1 1028-852X Pasteur Institute of Iran 49 Related Fields DNA Damage in Leukocytes from Fanconi Anemia (FA) Patients and Heterozygotes Induced by Mitomycin C and Ionizing Radiation as Assessed by the Comet and Comet-FISH Assay Mohseni Meybodi Anahita Mozdarani Hossein 1 1 2009 13 1 1 8 06 04 2010 26 03 2013 Background: Lymphocytes of Fanconi anemia (FA) show an increased sensitivity to the alkylating agents such as mitomycin C (MMC), but their responses to gamma-irradiation is controversial. The extent of DNA damage in leukocytes of FA patients following irradiation and MMC treatment was studied at cellular and single chromosome level. Methods: DNA damage induced by gamma-rays and MMC was measured in leukocytes of FA patients and carriers at whole genome level using the comet assay. Also, at the DNA level of specific chromosome involved in this disease using a modified comet-FISH protocol with whole chromosome painting probes (chromosomes 16 and 13), DNA damage in leukocytes of FA patients and heterozygotes were compared to healthy individuals. Results: Baseline DNA damage in leukocytes of patients and heterozygotes was higher than in controls. Net induced DNA damage by gamma-rays in leukocytes of FA cases was not significantly different from that of healthy donors and heterozygotes. Net induced DNA damage by MMC was statistically higher and significantly different (P<0.05) in patients than other groups. Hybridization of chromosome 16 reveals more signals in the tail but the number of spots in the tail was not significantly higher than the hybridization spots for chromosome 13 in both gamma-irradiated and MMC treated samples. Conclusion: Results indicate that DNA damage induced by MMC could be a better index for diagnosis of FA patients compared to gamma-rays. Results of comet-FISH showed no difference between the sensitivity of chromosome 16 and 13 to MMC and radiation. It may indicate that, although the FA-A gene is located on chromosome 16, this chromosome might have a similar sensitivity as other chromosomes.
50 Related Fields Cloning and Expression of Simian Rotavirus Spike Protein (VP4) in Insect Cells by Baculovirus Expression System Khodabandehloo Mazaher Shamsi Shahrabadi Mahmoud Keyvani Hossein Bambai Bijan 1 1 2009 13 1 9 18 06 04 2010 27 03 2013 Background: VP4 protein is as spikes on rotavirus outer capsid shell which is responsible for virus attachment to the host. VP4 induces production of neutralizing antibodies which could be used for serotyping of different isolates. Methods: Simian rotavirus SA11 gene 4 cDNA was cloned into a cloning plasmid pDONRTM by recombination reaction using clonase II enzyme mix. The resulting clone was called VP4-entry clone. In the second recombination reaction, cloned gene was inserted into the linear DNA of the Baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) downstream of the strong polyhedrin promoter. The recombinant AcNPV-VP4 DNA was transfected by lipofection into the insect cell line, Spodoptera frugiperda (Sf9) cells. Expression of VP4 in the Sf9 cells was confirmed by the immunofluorescence test using rabbit polyclonal anti-rotavirus and anti-rabbit FTIC-conjugated antibodies by Western immuno-blotting technique. The antigenicity of the expressed protein was determined by immunizing rabbits and testing the sera by Western-blotting and neutralization method. Results: The cloned VP4 gene was obtained and expressed in baculovirus system. The specificity of the expressed protein was confirmed by its reactivity with anti-rotavirus antibody. Antibody produced against the expressed protein showed neutralizing activity for rotavirus indicating that the protein was biologically active and could induce natural antibody response. Conclusion: the expressed protein from rotavirus VP4 gene has a potential for development of rotavirus vaccine. 