Cell Journal (Yakhteh)
Year:2005 | Volume:7 | Issue:27|Papers Count:12

Introduction: DNA damage, as an important initiator of neuronal cell death, has been implicated in numerous neurodegenerative conditions. Previously we have delineated several pathways that control embryonic cortical neuronal cell death evoked by the DNA-damaging agent, camptothecin. The camptothecin, topoisomerase I inhibitor, has been shown to induce cortical neuronal cell death in a reproducible manner. It has been shown that expression of p53, Bax and JNK is involved in this model. Accordingly, it has become important to further identify the mechanisms by which DNA damage evokes neuronal cell death. In this regard, we have investigated the role of P38 and ATF-2 expression in this model. Our results indicate that the expression of P38 MAPK is up-regulated and is involved in the death signaling following DNA damage.Material and Methods: In this experimental model, primary embryonic cortical neurons were prepared from E14-16 rat embryos. Then cells were transformed into suspensions.A density of 1.2x105 cells/well plated on the wells, pre-coated with poly-D-Iysine, and neurons were cultured in serum-free medium. Camptothecin (10 -5M) was added to neuronal culture after 24 hours. After 4, 6, 24 and 48 hours, expression of the P38 and ATF-2 was studied using primary antibody in the Immunocytochemistry technique. For evaluating nuclei, number of healthy and dead nuclei was counted by cell lysis buffer. Neurons were counted and studied using Fluorescent and light microscope. Then percentage of the healthy, dead and expression of the cells was analyzed by one way ANOVA followed by Tukey's post test.Results: After camptothecin treatment, percentage of the expression of P38 was 4%, 20%, 40% and 55%, and percentage of the survival was 95%, 85%, 64% and 50% for 4, 6, 24 and 48 hours, respectively. In the same manner, percentage of the expression of ATF-2 was also 3%, 20%, 30%, 45% and percentage of the survival was 97%, 85%, 64% and 50%, respectively.Percentage of the expression and survival of the P38 neurons for 24 hours were 40 and 64 percent and it was for ATF-2, 30 and 64 percent respectively. These results compared to control were increased significantly (p<0.05) Showed a significant increase.Expression of the proteins at 4 hours was not changed significantly.Conclusion: This study revealed that expression of the P38 and ATF-2 was increased simultaneously. Thus, in this model, Camptothecin induces neuronal death by stimulation P38-ATF-2 pathway.

Introduction: The aim of this study was to evaluate the changes in cAMP and some other cellular ingredients following adenyl cyclase activation in paragigantocellularis (PGi) neurons of morphine dependent and withdrawn rats.Material and methods: The 3.5 mm thick brainstem slice prepared from PGi area (-10.04 to -13.24 from bregma) of control and morphine dependent rats (n=5 in each group). Were treated by forskolin (50μM) and IBMX (500μM) bath. Then, tissue was extracted with perchloric acid and cAMP, ATP and phosphocreatine (PCr) were measured in the extract with 31p-NMR spectroscopy. Lactic acid, alanine, taurine, GABA, glutamate, glutamine and N-acetylaspartate were measured in the extract by 1H-NMR spectroscopy.Results: 31p-NMR spectroscopy revealed an increase of cAMP content and concurrent decrease in ATP and PCr following forskolin and lBMX applications (p<0.05, t-test). 1H-NMR spectroscopy showed a reduction in both lactic acid and alanine contents in dependent group after same treatment (p<0.05, t-test). Other cellular ingredients were not changed.In the dependent rats, cAMP increased and concurrently ATP and PCr decreased following forskolin/lBMX applications (p<0.05, t-test). Lactic acid and alanine decreased in the dependent group after forskolin/lBMX treatment (p<0.05, t-test).Conclusion: It is concluded that an increase in cAMP content results from enhancement of adenylyl cyclase activity and it is considered as one of the cellular basis of morphine dependence and withdrawal in PGi neurons. In addition, morphine dependence produces profound metabolic changes in PGi neurons.

