فهرست مطالب
Iranian Journal of Biotechnology
Volume:13 Issue: 1, Winter 2015
- تاریخ انتشار: 1394/06/29
- تعداد عناوین: 9
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Pages 1-10BackgroundMultifunctional core-shell magnetic nanocomposite particles with tunable characteristics have been paid much attention for biomedical applications in recent years. A rational design and suitable preparation method must be employed to be able to exploit attractive properties of magnetic nanocomposite particles.ObjectivesHerein, we report on a simple approach for the synthesis of magnetic mesoporous silica nanocomposite particles (MMSPs), consisted of a Fe3O4 cluster core, a nonporous silica shell and a second shell of the mesoporous silica of suitable sizes for biomedical applications and evaluate their cytotoxicity effects on human cancer prostate cell lines.Materials And MethodsClusters of magnetite (Fe3O4) nanoparticles were coated by a layer of nonporous silica using Stöber method. The coating step was completed by an outer layer of mesoporous silica via template-removing method. Structural properties of MMSPs were investigated by FTIR, HR-S(T)EM, BET, XRD techniques and magnetic properties of MMSPs by VSM instrument. MTT and LDH assays were employed to study the cytotoxicity of MMSPs.ResultsObtained results revealed that decreasing the precursor concentration and the reaction time at the nonporous silica shell formation step decreases the thickness of the nonporous silica shell and consequently leads to the formation of smaller MMSPs. The as-prepared MMSPs have a desirable average size of 180±10 nm, an average pore size of 3.01 nm, a high surface area of 390.4 m2.g-1 and a large pore volume of 0.294 cm3.g-1. In addition, the MMSPs exhibited a superparamagnetic behavior and a high magnetization saturation value of 21±0.5 emu/g. Furthermore, the viability tests of DU-145 cell lines exposed to various concentrations of these particles demonstrated negligible cytotoxicity effects of the as-prepared particles.ConclusionsThese results demonstrate interesting properties of MMSPs prepared in this study for biomedical applications.Keywords: Cytotoxicity, Nanocomposite magnetic particles, Silica shell
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Pages 11-16BackgroundA highly efficient genetic transformation system is essential for a successful genetic manipulation of the African violet (Saintpaulia ionantha Wendl.).ObjectivesDeveloping a particle bombardment-based genetic transformation system for the African violet.Materials And MethodsA local cultivar of the African violet from Guilan province was used for transformation experiments. The pFF19G and pBin61-Ech42 vectors were used for transient and stable transformation experiments, respectively. The PCR and RT-PCR techniques were used to verify transgene presence and transcript levels in candidate transgenic lines, respectively.ResultsUsing leaf explants as target tissues, we transferred an endochitinase gene cDNA into African violet. Transgenic plants were regenerated on selection medium at a reasonable frequency (in average, one stable transgenic line per shot). Molecular analysis of transgenic plants by PCR and RT-PCR techniques confirmed successful integration and expression of transgene in several independent transgenic lines.ConclusionsOur results provide an efficient stable transformation system for genetic transformation of African violet.Keywords: African violet, Biolistic, Genetic transformation, Saintpaulia ionantha
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Pages 17-25BackgroundPomegranate fruit (Punica granatum L.) is a rich source of anthocyanin pigments resulting in vibrant colours and anti-oxidant contents. Although the intensity and pattern of anthocyanin biosynthesis in fruit are strongly influenced by R2R3-MYB transcription factors, little is known about the regulation and role of MYB in anthocyanin pathway of pomegranate.ObjectivesThe present study was conducted to elucidate the relationship between the expression of MYB transcription factor and the anthocyanin accumulation during the colour development phase of pomegranate fruits.Materials And MethodsIn this work, R2R3-MYB transcription factor (PgMYB) was isolated and characterized from pomegranate skin through RACE-PCR. The expression of PgMYB gene was monitored in three distinct pomegranate accessions with distinctive skin colour and pattern by semi-quantitative RT-PCR.ResultsThe results indicated a strong association between skin colour in mature pomegranate fruits with the PgMYB transcripts. The highest expression level of PgMYB gene was observed in Poost Siyah Yazd (dark purple skin) throughout the ripening process. Furthermore, comparison of PgMYB amino acid sequences with those of R2R3-MYB family in grapevine, eucalyptus, peach, cacao, populus and Arabidopsis demonstrated that this protein shares high similarity (75-85% amino acid identity) with their conserved MYB domain. Computational structure prediction of PgMYB showed that the three conserved amino acids (Asn, Lys and Lys) are present in the same position of the MYB domain.ConclusionsIt is speculated that PgMYB gene influences the fruit colour and could be used to improve the accumula-tion of anthocyanin pigments in the pomegranate fruit.Keywords: Anthocyanin biosynthesis, MYB transcription factor, Pomegranate