51 Related Fields Enhancement of RNA Interference Effect in P19 EC Cells by an RNA-dependent RNA Polymerase Esmaeili Fariba 1 1 2009 13 1 19 25 06 04 2010 27 03 2013 Background: RNA interference (RNAi) is a phenomenon uses double-stranded RNA (dsRNA) to specifically inhibit gene expression. The non-specific silencing caused by interferon response to dsRNA in mammalian cells limits the potential of utilizing RNAi to study gene function. Duplexes of 21-nucleotide short interfering dsRNA (siRNA) inhibit gene expression by RNAi. In some organisms, siRNA can also function as a primer converting mRNA into dsRNA that are further cleaved to produce more siRNA. This activity involves the enzyme RNA-dependent RNA polymerase (RdRP). There are no known RdRP involved in RNAi in mammals. By using an RdRP from Caenorhabditis elegance named ego-1, investigators intend to enhance RNAi effect in mammalian cells. The aims of this project were: 1) to investigate the efficiency of siRNA to enhanced green fluorescent protein (eGFP) gene silencing and 2) to enhance the RNAi effect. Methods: We used a vector-based siRNA to target eGFP. Also we used a vector expressing ego-1 to test for a possible amplification effect of RNAi. The expression of eGFP in the cells was detected by using fluorescent microscopy, flowcytometry and Western-blotting. Results: Transfection of the plasmid into P19 cells significantly decreased eGFP fluorescence. In addition, eGFP protein was reduced. Preliminary data suggested that the presence of ego-1 enhanced the RNAi effect. Conclusion: The results indicated that use of hairpin siRNA expression vectors for RNAi is a promising method to inhibition of gene expression in mammalian cells. Also, introducing RdRP enzyme to mammalian cells might amplify the RNAi effect in the cells. 52 Related Fields Increased Cytotoxicity of Cisplatin in SK-MEL 28 Melanoma Cells upon Down-Regulation of Melanoma Inhibitor of Apoptosis Protein Mousavi-Shafaei Parisa Ziaee Abed-Ali Zangemeister-Wittke Uwe 1 1 2009 13 1 27 34 06 04 2010 27 03 2013 Background: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. Methods: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. Results: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. Conclusion: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status. 53 Related Fields Differentiation and Apoptosis of U937 Leukemia Cells by an Active Compound from Dendrostellera lessertii Meshkini Azadeh Yazdanparast Razieh Haidari Mojgan 1 1 2009 13 1 35 42 06 04 2010 27 03 2013 Background: Regarding the strong differentiation and anti-leukemic activity of Dendrostellera lessertii extract, an active agent with similar capabilities was isolated from the crude extract of the plant leaves. The aim of this study was to determine whether the anti-proliferative effect observed for this compound, is differentiation-dependent among the drug-treated cells, using U937 cells. Methods: Monocyte differentiation was evaluated by Wright-Giemsa staining, latex particle assay and flow cytometry. Induction of apoptosis was analyzed by Annexin-PI double staining. Results: The new compound, at 0.5-2.5 µg/ml inhibited proliferation of U937 cells by more than 70% and their viabilities were decreased by 47 ± 2.1% after 72 h of treatments. In addition, we found that the effect of the new compound on U937 cells was associated with differentiation toward monocyte/macrophage lineage based on nitroblue tetrazolium reduction assay, morphology change, phagocytic activity and expression of cell surface markers (CD14 and CD11b) as analyzed by flow cytometry. Moreover, our results indicated that the treatment of U937 cells with the new compound for 3 to 4 days induced apoptosis as assayed qualitatively by acridine orange/ethidium bromide and Annexin-V/PI double staining technique using flow cytometry. Conclusion: Based on these observations, it is concluded that the anti-proliferative function of the new compound is exerted through differentiation-dependent apoptosis among the treated cells, similar to the function of 3-hydrogenkwadaphnin previously characterized from D. lessertii crude extract. 54 Related Fields A Comparative Study of Therapeutic Benefits of Intraspinal and Intravenous Bone Marrow Stromal Cell Administration to Spinal Cord Injuries Khalatbary Ali Reza Tiraihi Taki 1 1 2009 13 1 43 48 06 04 2010 27 03 2013 Background: Recent reports demonstrated that intravenous route as a minimally invasive method, similar to direct injection, is suitable for bone marrow stromal cell (BMSC) transplantation. In this study, we made a comparison of intraspinal and intravenous route of BMSC administration to repair injured spinal cord tissue. Methods: Six groups of adult female rats were used in this study. Laminectomy and spinal cord injury (SCI) were carried out at first lumbar vertebra level (L1). Labeled stromal cells were administered intraspinally and intravenously in experimental groups