Iranian Journal of Biotechnology
Year:2013 | Volume:11 | Issue:3 (43)|Papers Count:10

Dear Editor, The witches’ broom disease of lime trees is a destructing disease caused by the bacterium Candidatus Phytoplasma aurantofolia (1). The disease is prevalent in Oman, United Arab Emirates and Iran. To restrict the spread of the infected trees, detection of the causative bacterium is crucial. Besides PCR-based methods, antibodies can be applied for detection of pathogen-infected cells.

Dear Editor, The recently published articles in the Iranian Journal of Biotechnology inspired us to write this short communication on nitrogen dioxide NO2-philic plants (1).To cope with environmental pollution, we thought (still think) that development of methods that mitigate pollution is an urgent issue, and that NO2-philic plants that can grow with NO2 as the sole nitrogen source are instrumental for the implementation of mitigation of air pollution.

Most recently, we read in the news about sending of ricin-exposed letter to some governmental important people. Ricin is a toxalbumin derived from caster bean (Ricinus communis; Euphorbiaceae), whose oil is employed in medicine. Ricin is a potent and highly dangerous protein, where its exposure can cause severe toxicity in different organs and then death. Because there are many possible ways to expose (including skin, mucosal, gastrointestinal and pulmonary routs) and also regarding to its importance, which is abused as a kind of biological killing weapons, and its potential to affect many people via extremely low dosages, the main headlines attributed to its toxicity signs and symptoms, various probable routs of exposure especially via skin and mucosal contacts have been reviewed here. Nevertheless ricin is an easily produced toxin with known application in biological weapons; it is hard to consider it as a quick bio-killing poison to terror someone via exposed letter.

Context: MicroRNAs (miRNAs) are a class of short, endogenously-initiated, non-coding RNAs that post-transcriptionally control gene expression via translational repression or mRNA turnover. MiRNAs have attracted much attention in recent years as they play critical roles in gene expression and are promising tools with many biotech and therapeutic applications. The molecular mechanisms underlying the translational control of mRNAs are not fully understood but emerging evidence point to a key role for microRNAs in this process.Evidence Acquisition: In this review, we discuss the potential role of miRNAs as regulators of mRNA traffic and translational control, focusing on molecular mechanisms of miRNA-mediated control of eukaryotic mRNA stability and translational efficiency.Results: Translational control by miRNAs is often associated with silencing and repression of mRNAs via accumulation within cytoplasmic processing bodies (P-bodies), the site of mRNA storage and/or decay. Specific miRNAs can interact with the 3’UTR or 5’UTR of target mRNAs and regulate their stability as well as translational efficiency. A better understanding of these mechanisms is critical in advancing our knowledge of the role of these regulatory RNAs in modulating protein synthesis and controlling metabolic pathways in health and disease.Conclusions: The discovery of miRNAs and their important role in controlling many aspects of cell function and metabolism have led to considerable interest in biotech applications of miRNAs and their application in modulating specific gene expression. We thus highlight the growing biotech and therapeutic applications of miRNAs.

Context: In this review, we discuss empirical and stoichiometric models, which have been developed recently in SSF processes and the influence of environmental conditions on the variables of these models. Additionally, new studies on modeling of product formation are also mentioned.Evidence Acquisition: Solid-state fermentation (SSF) is recognized as a cheap process for producing many valuable products like industrial enzymes and bioethanol. To develop, optimize, and scale-up this process, mathematical models are required. In this review, we collected all the papers regarding microbial growth and product formation modeling in SSF. The pros and cons of each model and confirmation with experimental data were also discussed. We discussed here the simple empirical growth kinetics models and the effect of environmental conditions on these models parameters, stoichiometric models and product formation models.Results: Simple empirical models are used widely in the kinetic modeling of SSF processes due to their simplicity and ease of use. However, more studies should be done in this field to make them more accurate, especially; the effect of environmental conditions, like temperature and moisture, on key variables of the model must be considered. Robust modeling methods, like stoichiometric models, are in their early stages in SSF processes and require more studies. Developing models in which transport phenomena models are coupled with the growth kinetics models can help better SSF bioreactor designing. On the other hand, to use SSF for producing valuable products, product formation models, which are not developed well in SSF processes, are necessary.Conclusions: To use SSF for producing valuable metabolites in large scales, more attention is required for modeling the SSF processes, especially for product formation models and using modern methods like stoichiometric models.

Background: Interferons with different functions such as antiviral, antiproliferative and immunomodulatory actions are effective medications for a number of diseases. One of these new interferons (IFNs) is Interleukin-29 (IL-29) belongs to the family of IFN-l has antiviral activity and its potent in accompanying with IFN-α in treatment of HCV infection has been evaluated (clinical trial phase II). Recombinant IL-29 has been previously produced in multiple expression systems but in this study we cloned and expressed this protein in an Iranian Lizard leishmania (I.L.L) for the first time.Objectives: The Main objective of this research was to evaluate expression of functional human IL-29 in domestic Lizard leishmania as an alternative eukaryotic expression system.Materials and Methods: The IL-29 expression cassette was constructed into a pLEXSY vector. The leishmania cells were transfected by electroporation. After selection of transfectants, the protein expression was evaluated at RNA and protein levels.Results: Expression cassette was successfully transfected to leishmania cells and expression of recombinant IL-29 was proved by RT-PCR and western blotting. Purified protein showed 20% activity compared to standard protein. Enzymatic removal of N-glycan resulted to the shift of protein mobility on SDS-PAGE.Conclusions: Easy handling and culture of this eukaryotic host and mammalian cell like posttranslational modifications are the main advantages of this expression host, but the expression yield of this protein is very low and it seems to be not economic for large-scale production.