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Pages 26-35BackgroundIn Algeria, date palm is currently confronted to the Bayoud disease. Biotechnological tools such as protoplasts fusion can appear as an alternative to ensure rapid multiplication and improvement of this species.ObjectivesCallogenesis induction in protoplasts isolated from embryogenic callus of three date palm cultivars.Materials And MethodsSome factors influencing the isolation and culture of protoplasts segregated from the calli of three date palm (Phoenix dactylifera L.) cultivars (Deglet Nour, Akerbouch and Degla Beida) were studied. Protoplasts of each cultivar were cultured on a semi-solid medium supplemented with various hormonal balances.ResultsMaceration with an enzymatic solution containing 1.5% cellulase and 1% macerozyme R10 in the presence of 0.5 M mannitol for more than 16 h with gentle agitation allows isolation of a great number of viable protoplasts. In addition, purification of protoplasts on a cushion of 21 or 25% sucrose was effective in cell debris removal and maximum recovery. The culture of isolated protoplasts on a semi-solidified Murashige and Skoog medium, with 0.3% agarose, 2 mg. L-1 2,4-D and 0.5 mg.L-1 BAP allowed good viable protoplast maintenance as well as cell wall regeneration. After more than two months of culture, cell divisions were still occurring and microcalli became visible to the naked eye, containing a large number of cells.ConclusionsThe developed protocol can be useful for application of somatic hybridization to improve date palm cultivars.Keywords: Date palm, Enzymatic solution, Hormonal balance, Microcallus, Phoenix dactylifera, Protoplasts
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Pages 36-42BackgroundSaffron (Crocus sativus L.) is a common but very expensive herbal medicine. As an important traditional medicine, it has an outstanding effect in treating irregular and painful menstruation. Recently, the over-demand tendency of saffron results in an unusual phenomenon in the medicinal markets. Adulterants and saffron-like substitutes are intentionally mixed into medicinal markets and pharmacies or online stores, affecting drug safety and food quality.ObjectivesOur study aimed to identify saffron from its adulterants via DNA barcoding.Materials And MethodsSamples (13 saffron + 4 others containing Carthamus tinctorius or Chrysanthemum x morifolium) obtained from 12 different provinces of China. Through DNA barcoding, samples were compared using three candidate markers, trnH-psbA, rbcL-a and ITS2.ResultstrnH-psbA and rbcL-a were capable of distinguishing different accessions. ITS2 could identify samples even at intra-specific level. According to these three barcodes, four samples were identified saffron-like substitutes.ConclusionsThe adulterant rate in Chinese markets reaches as high as 33.33% that may cause health risks and further may reduce saffron efficacy once is being used as herbal remedy. In order to make a distinction between C. sativus with other genera as adulterants, DNA barcoding is suggested.Keywords: Adulterant identification, Classification, Crocus sativus, DNA barcoding
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Pages 43-48BackgroundThe Osteopontin (OPN) is a highly phosphorylated glycoprotein in numbers of bovine tissues and milk. OPN has been reported to be associated with milk production in cattle.ObjectiveThe genotype and allelic frequencies for OPN and its association with milk production will be evaluated in Iranian Holstein Bulls.Materials And MethodsBulls DNA (100) was isolated. Oligo was used for primer design. Polymerase Chain Reaction was implemented to amplify a 826 bp fragment and the amplicon was digested by BsrI. Restricted Maximum likelihood (REML) method based on average information algorithm using ASRMEL programs (version 3.1) was employed to estimate the genetic parameters and variance of components. The association of OPN genotypes with milk production traits were analysed by the least square method as applied in the general linear model (GLM) procedure of SAS. Allele substitution effects were performed by regression analyses.ResultsAllele frequencies of T and C were 0.59±0.03 and 0.41±0.03, respectively. Genotype frequencies of TT, CT and CC were 34.69, 48.62, and 16.69, respectively. The chi-square test showed the deviation from Hardy-Weinberg equilibrium. Estimated heritability for milk yield, fat yield and its percent, protein yield and its percent were 0.28±0.0061, 0.21±0.0064, 0.22±0.0086, 0.32±0.0065 and 0.34±0.0096 respectively. Allelic substitution effects and differences between genotypes were not significant for milk production traits.ConclusionsThis study suggested that the C allele frequency of OPN was noticeable in Iranian proven bull Holstein population, but was not associated with milk production traits. However, before being practical for the breeding improvement of Iranian Holsteins a larger sample size is required.Keywords: Association, Iranian Holstein bulls, Milk production traits, OPN gene, Polymorphism
