2024-03-29T13:31:35+04:30 http://ibj.pasteur.ac.ir/browse.php?mag_id=62&slc_lang=en&sid=1
62-944 2024-03-29 10.1002
Iranian Biomedical Journal IBJ 1028-852X 2008-823X 10.61186/ibj 2013 17 2 Expression Analysis of RNA-Binding Motif Gene on Y Chromosome (RBMY) Protein Isoforms in Testis Tissue and a Testicular Germ Cell Cancer-Derived Cell Line (NT2) Mehdi Alikhani Mehdi Sharifi Tabar Shahab Mirshahvaladi Abolfazl Kheimeh Mohammad Ali Sadighi Gilani Marjan Sabbaghian marjan.sabbaghian@gmail.com a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer-derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages. Methods: Full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms. Results: Selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate. Conclusion: The results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility. Protein isoforms Spermatogenesis Male infertility 2013 4 01 54 61 http://ibj.pasteur.ac.ir/article-1-944-en.pdf 10.6091/ibj.1148.2013
62-910 2024-03-29 10.1002
Iranian Biomedical Journal IBJ 1028-852X 2008-823X 10.61186/ibj 2013 17 2 Bone Marrow Stromal Cell Transdifferentiation into Oligodendrocyte-Like Cells Using Triiodothyronine as a Inducer with Expression of Platelet-Derived Growth Factor α as a Maturity Marker Hojjat-Allah Abbaszadeh Taki Tiraihi takialtr@modares.ac.ir Ali Reza Delshad Majid Saghedi Zadeh Taher Taheri Background: The present study investigated the functional maturity of oligodendrocyte derived from rat bone marrow stromal cells (BMSC). Methods: The BMSC were isolated from female Sprague-Dawley rats and evaluated for different markers, such as fibronectin, CD106, CD90, Oct-4 and CD45. Transdifferentiation of OLC from BMSC was obtained by exposing the BMSC to DMSO and 1 µM all-trans-retinoic acid during the pre-induction stage and then induced by heregulin (HRG), platelet-derived growth factor AA (PDGFR-α), fibroblast growth factor and T3. The neuroprogenitor cells (NPC) were evaluated for nestin, neurofilament 68, neurofilament 160 and glial fibrillary acidic protein gene expression using immunocytochemistry. The OLC were assessed by immunocytochemistry for O4, oligo2, O1 and MBP marker and gene expression of PDGFR-α was examined by RT-PCR. Results: Our results showed that the fibronectin, CD106, CD90, CD45 and Oct-4 were expressed after the fourth passage. Also, the yield of OLC differentiation was about 71% when using the O1, O4 and oligo2 markers. Likewise, the expression of PDGFR-α in pre-oligodendrocytes was noticed, while MBP expression was detected in oligodendrocyte after 6 days of the induction. Conclusion: The conclusion of the study showed that BMSC can be induced to transdifferentiate into mature OLC. Bone marrow stromal cell Triiodothyronine Platelet-derived growth factor α 2013 4 01 62 70 http://ibj.pasteur.ac.ir/article-1-910-en.pdf 10.6091/ibj.11162.2013
62-947 2024-03-29 10.1002
Iranian Biomedical Journal IBJ 1028-852X 2008-823X 10.61186/ibj 2013 17 2 In vitro Labeling of Neural Stem Cells with Poly-L-Lysine Coated Super Paramagnetic Nanoparticles for Green Fluorescent Protein Transfection Salim Albukhaty Hossein Naderi-Manesh naderman@modares.ac.ir Taki Tiraihi 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 µg/ml SPION and in different concentrations of PLL (0.25, 0.50, 0.75, 1 and 2 µg/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 0.75μg/ml showed optimal results with 25 μg/ml SPION concentration compared with other PLL concentrations (0.25, 0.50, 1 and 2 μg/ml). The 18% efficiency with the transfected cells showed green fluorescence. Conclusion: Transfection with SPION is an efficient, non-viral gene transfere method. Neural Stem cells (NSC) Nanoparticles Transfection 2013 4 01 71 76 http://ibj.pasteur.ac.ir/article-1-947-en.pdf 10.6091/ibj.1114.2013
62-919 2024-03-29 10.1002
Iranian Biomedical Journal IBJ 1028-852X 2008-823X 10.61186/ibj 2013 17 2 Aberrant Promoter Methylation Profile of Niemann-Pick Type C1 Gene in Cardiovascular Disease Masoumeh Afzali Alireza Nakhaee alireza_nakhaee@yahoo.com Seyed Payman Tabatabaei Kourosh Tirgar-Fakheri Mohammad Hashemi Background: The protein of Niemann-pick type C1 (NPC1) gene promotes the egress of cholesterol from late endosomes and lysosomes to other cellular compartments and contributes to a process known as reverse cholesterol transport. This study aimed to examine whether promoter methylation of NPC1 is associated with risk of cardiovascular disease (CVD). Methods: Fifty CVD patients and 50 healthy subjects as the control group were recruited in this study. Promoter methylation of NPC1 gene was defined using a nested-methylation specific polymerase chain reaction method. Statistical analyses were done using the