, Hossein Danafar Pages 46-54
    Background
    Plumbagin is as an important bioactive secondary metabolite found in the roots of Plumbago spp. The only one species, Plumbago europaea L., grows wild in Iran. The therapeutic use of plumbagin is limited due to its insufficient supply from the natural sources as the plants grow slowly and take several years to produce quality roots.
    Objectives
    To develop an efficient protocol for the establishment of callus and cell suspension cultures of P. europaea and to evaluate production of plumbagin in callus and cell suspension cultures of P. europaea for the first time.
    Material and Methods
    Stems and leaves explants were cultured on agar solidified (7% w/v) MS media, supplemented with different combination of 2, 4-D and Kin or 6-Benzylaminopurin (BA) for callus induction. The rapid growing calli were cultured in liquid Murashige and Skoog (MS) media in agitated condition for establishing cell suspension cultures of P. europaea. Moreover, the effects of light and dark conditions on the cell growth, cell viability and plumbagin production in cell suspension cultures of P. europaea were assessed.
    Results
    Friable calli were successfully induced using stem segments of P. europaea in semisolid MS medium supplemented with 1 mg.L-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 0.5 mg.L-1of kinetin (Kin). Optimal cell growth was obtained when the cells were grown in MS liquid media supplemented with 1 mg.L-1 2, 4-D and 0.5 mg.L-1 kinetin with an initial cell density of ~3×105 cellsper ml incubated in the dark at 25 ± 1 °C. Growth curve revealed that the maximum cell growth rate (14.83×105 cellsper ml) achieved on the day 18 and the highest plumbagin content (0.9 mg.g-1 Dry Cell Weight (DCW)) in the cells was obtained at the late exponential phase under dark condition which determined by High Performance Liquid Chromatography (HPLC) technique. Based on the obtained results, cell viability remained around 82.73% during the 18 days of cell culture in darkness. These suspension cultures showed continuous and stable production of plumbagin.
    Conclusions
    Our study suggests that cell suspension cultures of P. europaea represent an effective system for biosynthesis and production of plumbagin as a valuable bioactive compound.
    Keywords: cell survival, light, Plumbagin
  • Zahra Sadat Shahmoradi, Masoud Tohidfar, Hasan Marashi *, Saeid Malekzadeh Shafaroudi, Ebrahim Karimi Pages 55-60
    Background
    In consideration for the increasing widespread use of genetically modified (GM) crops, one of the important issues for assessment is the effect of GM crops on soil microbial communities
    Objectives
    In this study, T2 chitinase-transgenic cotton (line #57) and its non-transgenic line were investigated for bacterial and fungal dynamics during its development stages.
    Material and Methods
    The assessments were performed by viable plate count and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) assays.
    Results
    Viable plate count analysis showed an increase in community structures and the number of culturable bacteria in rhizosphere of both transgenic and non-transgenic cultivars as compared to bulk soil. PCR-DGGE confirmed results of viable plate count assays of the changes in bacterial and fungal communities for all cotton development stages in rhizosphere and bulk zones. No significant differences in number of functional bacteria were observed between rhizosphere soil of chitinase transgenic and non-chitinase transgenic cotton at one particular stage.
    Conclusions
    The results indicated that T2 chitinase-transgenic cotton (line #57) might have no adverse effects on community structures and total number of culturable bacteria and fungi in the rhizosphere.
    Keywords: Microorganisms, Genetically-Modified, Microbiota, Rhizosphere
  • Maryam Zain *, Sumera Yasmin, Fauzia Hafeez Pages 61-70
    Background
    Plant Growth Promoting Rhizobacteria (PGPR) may be utilized to augment plant growth and suppress the plant pathogens.
    Objective
    The present study was conducted to isolate and characterize the antagonistic bacteria indigenous to cotton and sugarcane rhizosphere in Pakistan, and to evaluate their ability to suppress phytopathogenic Fusarium spp. Out of 63 isolates 37 different morphotypes were studied for their antagonistic activity against Fusarium monoliformae, Fusarium oxysporum and Fusarium solani. Among these 31 strains showed the percentage suppression ranging from 40 to 66% against Fusarium spp.
    Objectives
    The antagonistic bacteria having antifungal activity were studied for different morphological and physiological characteristics using Gram staining and light microscopy. Most of them were Gram negative and tentatively identified as Pseudomonas spp. The selected strains were screened in vitro for plant growth regulation and antifungal traits.
    Material and Methods
    Our study included 1000 premature CAD patients that classified into two groups with history of MI (n = 461) and without of MI (n = 539). The polymorphism variants in 10% of samples were determined by PCR-RFLP technique and genotyping of the polymorphism in all subjects was conducted by High Resolution Melting method. Given the two conditions of patients residing in Tehran and also faced with their first episode of MI, 640 out of 1000 study samples that had been previously followed-up were assessed in a retrospective cohort phase regarding long-term major adverse cardiac events (MACE).
    Results