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Pages 49-54BackgroundIn prostate cancer, mutated p53 alleles typically contain missense single-base substitution in codon 72 that resides within exons 5-8. Stable p53 proteins in tumor cell nuclei have been associated with malignancy. A role of p53 is the regulation of drug transporters like ABCC1 (MRP1) by an effect on promoter region.ObjectivesThe objective of this study was to identify association of mutations of p53 at codon 72 and 282 and promoter region of ABCC1 with increased risks of prostate cancer.Materials And MethodsFormalin fixed, paraffin-embedded malignant tissues of 45 patients and 45 control samples were evaluated. PCR-RFLP using BstUI for codon 72 and HpaII restriction enzyme for codon 282 p53 gene, and G-1666A promoter region of ABCC1 gene was performed. To assess the frequency of these mutations and to detect new mutations in cancerous samples, PCR-SSCP analysis was performed.ResultsThe frequencies of CC, GC and GG genotypes of codon 72 of p53 were 33.33%, 46.67% and 20.00% in patients with cancer and 15.56%, 48.89% and 35.55% in controls, respectively. The relative allele frequencies of ABCC1 promoter polymorphism were 60.00% A and 40.00% G in patients as opposed to 37.78% for A and 62.22% for G in controls. Genotypic frequencies of p53 codon 72 and G1666A of ABCC1 in patients vs. Controls were statistically significant(p<0.05). The study of these samples with PCR-SSCP displayed some new banding patterns.ConclusionsThe present findings suggest that CC homozygosity in codon 72 of p53 gene and AA genotype in G-1666A of ABCC1 gene may play a role in combination in prostate cancer and increased susceptibility for this malignancy in the Iranian Kurdish population.Keywords: ABCC1, Codon 72 p53, PCR, RFLP, PCR, SSCP, Polymorphism, Prostate cancer
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Pages 55-62BackgroundThe poor permeability of the plasma and nuclear membranes to DNA plasmids are two major barriers for the development of these therapeutic molecules. Therefore, success in gene therapy approaches depends on the development of efficient and safe non-viral delivery systems.ObjectivesThe aim of this study was to investigate the in vitro delivery of plasmid DNA encoding HPV16 E7 gene using cell penetrating peptide delivery system to achieve the best conditions for cell transfection and protein expression. For this purpose, we have used a cationic peptide delivery system, MPG which forms stable non-covalent complexes with nucleic acids for delivery of pEGFP-E7 as a model antigen in vitro.Materials And MethodsDNA construct encoding HPV16 E7 (pEGFP-E7) was prepared in large scale with high purity. MPG peptide/ DNA complexes were prepared at different N/P (nitrogen/phosphate) ratios and physicochemical characterization and stability of nanoparticles were investigated. In vitro peptide-mediated E7-GFP DNA transfection, and its expression was evaluated in three cell types. To quantify the transfection efficiency of this delivery system, transfected cells were harvested and assessed for GFP-positive cells by flow cytometry. Furthermore, E7-GFP expression was confirmed by western blot analysis.ResultsThe cellular uptake of MPG based nanoparticles was shown to be comparable with standard reagent PEI. The COS-7 cells transfected by MPG-based nanoparticles at an N/P ratio of 15:1 showed the highest transfection efficiency and gene expression.ConclusionsThe results indicated that the efficient gene expression depends on both cell type and N/P ratio applied, in vitro. The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPG-based nanoparticles as a potent gene delivery system.Keywords: Cell penetrating peptide, E7, Gene delivery, Human papillomavirus, MPG, based nanoparticles
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Pages 63-67BackgroundAcinetobacter baumannii, is an opportunistic pathogen and is responsible for numerous nosocomial infections. In recent years, this microorganism has been resistant to a wide range of antibiotics. One of the most important mechanisms of resistance in this microorganism is production of metallo-beta-lactamases (MBLs).ObjectivesThe aim of this study was to detect VIM- and IMP-type metallo-beta-lactamase genes in Acinetobacter baumanniiisolates from patients in two Hospitals in Tehran.Materials And Methods104 isolates were tested using the PCR method for the identification of VIM- and IMP-type genes.Resultsvim1, vim2, imp1and imp2 genes were detected in 6.7%, 41.7%, 50% and 1.7% of the isolates from Tehran Heart Center, and in 29.5%, 38.6%, 4.5% and 4.5% of the isolates from Shahid Mutahhari Hospital respectively.DiscussionOur analysis revealed that the majority of the isolates had at least one of these genes, indicating that MBLs production is an important resistance mechanism in Acinetobacter baumannii.Keywords: Acinetobacter baumannii, Imp1, Imp2, Metallo, beta, lactamase, Vim1, Vim2