Cell Journal (Yakhteh)

161-170

Authors: Mohammad Hussein Abnosi , Zahra Jafari Yazdi

Objective: Although arsenite is toxic it is currently recommended for the treatment of malignancies. In this study the effects of sub-micromolar concentrations of sodium arsenite on the viability, morphology and mechanism of cell death of rat bone marrow mesenchymal stem cells (BMCs) over 21 days was investigated. Materials and Methods: In this experimental study, BMCs were extracted in Dulbecco’s Modified Eagles Medium (DMEM) containing 15% of fetal bovine serum (FBS) and expanded till the 3rd passage. The cells were treated with 1, 10, 25, 50, 75 and 100 nM of sodium arsenite for 21 days and the viability of the cells estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium (MTT) and trypan blue staining. Cells were then treated with the selected dose (25 nM) of sodium arsenite to determine their colony forming ability (CFA) and population doubling number (PDN). Morphology of the cells was studied using florescent dyes, and the integrity of the DNA was investigated using the comet assay and agarose gel electrophoresis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the caspase 3 assay were then applied to understand the mechanism of cell death. Data was analyzed using one way ANOVA, Tukey test. Results: A significant reduction of viability, PDN and CFA was found following treatment of BMCs with 25 nM sodium arsenite (p<0.05). Cytoplasm shrinkage and a significant decrease in the diameter of the nuclei were also seen. Comet assay and agarose gel electrophoresis revealed DNA breakage, while positive TUNEL and activated caspase 3 confirmed the apoptosis. Conclusion: A low concentration of sodium arsenite (25 nM) caused reduction of viability due to induction of apoptosis. Therefore, long term exposure to low dose of this chemical may have unwanted effects on BMCs.

177-184

Authors: Mehdi Mehdizadeh , Fataneh Dabaghian , Akram Nejhadi , Hassan Fallah-huseini , Samira Choopani , Nima Shekarriz , Nima Molavi , Arghavan Basirat , Farzaneh Mohammadzadeh Kazorgah , Alireza Samzadeh-Kermani , Sara Soleimani Asl

Objective: The spice Zingiber officinale or ginger possesses antioxidant activity and neuroprotective effects. The effects of this traditional herbal medicine on 3,4-methylenedioxymethamphetamine (MDMA) induced neurotoxicity have not yet been studied. The present study considers the effects of Zingiber officinale on MDMA-induced spatial memory impairment and apoptosis in the hippocampus of male rats. Materials and Methods: In this experimental study, 21 adult male Sprague Dawley rats (200-250 g) were classified into three groups (control, MDMA, and MDMA plus ginger). The groups were intraperitoneally administered 10 mg/kg MDMA, 10 mg/kg MDMA plus 100 mg/kg ginger extract, or 1 cc/kg normal saline as the control solution for one week (n=7 per group). Learning memory was assessed by Morris water maze (MWM) after the last administration. Finally, the brains were removed to study the cell number in the cornu ammonis (CA1) hippocampus by light microscope, Bcl-2 by immunoblotting, and Bax expression by reverse transcription polymerase chain reaction (RT-PCR). Data was analyzed using SPSS 16 software and a one-way ANOVA test. Results: Escape latency and traveled distances decreased significantly in the MDMA plus ginger group relative to the MDMA group (p<0.001). Cell number increased in the MDMA plus ginger group in comparison to the MDMA group. Down-regulation of Bcl-2 and up-regulation of Bax were observed in the MDMA plus ginger group in comparison to the MDMA group (p<0.05). Conclusion: Our findings suggest that ginger consumption may lead to an improvement of MDMA-induced neurotoxicity.

193-202

Authors: Mahboobeh Ashrafi , Seyedeh Zahra Bathaie , Saeid Abroun

Objective: N-nitroso-N-methylurea (NMU) induces breast cancer in rodents, particularly in rats. This model of breast cancer is very similar to human breast cancer. As a continuation of our recent work, we investigated the expressions of cyclin D1 and p21 in NMU-induced breast cancer of Wistar Albino rats. Materials and Methods: In this experimental study, mammary carcinoma was induced in female Wistar Albino rats by a new protocol which included the intraperitoneal injection of NMU (50 mg/kg) at 50, 65, and 80 days of the animal’s age. The animals were weighed weekly and palpated in order to record the numbers, location, and size of tumors. Subsequently tumor incidence (TI), latency period (LP), and tumor multiplicity (TM) were reported. About four weeks after the tumor size reached 1.5 cm3, rats were sacrificed. Cyclin D1 and p21 expressions in tumors and normal mammary glands from normal rats were measured by reverse-transcription polymerase chain reaction (RT- PCR) and Western blot analysis. Statistical analysis of the data was performed using SPSS software version 16.0. Results: The efficiency of tumor induction was 65%, LP was 150 days, and a TM of 1.43 ± 0.53 per rat was noted. RT-PCR and Western blot data indicated significant (p<0.05) induction of both cyclin D1 and p21 expressions in rat mammary tumors compared with normal tissue from the control group. Conclusion: These results indicate an efficient mammary tumor induction protocol for this type of rat, which is accompanied by an increase in cyclin D1 and p21 expressions.

209-214

Authors: Shaghayegh Anvari , Shahin Najar Peerayeh , Mehrdad Behmanesh , Mina Boustanshenas

Objective: Vibrio cholerae (V. cholerae) causes a potentially lethal disease named cholera. The cholera enterotoxin (CT) is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin (Zot) and accessory cholera enterotoxin (Ace). The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli (E. coli) and determination of some characteristics of the recombinant Ace protein. Materials and Methods: In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli (DH5 α) host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-β-D-galctoside (IPTG) at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). Results: The recombinant Ace protein with a molecular weight of 18 kDa (dimeric form) was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of ≥200 μg/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test. Conclusion: This study described an E. coli cloning and expression system (E. coli BL21- pET-28a-ace) for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein.

225-230

Authors: Bahar Movaghar , Saeedeh Askarian

Objective: The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors (progesterone receptor), cytokines [leukemia inhibitory factors (LIF)], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor (PgR) genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles. Materials and Methods: In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice (6- 8 weeks old) were injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). Their blastocysts (approximately n= 120) were flushed out after 3.5 days, following administration of human chorionic gonadotropin (hCG). The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Data were analyzed by the student’s t-test (one sample t-test). Results: Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts (p<0.05). Conclusion: Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from the natural cycle. The results observed in this study have shown the possibility of an unfavorable effect on implantation and pregnancy rate.