    Four bacterial strains were able to produce the chitinase enzyme while four other bacterial strains showed protease production. Ten strains were positive for HCN production. Out of 37, eight strains showed phosphate solubilization ranging from 13 to 24 µg/ml. eighteen strains produced indole acetic acid ranging from 5 to 19 µg/ml.
    Conclusions
    This study identified specific traits in the isolated rhizobacteria which make them good candidates as PGPR and might contribute to enhance growth of crop plants. This information is of general interest and also helpful for devising strategies to manage diseases caused by Fusarium in cotton and sugarcane.
    Keywords: Fusarium monoliformae, Fusarium solani, Fusarium.oxysporum, Pseudomonas spp, Biocontrol
  • Bahareh Nowruzi *, Matti Wahlsten, Juoni Jokela Pages 71-78
    Background
    Cyanobacteria have a worldwide distribution in the terrestrial habitats, occurring predominantly on the surface of the soils, stones, rocks, and trees, practically in moist, neutral or alkaline aeries. The unique natural and bioactive compounds from cyanobacteria with various biological activities and an extensive range of chemical classes have a significant capability for expansion of the pharmaceuticals and other biomedical purposes.
    Objectives
    Regardless of the progresses in our knowledge on cyanobacteria, however, cyanobacteria are still viewed as an unexplored source of potential drugs. In this study presence of bioactive compounds among the cyanobacteria culture collection of Iran, where a wide variety of strains can be found, was investigated.
    Material and Methods
    We explored one Nostoc strain isolated from rice fields in Golestan province of northern Iran for searching for novel products. The chemical construction of the new bioactive compound was clarified by application of liquid chromatography-mass spectrometer (LC-MS) and Marfey’s analysis of the degradation products.
    Results
    We found a novel peptide aldehyde compound from a hydrophilic extract of the Nostoc sp. Bahar_M, which is composed of the three subunits, 2-hydroxy-4-(4-hydroxyphenyl) butanoic acid (Hhpba), L-Ile, and L-argininal. According to the structural information, we predicted that the novel peptide-aldehyde compound probably to be trypsin inhibitors.
    Conclusions
    Results demonstrated that terrestrial cyanobacteria are a promissing resource of bioactive natural products.
    Keywords: bioactive compounds, Cyanobacteria, Nostoc
  • Mehrdad Sheikhvatan, Mohammad Ali Boroumand *, Mehrdad Behmanesh, Shayan Ziaee, Sara Cheraghee Pages 79-88
    Background
    Contradictory results have been obtained regarding the role of integrin, beta 3 (ITGB3) gene polymorphisms in occurrence of myocardial infarction (MI).
    Objectives
    We aimed to assess the association between 1565C/T polymorphism of ITGB3 gene and increased risk for acute MI in patients with premature coronary artery disease (CAD).
    Material and Methods
    Our study included 1000 premature CAD patients that classified into two groups with history of MI (n = 461) and without of MI (n = 539). The polymorphism variants in 10% of samples were determined by PCR-RFLP technique and genotyping of the polymorphism in all subjects was conducted by High Resolution Melting method. Given the two conditions of patients residing in Tehran and also faced with their first episode of MI, 640 out of 1000 study samples that had been previously followed-up were assessed in a retrospective cohort phase regarding long-term major adverse cardiac events (MACE).
    Results
    There was no significant difference in the frequency of 1565C/T polymorphism between the MI and non-MI groups. The frequency of wild genotype was 69.2% and 72.2%, the frequency of homozygous genotype was 21.3% and 18.4%, and the frequency of mutant genotype was 9.5% and 9.5%, respectively (P = 0.505). No significant difference was also found in total-MACE free survival rate between the patients with different genotypes of 1565C/T polymorphism in both MI and non-MI group.
    Conclusions
    The carriage of the 1565C/T polymorphism of ITGB3 gene seems unlikely to be a significant risk factor for the development of MI in Iranian patients with premature CAD.
    Keywords: Coronary Artery Disease, myocardial infarction, genetics, Atherosclerosis
  • Fajun Li *, Chunpeng Fu, Qunfeng Li Pages 89-93
    Background

    Genome walking is a DNA-cloning methodology that is used to isolate unknown genomic regions adjacent to known sequences. However, the existing genome-walking methods have their own limitations.

    Objectives

    Our aim was to provide a simple and efficient genome-walking technology.

    Material and Methods

    In this paper, we developed a novel PCR strategy (termed SLRA PCR) that uses a single long primer (SLP), a set of gene specific primers (GSP), and a random amplified polymorphic DNA (RAPD) primer for genome walking. SLRA PCR consists of two processes: the first amplification using SLP, and three successive rounds of nested PCR amplified by GSP and RAPD primer. The novelty of the approach lies in the use of long primers (SLP and GSP) and same annealing and extension temperature 68℃ in combination. This method offers higher amplification efficiency, superior versatility, and greater simplicity compared with conventional randomly primed PCR methods for genome walking.

    Results

    The promoter regions and the first introns of theinsulin-like androgenic gland hormone (IAG) gene and the hemocyanin gene of Macrobrachium nipponense were cloned using SLRA PCR, respectively.

    Conclusions

    This genome walking strategy can be applied to a wide range of genomes.

    Keywords: Hemocyanins, Polymerase Chain Reaction, DNA Primers