chi-square, t-test or ANOVA tests. Results: Our study showed that the frequency of semi-methylated promoter (methylated/unmethylated status) was significantly higher in CVD patients than that in controls (OR = 6.521, 95% CI = 2.211-19.215, P = 0.008). However, a completely methylated promoter (methylated/methylated status) was not detected in any subjects in either of the two groups tested. Additionally, the analysis of clinical data according to the methylation status of NPC1 gene demonstrated that serum levels of total cholesterol, total triglycerides, high low-density lipoprotein cholesterol (LDL-C) and low high-density lipoprotein cholesterol (HDL-C) are influenced by NPC1 methylation, so that subjects with a completely unmethylated promoter (Unmethylated/unmethylated status) held lower levels of total triglycerides, total cholesterol, LDL-C and higher levels of HDL-C. Conclusion: Our findings propose that the NPC1 promoter methylation is a probable mechanism that can result in reduced/impaired NPC1 expression/activity and may thus contribute to progression of CVD. Niemann-pick type C1 (NPC1) Promoter methylation cardiovascular disease 2013 4 01 77 83 http://ibj.pasteur.ac.ir/article-1-919-en.pdf 10.6091/ibj.11432.2013
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Iranian Biomedical Journal IBJ 1028-852X 2008-823X 10.61186/ibj 2013 17 2 Compare the Effect of Eicosapentaenoic Acid and Oxidized Low-Density Lipoprotein on the Expression of CD36 and Peroxisome Proliferator-Activated Receptor Gamma Hossein Babaahmadi Rezaei Mahmood Doosti Doostimd@sina.tums.ac.ir Mahdi Aminian Parisa Shabani Background: There is evidence that CD36 promotes foam cell formation through internalizing oxidized LDL (ox-LDL) into macrophages therefore, it plays a key role in pathogenesis of atherosclerosis. In addition, CD36 expression seems to be mediated by nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-γ). The aim of the present study was to evaluate and compare the effect of PPAR-γ ligands, eicosapentaenoic acid (EPA) as an anti-atherogenic factor and ox-LDL as an atherogenic factor on CD36 expression. Mechanism of PPAR-γ action and its ligands in CD36 expression were also investigated. Methods: Raw 264.7 macrophage cell line was treated with ox-LDL (100 and 150 μg protein/LDL) and EPA (100 and 200 μM) for 24 and 48 hours in absence or presence of PPAR-γ inhibitor, T0070907. Quantitative real-time PCR and Western-blotting were used for analysis of gene and protein expression, respectively. Results: Raw 264.7 exposures to ox-LDL and EPA resulted in increased expression of CD36 mRNA and protein however, mRNA and PPAR-γ protein were not up-regulated significantly. Pre-incubation of cells with T0070907 led to decreased expression of CD36 when treated with ox-LDL and EPA. Conclusion: It was confirmed that both EPA and ox-LDL increased CD36 expression but not PPAR-γ, and also co-treatment with PPAR-γ inhibitor decreased CD36 expression. We concluded that up-regulation of CD36 depends on PPAR-γ activation and is not related to increased expression of PPAR-γ. Induction of CD36 by EPA showed that CD36 suppression is not the means by which ω-3 fatty acids (EPA) provide protection against formation of atherosclerotic plaque. Atherosclerosis proliferator-activated receptor gamma (PPAR-γ) Oxidized low density lipoprotein (ox-LDL) Eicosapentaenoic acid (EPA) 2013 4 01 84 92 http://ibj.pasteur.ac.ir/article-1-933-en.pdf 10.6091/ibj.11322.2013
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Iranian Biomedical Journal IBJ 1028-852X 2008-823X 10.61186/ibj 2013 17 2 Homocysteine Induces Heme Oxygenase-1 Expression via Transcription Factor Nrf2 Activation in HepG2 Cells Monireh Mani Taghi Golmohammadi golmoham@sina.tums.ac.irgolmoham@sina.tums.ac.ir Shahnaz Khaghani Zahra Zamani Kayhan Azadmanesh Reza Meshkani Parvin Pasalar Background: Elevated level of plasma homocysteine has been related to various diseases. Patients with hyperhomocysteinemia can develop hepatic steatosis and fibrosis. We hypothesized that oxidative stress induced by homocysteine might play an important role in pathogenesis of liver injury. Also, the cellular response designed to combat oxidative stress is primarily controlled by the transcription factor Nrf2, a principal inducer of anti-oxidant and phase II-related genes. Methods: HepG2 cells were treated with homocysteine in different time periods. Glutathione content was measured by flowcytometry. Using electrophoretic mobility shift assay (EMSA) and Western-blotting, anti-oxidant response element (ARE)-binding activity of Nrf2 for heme ocygenase-1 (HO-1) was demonstrated. Real time RT-PCR and Western-blotting were performed to evaluate whether