Introduction: Ischemia-reperfusion (IR) injury involves a complex, interrelated sequence of events. Generation of reactive oxygen species (ROS) especially superoxide anion, during IR is reported. The present study was designed to investigate the effect of Mn (III) tetrakis (4- benzoic acid) porphyrin (MnTBAP), an active, stable, nontoxic, and cell permeable SOD (super oxide dismutase) mimetic in prevention of renal IR injury. It is very important that MnTBAP, possesses both SOD and catalase activity.Material and Methods: Experiments were performed on pentobarbital anesthetized rats. After tracheotomy, the femoral artery was cannulated and mean arterial pressure and heart rate were recorded for the duration of the experiment using a PowerLab/4sp data acquisition system. All groups received a continuous saline infusion with a rate of 6 ml/kg/h (through the femoral vein). Bladder was cannulated and urine was collected throughout the reperfusion (6h) periods. A midline laparatomy was performed and the renal arteries were carefully separated from around tissues. After surgery and stabilization period (60 min), animals were randomly assigned to the four groups: Sham operated Sham+ MnTBAP, IR, IR + MnTBAP. In the IR groups, rats were subjected to bilateral renal artery occlusion for 40 min followed by 6 h reperfusion. Rats were administered either MnTBAP (10 mg/kg IV bolus 15 min prior to IR) or saline. Data were analyzed by one way ANOVA. Results: Renal IR resulted in significant increases in creatinine, BUN, aspartate aminotransferase (AST), Fractional excretion of Na+ (FENA+) and urinary N-acetyl-β-D-glucosaminidase (NAG) activity and MnTBAP significantly reduced the IR mediated increases in creatinine, BUN, FENa+, AST and NAG.Conclusion: These results suggest that MnTBAP reduce the renal dysfunction and injury associated with IR. Thus, a SOD-mimetic like MnTBAP, should be considered in the treatment of renal I/R injury.  

Introduction: Lactate dehydrogenase is one of the cardiac enzyme markers, studied in vivo. In the present study, activity and kinetic properties of enzyme extracted from cardiomycytes derived from mouse embyronic stem cell in vitro were compared with enzyme extracted mouse neonatal from cardiomyocytes in vivo.Material and Methods: Mouse embryonic stem cells were cultured and differentiated and there after cardiomyocytes were separated from mouse neonatal heart and studied by immunocytological methods. Both cells were sonicated, total protein was extracted and kinetic properties were determined by enzyme assay. Results: Immunocytological study confirmed existence of two purified cell types. With spectrophotometic methods, enzyme activity was determined and kinetic constants for NAD+ were calculated. In the embryonic stem cell-derived cardiomyocytes specific activity value was 16.78±1.02 μmol min -1 mg-1 and Km value was 0.37±0.03mM. The neonatal cardiomyocyte specific activity value was 29.41±1.87 μmol min -1 mg-1 and Km value was 0.69±0.04 mM (r2>0.95). The optimum temperature for embyronic stem cell-derived cardiomyocyte and natural cardiomyocyte derived enzymes were 65°C, 60°C, respectively. The optimum pH for forward reaction was 8 for both enzyme preparations.Conclusion: Differences between kinetic properties of both enzymes were observed.

Introduction: Daphne species has both pharmaceutical applications and anti tumor activity. It has been shown that the extract of Daphne mucronata is effective in decreasing the size of breast adenocarcinoma in rats. We investigated the mechanism of action and the effects of the extract on TNF-α and TNF-α receptors on cultured human monocytes.Material and Methods: The breast tumors were induced in rats by DMBA. The effect of oral administration of the extract on tumor size was studied. Human monocytes were isolated by adhesion and density gradient procedures. TNF- α in cultured media were measured by sensitive biotin-streptoavid in ELlSA method, with and without the presence of LPS and radio iodination was used for TNF-α receptor determination on human cultured monocytes.Results: Oral administration of Daphne Mucronata extract during 20 consecutive days reduced significantly the diameter of tumor or eliminated it totally. On the basis of Hest analysis, our data indicated that the plant extract at various doses did increase TNF- α release and in a time dependent manner decreased the TNF-α receptors in cultured human monocytes in less than 2 hours significantly (P<0.001).Conclusion: Daphne Mucronate extract (H2O/EtOH) decreases the size of breast tumors in rat adenocarcinoma. Our data support that the extract probably acts through increasing TNF- α release and decreasing TNF- α receptors on cultured human monocytes.

Introduction: Different methods have been used to evaluate the beneficial effect of varicocelectomy; these include semen parameters and pregnancy rate. Due to high biological variability of semen parameters, sperm functional test has been considered an efficient end point in assessment of fertility. Therefore, the aim of this study was to evaluate the effect of varicocelectomy on semen parameters and sperm chromatin status.Material and Methods: Over a 2 year period a total of 35 patients were evaluated for protamine deficiency, presence of excessive histone, chromatin stability, ability of sperm to undergo decondensation by chromomycin A3, aniline blue staining, SDS, and SDS+EDTA respectively.Results: The results of this study showed that among semen parameters only sperm motility and sperm chromatin integrity showed significant improvement post surgery (P<0.05). The cumulative pregnancy rate was 25.7%. Comparing the results of the aforementioned parameters between patients whom became pregnant with those who did not benefit from varicocelectomy showed that patients may benefit from varicocelectomy that have improved sperm morphology, decreased sperm protamine deficiency and decreased presence of excessive histone at 3 months post surgery (P<0.05). Conclusion: These results suggest that the initial values and 3 months post varicocelectomy values of some of these functional tests may help special foresee the outcome of surgery and may help in patient management.  

