1 1028-852X Pasteur Institute of Iran 104 Related Fields Induction of Apoptosis on K562 Cell Line and Double Strand Breaks on Colon Cancer Cell Line Expressing High Affinity Receptor for Granulocyte Macrophage-Colony Stimulating factor (GM-CSF) Habibi Roudkenar Mehryar Bouzari Saeid Kuwahara Yoshikazu Mohammadi Amaneh Baba Taisuke Oloomi Mana Fukumoto Manabu 1 1 2008 12 1 1 6 13 04 2010 27 03 2013 Background: Immunotoxins are comprised of both the cell targeting and the cell killing moieties. We previously established a new immunotoxin, i.e. Shiga toxin granulocyte macrophage-colony stimulating factor (StxA1-GM-CSF), comprises of catalytic domain of Stx, as a killing moiety and GM-CSF, as a cell targeting moiety. In this study, the ability of the immunotoxin to induce apoptosis and double strand breaks (DSB) on different cell lines was investigated. Methods: The recombinant hybrid protein was expressed in bacterial expression system and purified with nickel-nitrilotriacetate acid resin. The K562 (erythroid leukemia) cell line and LS174 (colon carcinoma) were used in this study. The neutral comet assay was carried out for the detection of DSB and Hoechst staining was performed for apoptosis. Results: StxA1-GM-CSF effectively induced apoptosis on K562 cell line and DNA Double Strand Break (DSB) were observed on colon cancer cell line treated with StxA1-GM-CSF. Conclusion: This novel action i.e. DNA damage might be a relevant mechanism of action for StxA1-GM-CSF that is designed to act as immunotoxin, although further investigation is required.
111 Related Fields Evaluation of Growth Inhibitory and Apoptosis Inducing Activity of Human Calprotectin on the Human Gastric Cell Line (AGS) Zali Hakimeh Rezaei-Tavirani Mostafa Kariminia Amina Yousefi Reza Shokrgozar Mohammad Ali 1 1 2008 12 1 7 14 13 04 2010 27 03 2013 Background: Calprotectin is cytotoxic agent that its anti-tumor effects are governed through suppression of topoisomerase II a key enzyme in apoptosis. In previous studies, cytotoxicity and apoptotic effects of calprotectin are shown on different cancer cell lines, but not human gastric cancer cell lines. In the present study, cytotoxicity and apoptotic effects of calprotectin on human gastric adenocarcinoma cancer cell line (AGS) were evaluated. Methods: The AGS cells were exposed to the different concentrations of calprotectin for 24, 48 and 72 hours. Cell proliferation was assessed using dimethylthiazol diphenyl tetrazolium bromide assay and dye exclusion tests. For evaluation of cytotoxic mechanism in calprotectin on AGS cells, flow cytometric analysis was performed. Results: Our results revealed that calprotectin induces growth inhibition of AGS in a dose- and time-dependent manner. Results of this investigation showed that sensitivity of AGS cells to cytotoxic effect of human calprotectin was highly remarkable. In addition, growth inhibitory effect of this cytotoxic agent mostly was governed through induction of apoptosis in the AGS cells. Conclusion: These findings indicated that calprotectin induces growth inhibition and apoptosis in the AGS cells. 105 Related Fields Rapid Screening of Toxigenic Vibrio cholerae O1 Strains from South Iran by PCR-ELISA Mousavi Seyed Latif Nazarian Shahram Amani Jafar Karimi Rahgerdi Ahmad 1 1 2008 12 1 15 21 13 04 2010 27 03 2013 Background: The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. Methods: The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. Results and Conclusion: The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains. 110 Related Fields The Full Length Hepatitis C Virus Polyprotein and Interactions with the Interferon-Beta Signalling Pathways in vitro Sabouri Ghannad Masoud Zamani Alireza 1 1 2008 12 1 23 34 13 04 2010 27 03 2013 Background: Hepatitis C is a global health problem. The exact mechanisms by which hepatitis C virus (HCV) can evade the host immune system have become controversial. Whether HCV polyproteins modulate IFN signalling