homocysteine was able to induce mRNA and protein expression of HO-1. Results: The role of Nrf2 in cellular response to homocysteine is substantiated by the following observations in HepG2 cells exposed to homocysteine (i) Western-blotting revealed that Nrf2 is strongly stabilized and became detectable in nuclear extracts. (ii) EMSA demonstrated increased binding of Nrf2 to oligomers containing HO-1 promoter-specific ARE-binding site. (iii) Real time RT-PCR and Western-blotting revealed increased mRNA and protein expression of inducible gene HO-1 after treatment with homocysteine. Conclusion: Data presented in the current study provide direct evidence that the immediate cellular response to oxidative stress provoked by homocysteine is orchestrated mainly by the Nrf2-ARE pathway. Therefore, induction of Nrf2-ARE-dependent expression of HO-1 could be a therapeutic option for hepatic cells damage induced by homocysteine. Heme oxygenase-1 HepG2 cells Oxidative stress 2013 4 01 93 100 http://ibj.pasteur.ac.ir/article-1-951-en.pdf 10.6091/ibj.1158.2013
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Iranian Biomedical Journal IBJ 1028-852X 2008-823X 10.61186/ibj 2013 17 2 Antibiotic Supplements Affect Electrophysiological Properties and Excitability of Rat Hippocampal Pyramidal Neurons in Primary Culture Farideh Bahrami Mahyar Janahmadi mjanahmadi@yahoo.com Introduction: Antibiotic supplements are regularly used in neuronal culture media to control contamination however, they can interfere with the neuronal excitability and affect electrophysiological properties. Therefore, in this study, the effect of penicillin/streptomycin supplements on the spontaneous electrophysiological activity of hippocampal pyramidal neurons was examined. Methods: Electrophysiological whole-cell patch-clamp recordings from rat hippocampal pyramidal cells in primary culture were performed to investigate the effects of antibiotic supplements on the intrinsic excitability of cultured cells. Results: The present findings indicated that presence of antibiotic supplements (penicillin/streptomycin) in the culture medium altered the intrinsic electrical activity of hippocampal pyramidal neurons in primary culture. These alterations included: 1) depolarized resting membrane potential 2) a significant enhancement in the after-hyperpolarization amplitude 3) a significant increase in the area under the action potential and in the decay and rise time of the action potential 4) a significant broadening of action potential and 5) a significant reduction in the firing frequency. Conclusion: These findings suggest that addition of antibiotic supplements to culture media influences the neuronal excitability and alters the electrophysiological properties of cultured neurons, possibly through changing the ionic conductance underlying neuronal excitability. Primary cell culture Patch-clamp techniques Hippocampus 2013 4 01 101 106 http://ibj.pasteur.ac.ir/article-1-903-en.pdf 10.6091/ibj.11242.2013
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Iranian Biomedical Journal IBJ 1028-852X 2008-823X 10.61186/ibj 2013 17 2 Nucleolar Organizer Regions of Oral Epithelial Cells in Crack Cocaine Users Magna Carvalho de M. Thiele Carlos Bohn Carlos Bohn Cassiano Lima Chaiben Ana Maria Trindade Grégio Maria Ângela Naval Machado Antonio Adilson Soares de Lima antollima@hotmail.com Background: The health risks of crack cocaine smoking on the oral mucosa has not been widely researched and documented. Objective: The purpose of this study was to analyze the proliferative activity of oral epithelial cells exposed to crack cocaine smoke using silver nucleolar organizer region (AgNOR) staining. Methods: Oral smears were collected from clinically normal-appearing buccal mucosa by liquid-based exfoliative cytology of 60 individuals (30 crack cocaine users and 30 healthy controls matched for age and gender) and analyzed for cytomorphologic and cytomorphometric techniques. Results: Crack cocaine users consumed about 13.3 heat-stable rocks per day and the time consumption of the drug was of 5.2 (± 3.3) years. Mean values of AgNOR counting for case and control groups were 5.18 ± 1.83 and 3.38 ± 1.02 (P<0.05), respectively. AgNOR area and percentage of AgNOR-occupied nuclear area were increased in comparison with the control (P<0.05). There was no statistically significant difference in the mean values of the nuclear area between the groups (P>0.05). Conclusion: This study revealed that crack cocaine smoke increases the rate of cellular proliferation in cells of normal buccal mucosa. Crack-Cocaine Mouth mucosa Cell proliferation 2013 4 01 107 111 http://ibj.pasteur.ac.ir/article-1-934-en.pdf 10.6091/ibj.11152.2013