Introduction: In the present investigation, we aimed to study the effect of different concentrations of ascorbate/vitamin C, on membrane integrity, viability, and acrosome reaction of human spermatozoa in presence of ferrous (Fe2+) ions as lipid peroxidation (LPO) promoter. Material and Methods: We used 12 semen samples from healthy donors in Shahrekord. The samples were then washed twice with Tyrode's ringer and centrifuged (500 xg, for 10 min) to separate semen plasma from sperm cells (dissolved in Tyrode's ringer). LPO test was performed to assess generated MDA in media spectrophotometrically. Eosin staining method was used to detect dead or alive sperm cells. To assess the ability of sperm cells undergoing acrosome reaction, the gelatin digestion test was performed in which iron and ascorbate were added to sperm cells and stained with comassie blue dye. Number of sperm cells with/without halo around their heads was recorded. We used Hest as statistical analysis.Results: Ascorbate in concentrations below 1000 micromolar protects spermatozoa from free radical damages as evidenced by improvement in their membrane integrity (data of LPO test), elevated acrosome reactions, and increased viability. Concomitantly, there is also witnessed depletion of malondialdehyde (MDA) (an end product of LPO) generation following ascorbate supplementation. Ascorbate at 1000 micromolar concentration and above, however, is not protective, as evidenced by abrupt fall in sperm membrane integrity and rate of acrosome reactions and lowered percent of alive sperm cells.Conclusion: Collectively, ascorbate acts as a double-edged sward in different concentrations and researchers must keep in mind this negative side of ascorbate application in their research fields like in vitro fertilization, cell culture, and preservation of gametes to minimize its deleterious impacts on biological systems.

Introduction: Embryonic stem (ES) cells are pluripotent cells derived from inner cell mass (ICM) of blastocyst stage and are able to differentiate in vitro into different cells.Purpose: This study was initiated to evaluate the effect of basic fibroblast growth factor (bFGF) on differentiation of ES cells into cardiomyocytes and to evaluate their response to cardiac drugs.Material and Methods: The mouse embryonic stem cells (Royan B1) were cultured as 800 cells per 20μ1 medium. After two days ES cells in each drop aggregated to form embryoid bodies (EBs). EBs were transferred to bacterial dishes for 5 days. In the first two days, cells were treated with 10ng/ml bFGF. After 5 days EBs were plated in 24-well plates and cardiomyocytes were differentiated. Beating rate of EBs were recorded in control and bFGF groups daily. The chronotropic effects of isoprenaline and phenylephrine on ES cells-derived cardiomyocytes at 3th, 7th and 14th days after plating were investigated. The expression of cardiac specific genes included: α-MHC (Myosin Heavy Chain), β -MHC, MLC-2V (Myosin Light Chain), β-tubulin , ANF (Atrial Natriuretic Factor) and oct-4 in control and bFGF groups with RT-PCR at 7+14 day. Moreover immunostaining was done by staining the cardiomyocytes with anti α-actinin.Results: Our results showed that compared to bFGF, the beating rate was higher on EBs of the control group, when no drugs was used and treatment of cardiomyocytes with isoprenaline and phenylephrine increased the rate of beating in both groups, and in early stage of development, this enhancement rate was significantly higher in bFGF group compared to control (p<0.035 and p<0.019, respectively). As RTPCR showed, cardiomyocytes on 7+14 day expressed α -MHC, β -MHC, MLC-2V, β-tubulin and ANF in bFGF and control groups but there was no difference between these groups. Immunoassaying showed presence of cardiac-specific proteins and their spatial organization. Conclusion: These results showed that although bFGF probably exerts its effects on the expression or function of a-adrenergic and β- adrenergic receptors, but individually it has no influential effect on cardiogenesis.

Embryonic stem cells (ESCs) are undifferentiated cells which are typically derived from the inner cell mass of a blastocyst-stage embryo.In culture, these cells have the capacity to self-renew in an undifferentiated state but also may differentiate into cell types representing the three embryonic germ lineages, thus revealing their pluripotent potential. These cells could be of value for creating in-vitro culture systems and animal models that may be used to study human genetic disease, gene function, drug discovery, developmental biology and differentiation events. This review summarizes a simple description about embryonic stem cell concepts and their potential applications.