pathways or HCV proteins are responsible for such a property is the subject of interest. Therefore, an efficient baculovirus delivery system was developed to introduce the whole genome of HCV1B minus 3’untranslated region (UTR) (HCV1BΔ3’UTR) into hepatoma cells. Methods: The whole genome of HCV genotype 1b was developed into hepatoma cells. Also, two replicon constructs were used in this research: a recombinant baculovirus containing the culture adapted sub-genomic replicon (FK5.1) derived from HCV genotype 1b, and a mutant form containing an inactivating mutation within the non-structural protein 5B (NS5B). Results: As expected, the baculovirus carrying the FK5.1 replicon induced the production of IFN-gamma as judged by the use of an IFN- gamma promoter luciferase reporter construct, whereas the GND baculovirus (a control polymerase knock-out replicon) and the full-length 3’UTR deletant failed to induce luciferase expression. The activation of both IFN regulatory factor 3 (IRF3) and nuclear factor κB (NFκB), two transcription factors induced by dsRNA signalling were examined. Both the wild type and GND-mutant replicon blocked the dsRNA-induced activation of IRF3 and NFκB. Conclusion: Inhibition of the transcriptional response to IRF3 and NFκB seems to be one of the multiple mechanisms which HCV employs to escape the host immune defence. In contrast, the full length 3’UTR deletant had no significant effect on either transcription factor. These results may be attributed to the function of HCV subgenomic replicons when compared with full length 3’UTR deletant. 107 Related Fields The Morphological Changes of Adult Mouse Testes after 60Co γ-Radiation Koruji Morteza Movahedin Mansoureh Mowla Seyed Javad Gourabi Hamid Jabbary Arfaee Ali 1 1 2008 12 1 35 42 13 04 2010 27 03 2013 Background: Cytotoxic therapy can lead to prolonged azoospermia or even sterility. In the present study, we investigated the morphological changes of mouse testes after gamma-Radiation. Methods: After anaesthetizing of NMRI mice, testes and their surrounding tissues were irradiated using a cobalt therapy machine. Four experimental groups were irradiated with fractionated doses of: 1.5+8, 1.5+12 and 1.5+16 Gy (with an interval of 24 h) and single dose of 14 Gy. Non-irradiated mice were considered as control group. Testes were removed 4, 6 and 8 weeks following irradiation, weighed and processed for light microscopic study. Diameters of seminiferous tubules and their lumens, epithelium thickness, percentage of different types of tubules and number of spermatogenic cell were measured. Moreover, sperm count motility and viability rates were evaluated in epididymis. Results: Number of normal tubules, epithelium thickness, tubules diameter and lumen diameter were significantly reduced with high dose irradiation in comparison with control testes. The recovery was observed after 8 weeks. Epididymal sperm count, motility and viability rates were significantly decreased in the irradiated mice comparing non-irradiated ones. These parameters were increased after 8 weeks. Conclusion: According to the results, irradiation can cause temporary azoospermia in mouse and this effect is reversible after 8 weeks. 108 Related Fields The Assessment of Feto-Maternal Hemorrhage in an Artificial Model Using Anti-D and Anti-Fetal Hemoglobin Antibody by Flow Cytometry Pourazar Abassali Homayouni Vida Rezaei Abbas Andalib Alireza Oreizi Farzad 1 1 2008 12 1 43 48 13 04 2010 27 03 2013 Background: When fetal red cells enter the maternal circulation from placenta, an event would be happened that is described as feto-maternal hemorrhage (FMH). This life-threatening condition could be detected by using RBC antigens (surface antigens and intracellular antigens). Therefore, the measurement of fetal RBC in an artificial model would be useful to calculate FMH and consequently the dosage of Rhogam for prophylaxis. The aim of the present study was to evaluate FMH in an artificial