one week after SCI. In control groups, serum was administered in the same way. Another groups consisted of laminectomy alone and SCI. Behavioral testing was performed weekly to 5 weeks post injury. Tissue processing and immune-histochemical studies were performed four weeks after cell transplantation. Results: Mean of Basso-Beattie-Bresnehan (BBB) scale scores in intraspinal and intravenous groups were 15.8 ± 0.44 and 15.6 ± 0.54, while in their controls were 10.6 ± 0.33 and 10.6 ± 0.56, respectively. BBB scale in laminectomy and SCI groups were 21 and 10.5 ± 0.36, respectively. Immuno-histochemical staining visualized BMSC in the site of injury. Differentiation of a few implanted cells to neuron and glial cell was detected in intravenous group, while only differentiation to glial cells was detected in intraspinal group. Conclusion: The results of this study suggest that intravenous administration of BMSC, such as intraspinal method, provides therapeutic benefits for SCI. 55 Related Fields Effect of Spatial Learning on Hippocampal Testosterone in Intact and Castrated Male Rats Mohaddes Gisou Naghdi Nasser Khamnei Saeed Khatami Shohreh Haeri Ali 1 1 2009 13 1 49 58 06 04 2010 26 03 2013 Background: Sex steroids and their receptors exist in hippocampus and affect spatial learning and memory. This study was designed to measure testosterone level of CA1 and to assess the effect of spatial learning on its amount in left and right hippocampus of adult male rats. Methods: Sixteen rats were divided into two intact and castrated groups, and then trained in Morris water maze (MWM). Another 40 animals were divided into four groups and their right or left hippocampus cannulated. Half of the animals in each group were castrated simultaneously. All the animals were trained in MWM. Microdialysis was performed and steroid contents of hippocampal dialysate were analyzed through HPLC/ultraviolet detection device method. Results: Results showed no significant differences between control and castrated animals in spatial learning after four days of training. Gonadectomy did not change testosterone level in CA1 region of hippocampus. Spatial learning decreased testosterone levels in CA1 region of hippocampus in right hippocampus of the non-castrated group. Significant differences were indicated in testosterone level between left and right hippocampus, in favor of left side in all groups. Conclusion: Castration does not affect learning. Testosterone, as a neuromodulator, exists in CA1 region of hippocampus and training can decrease its level only in right hippocampus significantly. Lesser testosterone content of right hippocampus may show the conversion of it to other metabolites. 56 Related Fields Glucose Influence on Copper Ion-Dependent Oxidation of Low Density Lipoprotein Ghaffari Mohammad Ali Mojab Samad 1 1 2009 13 1 59 64 06 04 2010 26 03 2013 Background: It is well established that oxidative modification of low density lipoprotein (LDL) plays a causal role in human atherogenesis and the risk of atherosclerosis is increased in patients with diabetes mellitus. We examined the in vitro effect of glucose on native and glycated LDL oxidation using copper ion dependent oxidation system. Methods: In this study, LDL was isolated from plasma by ultracentrifugation using a single step discontinuous gradient. Native LDL preparations were glycated by glucose and also were oxidized by copper ions. LDL glycation and oxidation levels were estimated by sodium periodate assay and thiobarbituric acid reactive substances (TBARS), respectively. Then, native LDL was incubated with glucose and copper and LDL oxidation was estimated by TBARS. Finally, oxidation of glycated LDL was studied in presence of copper ions by TBARS and relative electrophoretic mobility on polyacrylamide gel. Results: This study showed that glucose considerably decreased the oxidation of native LDL by copper ions. But oxidation of glycated LDL elevated with presence of copper ions. Conclusion: The results of this investigation show that LDL glycated in vitro is prone to oxidation by copper ions. Thus, promotion of glycated LDL oxidation by glucose is specific for copper ion dependent oxidation and involves increased copper ion reduction. These results provide one mechanism that may enhanced LDL oxidation in diabetes and thus contribute to the pathogenesis of atherosclerosis in diabetic patients.