Cloning and expression of TNF related apoptosis inducing ligand in Nicotiana tabacum

Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand [TRAIL] as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV [Cauliflower Mosaic Virus] helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay [Methylthiazol Tetrazolium Assay]. The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable

effect of iron- gold core shell magnetic nanoparticles on the sensitization of breast cancer cells to irradiation

Herein, iron-gold core shell magnetic nanoparticles Fe[commercial at]Au NPs was investigated as contrasting agent in radiation therapy in the breast cancer. Assessment of cytotoxic and radio sensitizing potential was done by MTT method and Flow cytometry. Radiation was done using Co 60 source. The response of cells to treatment with radiation alone and radiation with nanoparticles was assessed. The study demonstrates that Fe[commercial at]Au nanoparticles do not have considerable cytotoxic effects, but they increase the effectiveness of radiation that means the survival of the group without nanoparticles exposed to 5 Gy radiations is 75%while the group with nanoparticles is 33%. With 2 Gy radiations the survival of the two groups are 87% and 80% respectively

In vitro labeling of neural stem cells with poly-L-lysine coated super paramagnetic nanoparticles for green fluorescent protein transfection

Background: the magnetic nanoparticle-based transfection method is a relatively new technique for delivery of functional genes to target tissues. We aimed to evaluate the transfection efficiency of rat neural stem cell [NSC] using poly-L-lysine hydrobromide [PLL]-coated super paramagnetic iron oxide nanoparticles [SPION]

Methods: the SPION was prepared and coated with PLL as transfection agent and the transfection efficiency was evaluated in rat NSC using enhanced green fluorescent protein-N1 plasmid containing GFP as a reporter gene. NSC was incubated for 24 h in cell culture media containing 25 [micro]g/ml SPION and in different concentrations of PLL [0.25, 0.50, 0.75, 1 and 2 [micro]g/ml]. Cell viability was determined before and after transfection for every concentration using Trypan blue assay. Characterization of prepared uncoated [SPION] and coated [SPION-PLL] complexes were evaluated by a transmission electron microscope and the zeta potential

Results: PLL at 0.75 [micro]g/ml showed optimal results with 25 [micro]g/ml SPION concentration compared with other PLL concentrations [0.25, 0.50, 1 and 2 [micro]g/ml]. The 18% efficiency of the transfected cells showed green fluorescence

Conclusion: transfection with SPION is an efficient, non-viral gene transfere method. Iran. Biomed. J. 17 [2]: 71-76, 2013

[Antifungal effect of zinc oxide nano_particlees of the standard strains of candida albicansbiofim growth on catheters]

Candida albicans is an important human pathogen that can causes systemic infections, predominantly among populations with weak immune systems. Candida spp. produces biofilms on synthetic materials such as catheter, which facilitates adhesion of the organisms to devices. Since the frequent replacement of these devices is difficult and expensive to achieve solutions for rapid identification and control of biofilm infections is essential. The purpose of this study is, the use of zinc oxide nanoparticles in the manufacture catheter and evaluation of its inhibitory effect in preventing of the biofilm formation. In this experimental study, the catheters in making poly vinyl chloride, Znonano particles with aconcentration 2.027 microg/ml was used. Biofilms were formed over a series of time intervals [2, 4, 6, 8, 24 and 48 h]. At each time interval, biofilm formation was measured with the XTT and biomass assay and concurrently assessed by light microscopy. Results showed that catheters containing zinc oxide increases with time in cell density [absorbance increase] were lower than those without catheters. Similar results were also measured in dry weight. This study demonstrated antifungal activity of ZnO. ther for biofilm formation on medical equipment such as catheters, implants, dentures, artificial heart valves, etc can be inhibited by ZnO nanoparticles

Comparative proteomics analysis of a novel gamma-radiation-resistant bacterium wild-type Bacillus megaterium strain WHO DQ973298 recovering from 5 KGy gamma-irradiation

In order to examine radiation-induced proteins in an extremely radio-resistant bacterium, it became possible to perform comparative proteomic analysis on radio-resistance Bacillus megaterium WHO as a wild-type strain for the first time. Variation in cellular proteins profiles of the Bacillus megaterium WHO after 5 KGy gamma -irradiation were analyzed by two-dimensional poly acryl amide gel electrophoresis and silver staining. Although many spots were decreased in density, our primary focus was on the induced spots. The expression level of 48 protein spots showed significant increase under radiation stress. Of these spots, 45 were identified with MALDI TOF-TOF [peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry] after tryptic in-gel digestion. These proteins exhibited various interesting cellular functions including: [i] transcription [ii] translation [iii] signal transduction [iv] carbohydrate transport and metabolism [v] energy production and conversion [vi] nucleotide transport and metabolism [vii] posttranslational modification, protein turnover and chaperones [viii] DNA replication, recombination and repair [ix] bacterial general stress response and [iix] different and some still unknown functions. The appearance of four spots [24, 27, 30 and 36] in response to gamma -irradiation was the distinct result of present study. These proteins appear to mediate processes related to ionizing radiation resistance and clearly demonstrate that Bacillus megaterium WHO, significantly has mechanisms contribute to the ionizing radiation resistance

anticancer activity compared between triptorelin and a new gonadotropin releasing hormone analogue

Gonadotropin releasing hormone [GnRH] plays a key role in reproduction. This decapeptide is synthesized and released by hypothalamus and induces the pituitary gonadotrop cells to release pituitary gonadotropin hormones. In some extrapituitary compartments GnRH and its receptor act as part of the autocrine regulatory system of cell proliferation. The anticancer activity of GnRH and its analogues has been observed by many researchers. In this study the anticancer activity of a new analogue of GnRH and triptorelin was investigated by cell proliferation assay. Results indicate that proliferation of human breast and ovarian cancer cell lines are dose-dependently inhibited. The inhibitory efficiency of the new analogue is proved to be higher than the original triptorelin. In addition to its antimitogenic activity, evidence was found for the involvement of the apoptotic mechanism in the action of the new analogue and triptorelin. In conclusion, the new analogue can be considered as a good pharmaceutical candidate