mixture model. Methods: A series of 40 artificial specimens were prepared consisting of Rh(D) negative adult blood (non-immunized) spiked with varying amounts of Rh(D) positive cord blood from mothers between 20-30 years old in Shahid Beheshti Hospital, Tehran, Iran. Monoclonal anti-D and anti-HbF (fetal hemoglobin) were used for detection of fetal RBC in artificial mixture sample modeling. Results: This study showed that the percentage of fetal cells in artificial sample for anti-D antigen is in ranges of 0.28%-0.32% for a 0.25% dilution mixture, and 1.3%-2.05% for the mixture with dilution 2%. In addition, the ranges of data for anti-HbF staining was obtained 0.2%-0.34% for the 0.25% dilution sample, and the ranges of 1.04-1.8% for the 2% dilution. The regression analysis indicated that the correlation of anti-D assessment with expected standard method was r2 = 0.9672 and anti-HbF assessment was r2 = 0.8842. Conclusion: Although both molecule targets could be used for detection of fetal RBC, in this model, anti-D staining was more accurate than anti-HbF staining. However, since anti-D can not be utilized for low-density or weak phenotype and other incompatibility, the anti-HbF labeling could be used for all FMH. 109 Related Fields The Frequency of Eight Common Point Mutations in CYP21 Gene in Iranian Patients with Congenital Adrenal Hyperplasia Ramazani Ali Kahrizi Kimia Razaghiazar Maryam Mahdieh Nejat Koppens Paul 1 1 2008 12 1 49 53 13 04 2010 27 03 2013 Background: Congenital Adrenal Hyperplasia (CAH, the inherited inability to synthesize cortisol) is one of the most common (1 in 10000 to 1 in 15000) autosomal recessive disorders. More than 95% of cases of CAH are caused by 21-hydroxylase deficiency (21-OHD). Females with severe, classic 21-OHD are exposed to excess androgens prenatally and are born with virilized external genitalia. Most patients cannot synthesize sufficient aldosterone to maintain sodium balance and may develop potentially fatal salt wasting crisis if not treated. Methods: We applied allele specific PCR to detect the eight common mutations in the CYP21 gene in patients. Fifty unrelated patients with symptoms of classical CAH were studied. Results and Conclusion: Seventy percent of our subjects had these mutations. The most frequent mutations were found to be I2G and del-8bp (28% and 13%, respectively). The frequencies of other alleles were as following: I172N, 9% V281L, 3% exon 6 cluster (I236N, V237E and M239K), 4% Q318X, 9% R356W, 5% and P30L, 0%. The frequency of mutations did not differ substantially from other ethnics, however, a higher rate of del-8 bp (13%) was found in our population. The aim of this study was to detect common mutations for setting up a molecular method for prenatal diagnosis. 106 Related Fields Molecular Characterization of a Squalene epoxidase Gene in Dermatophyte Pathogen Trichophyton tonsurans Motavaze Kamyar Namvar Zahra Emami Massoud Noorbakhsh Fatemeh Rezaie Sassan 1 1 2008 12 1 55 58 13 04 2010 27 03 2013 Background: Trichophyton tonsurans is one of the dermatophyte fungi which invades the skin and hair of human. Several properties of this fungus have been investigated so far. However a few studies were carried out in the field of molecular biology of this fungus. In the present study, we tried to identify the Squalene epoxidase gene which is related to synthesis of ergosterol in this fungus. Methods: Pairs of 23 and 24 nucleotides primers were designed from highly conserved regions of the similar genes in other fungi. Mentioned primers were utilized in PCR by using isolated genomic DNA of T. tonsurans whereas the PCR fragments were then sequenced. Results and Conclusion: Nucleotides (n = 558) have been sequenced from this new gene which encodes a polypeptide with 186 amino acids. Sequences comparison in gene data banks (NCBI, NIH) for this part of DNA and its deduced amino acid revealed significant homology with members of the eukaryotic Squalene epoxidase.