Background: Confirmation of olive oil authenticity and particularly virgin olive oil has a great importance. Several advanced chemical and genetic analyses have been used to monitor especial components; however, each has its limitations especially when detecting hazelnut-adulterated olive oil.Objectives: The objective of this research was to assess the presence of trace amount of hazelnut oil in olive oil (less than 10%) by Single Base Extension (SBE) and Fourier Transform InfraRed Spectroscopy (FTIR).Materials and Methods: The study was based on the analysis of chloroplast DNA sequences using SBE to detect Single Nucleotide Polymorphisms (SNPs) in highly preserved DNA regions among olive and hazelnut species to differentiate pure and adulterated olive oil by means of two parallel tools; ABI PRISM sequencing and AcycloPrime Single Nucleotide Polymorphism Detection. Fourier -Transform InfraRed technique was used for FTIR spectrum comparisons of pure olive oil and hazelnut-adulterated one, as well.Results: Total DNA was extracted successfully from pure and hazelnut-adulterated olive oil, and it provided properly acceptable amplification with the primers designed on chloroplast region of both species and their admixture oil in different ratios; 50: 50, 70: 30, and vice versa. However, for lesser than 10% hazelnut oil in olive oil only SBE analysis provided recognizable results. FTIR spectra of oil samples were assessed at frequency regions of 4000 - 700 cm -1. Eight wave numbers (3007, 1373, 1237, 1120, 1098, 1032, 965, and 722 cm -1) of eleven differentiating ones were selected as candidate wave-numbers to distinguish pure and adulterated olive oil.Conclusions: SBE technique proved to be an effective strategy to verify olive oil authenticity, especially from hazelnut-adulterated olive oil. However, FTIR technique provided trustable results only when higher than 10% hazelnut oil is present in olive oil.

Background: The Fc receptor like (FCRL) molecules belong to the immunoglobulin (Ig) superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identified for the human FCRL1, 2 and 4 molecules.Objectives: Cloning, expression, purification and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins.Materials and Methods: In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b (+) and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fine adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purified by metal affinity chromatography using Ni-NTA resin. Purified FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specific polyclonal antibodies.Results: Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b (+) vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37oC resulted in the highest expression levels of FCRL proteins ranging from approximately 15% (FCRL1) to 25% (FCRL2 and 4) of the total bacterial lysate proteins.Conclusions: These purified recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specific mAbs for immunotherapeutic interventions.

Background: Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the development of a vaccine against Pseudomonas aeruginosa infection.Objectives: In this study, recombinant exotoxin A (domains I and II), (ExoA I-II) protein was expressed, purified and its immunological characteristics were evaluated in a mouse model.Materials and Methods: The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II of exotoxin A was amplified by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 and the protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A (Domains I, II). Antibody production was evaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 × LD50 (7.5 × 107 CFU) of clinical strain of mucoid P. aeruginosa.Results: Sequencing of the cloned gene showed that the sequence of ExoA I-II gene was in accordance with ExoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-II produced a significant amount of specific IgG antibodies in mice. Also immunization of mice with ExoA I-II increased survival times against intra-peritoneal challenge with an approximate 7.5 × 107 CFU (2 × LD50) of clinical strain of P. aeruginosa.Conclusions: Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a new vaccine candidate against P. aeruginosa.

Background: Lung cancer is considered as one of the most frequent cancers worldwide, and has been the cause of more than one million mortalities each year. Exposure to tobacco smoke is the primary cause of most lung cancers, since it contains several thousand compounds, including more than 50 known carcinogens. However, a small fraction of individuals who are exposed to tobacco smoke develop lung cancer, therefore genetic factors may render some tobacco smokers more susceptible to cancer.Objectives: Genetic polymorphism in genes that encode metabolizing enzymes may be related to differentiated susceptibility of malignancy. CYP1B1 protein is a member of the more significant CYP1 subfamily enzymes, involved in environmental carcinogen metabolic activation. The most studied polymorphism in CYP1B1 gene includes 4325 C®G, resulting in an amino acid change from leucine to valine amino acid.Materials and Methods: A case-control study (included 65 lung cancer cases and 80 healthy controls) was designed based on the RFLP-PCR method to estimate the possible association of this polymorphism with lung cancer susceptibility in the Iranian population.Results: Regarding the distribution of CYP1B1 L432V genotypes, there were no meaningful differences among controls and lung cancer patients, however among patients carrying the CC genotype, tobacco smokers had a considerable elevated risk for lung cancer compared to those who had the GG genotype.Conclusions: CYP1B1 L432V polymorphism has an important role in lung cancer risk. Therefore, further studies are recommended for investigation of other related CYP1B1 gene polymorphisms, their association with affective genes and regulatory factors in the Iranian population.