[Evaluating the antibacterial, hemolytic and cytotoxic activities of the Iranian Rana ridibunda skin extract as a new source of antimicrobial compound]

Amphibian skins possess various antibacterial compounds that are effective against some microbial pathogens and are mostly released in response to environmental stress. In fact, the skin of Rana ridibunda, a large green frog, is a rich source of antimicrobial compounds that can be developed for therapeutic use. In the present study, the skin extract of Iranian Rana ridibunda was evaluated for its antimicrobial, hemolytic and cytototoxic activities. The frog specimens were collected from Minoodasht located in Golesten province in Iran, during 2009. Subsequently, their skins were removed and the intended compounds were extracted. The crude extract was partially purified by gel filtration chromatography. The antimicrobial effects of skin extract were assessed against various microorganisms such as Escherchia coli, methicillin-resistant and-sensitive Staphyloccus aureus, vancomycin-resistant and-susceptible Enteroccus fecalis, Pseudomonas aeroginosa and Candida albicans. In addition, its minimum inhibition concentration, cytotoxic and hemolytic activities were determined. The crude extract of Rana ridibunda skin had valuable antimicrobial effects against methicillin-resistant and-susceptible S. aureus in comparison with E.coli and vancomycin-resistant and-susceptible E. fecalis. Besides, no antimicrobial activities were seen against P. aeroginosa or C. albicans. Moreover, the hemolytic and cytotoxic activities of the skin extract were minimal. The antimicrobial activity of Iranian Rana ridibunda was comparable to those isolated from other Rana species. In conclusion, the skin extract of Rana ridibunda had the potential for a new therapeutic agent against the emerging drug-resistant bacteria, particularly methicillin-resistant and-sensitive S. aureus

[Evaluation of knocking down of the RNAi mediated gamma globin repressor into K562 cells, for gene therapy of beta-thalassemia]

Background and Aim: Beta-thalassemia is a genetic disorder manifested by the presence of anemia in adult patients. One approach to treatment of beta-thalassemia is induction of the fetal gamma-globin gene. One of the gamma-globin repressors is a complex called DRED [Direct repeat erythroid-definitive]. DRED is composed of TR2 and TR4 DNA binding subunits. The aim of this study was to set up the RNAi system to increase the expression of the gamma-globin gene

Materials and Methods: In this study, lipofectamin[Trade Mark] 2000 was used to transfect siRNA molecules and pSV-beta-Galactosidase vector was used as a reporter to monitor transfection efficiency. Real-time PCR method was used to measure TR4 knockdown and gamma-globin expression levels

Results: Our findings showed that K562 cells were transfected by 40% using Lipofectamine[Trade Mark] 2000. Also, the level of TR4 expression decreased by 44% after using TR4siRNA, even though the expression of gamma-globin gene was induced by 1.18 times

Conclusion: Despite a 44% knockdown of TR4, no increase in gamma-globin mRNA was observed. Two factors may count for this observation; first, TR4 knockdown may have been limited by our transfection efficiency. Second, simultaneous knockdown of TR2 and TR4 may lead to increased gamma-globin levels. In conclusion, although knocking down of TR4 expression occurred by using RNAi system, this can not be a solitary efficient way to induce the gamma-globin expression

[Determination of gamma-globin induction levels by different concentrations of hydroxyurea in K 562 cell lines for beta thalassemia treatment

Hydroxyurea [HU] is currently used for beta thalassemia treatment through induction of fetal gamma-globin, In this study, effects of different concentrations of hydroxyurea on induction of gamma-globin gene in K562 cells, and inhibitory effect of siRNA against candidate gene which may be involved in HU mediated gamma-globin induction were assessed. In this eperimantal study, K562 cells were treated by different concentrations of HU [0, 50, 100 and 200 microM]. siRNA against candidate gene in HU mediated gamma-globin gene induction was transfected to K562 cells using lipofectamine 2000. The level of gammal-globin gene induction and inhibition were determined by quantitavie real-time PCR. There were 1.75-, 2.45- and 2.55- fold inductions in gamma-globin gene using 50, 100 and 200 microM HU, respectively. There has been 79.5% down regulation on candidate gene in siRNA transfected K562 cells. This study showed that gamma-globin induction in 100 microM HU is similar to 200 micro M HU treated K562 cells. There was also efficient inhibitory effect on candidate gene which may be involved in HU mediated gamma-globin induction

[Assessing the levels of cAMP response element binding protein 1 [CREB1] after siRNA mediated knockdown in K562 cells]

CREB1 is an important downstream protein for many signaling pathways. By designing efficient siRNAs against CREB1, it may be possible to assess the role of molecules involved in signaling pathways in different cell types. In this research the efficiency of CREB1 knockdown by two different siRNAs in K562 cells has been studied. siRNAs have been designed according to the criteria suggested by Reynolds et al. K562 cells were transfected by siRNA using Lipofectamine 2000. The efficiency of CREB knockdown has been assessed by quantitative relative Real-time PCR. Our results have shown that only one of the siRNAs has a high level of inhibitory effect on CREB1 gene expression. The expression of CREB1 by this siRNA was knocked-down by 87% in K562 cells. In this research, although two siRNAs were designed according to the Reynolds et al. criteria, only one showed an inhibitory effect. Reasons other than the aforementioned criteria may be involved in effectiveness of siRNAs