Cell Journal (Yakhteh)
Year:2005 | Volume:6 | Issue:24|Papers Count:15

Introduction: Considering the anticonvulsant effects of A1 Adenosine Receptors and the anatomical connections between pJriform Cortex and Amygdala, in this study, the role of A1 adenosine receptors activity of piriform cortex neurons on amygdala kindled seizures was investigated in rats. Material and Methods: The rats were fully kindled by daily electrical stimulation of amygdala. N6-cyclohexyleadenosine (CHA; 1,10 and 100 /lM), as a selective A1 agonist and 1,3-dimethyl-8-cyclohexylexantine (CPT; 20 and 10 /lM), as a selective A1 antagonist were microinjected (0.5/l1, 0.25 /ll/min) into the piriform cortex. Animals were stimulated at 5, 15 or 90 min after drug microinjection and seizure parameters were measured. Results: Intra-piriform CHA (10 or 100 /lM) reduced afterdischarge duration and stage 5 seizure duration and prolonged stage 4 latency significantly. Pretreatment with CPT (10 /lM) 5 min before CHA (100 /lM) eliminated the effects of CHA. Conclusion: These observations suggest that A1 adenosine receptors activity in piriform cortex reduced the seizure severity and attenuated the distribution of seizure activity to other brain regions.

Introduction: Although tolerance to and dependence on opioids are characterized by excessive activity of cAMP pathway in some brain stem nuclei, the impact of morphine dependence on activity of cAMP pathway in paragigantocellularis nucleus (PGi), located in the rostral ventrolateral medulla, remains unclear. Therefore, the effect of adenyl cyclase activator forskolin on spontaneous firing rate of PGi neurons anp precipitation of withdrawal signs in morphine dependent rats was studied. Material and Methods: Electrophysiologic extracellular single unit activity was recorded from PGi of urethane anesthetized NMRI male rats (200-350 g). Forskolin (100 nM /300-400 nL / 3-4 min) was microinjected into PGi. To assess behavioral signs, frequency analysis was used. Results: The results showed that spontaneous PGi neuronal firing rate in morphine dependent rats was lower than that of control ones, which confirms the tolerance to morphine. Forskolin caused an increase in PGi neuronal firing rate in control rats and completely suppressed spohtaneousfiring rate of PGi neurons in morphine dependent ones (p<0.001). In freely moving control rats, forskolin induced wet-dog shake and chewing sings but not in morphine dependent ones. Conclusion: It is concluded that adaptive change in activity of cAMP pathway in PGi neurons following chronic morphine exposure may play a role in the development of tolerance to and dependence on morphine.

Introduction: One of the goals of modern genetics is to develope safe prenatal diagnostic tests which do not constitute any risk to the fetus. One potentially non-invasive approach for doing so is to obtain fetal material from the maternal circulation. In this study we obtained Rlaternal blood from the pregnant women and tried to determine sex of fetus by nested-PCR. Material and Methods: To determine the sensitivity of the PCR methods, artificial samples were prepared first by mixing whole blood of adult males with that of the adult females. After performing DNA extraction, PCR was optimized on these samples. Whole blood samples were then obtained from 70 pregnant women in 8 to 12 weeks of gestation after considering the medical ethics. In this study we tried to extract the DNA of the white blood cells (WBC) as well as free DNA of serum and plasma of the maternal bloods and performed Nested-PCR using primers flanking the specific-sequence of V-chromosome or Sex determining region on Y (SRY). If the fetus was male, the size of amplified fragment of the first round product was about 470 base pairs and second round was 254 base pairs. Results: After delivery, Only 32 out of 70 of the samples could be followed and results showed that 18 were male. However, the results of the nested-PCR examination of the serums indicated that 21 of the fetas were male and examination of the WBCs showed that 22 were male, in each group 3 and 4 cases were misinterpreted respectively. So the accuracy of sex determination of the samples in serums and WBCs were 90.62 % and 81.25% respectively which is almost similar to the recent reports (91.5%). We could not obtain good results from the free DNA of the plasma. Conclusion: We have obtained relatively positive results from the analysis of maternal blood and if we could follow all cases to see the results of all deliveries, we could then reconfirm the results of our research. Anyway we hope this project could be able to introduce this new approach as means of noninvasive prenatal diagnosis for detection of common inherited diseases in Iran.

Introduction: The tumor suppressor p53 protein can induce apoptosis in some cellular contexts. Among the apoptosis-inducing genes, p53 has received the most attention for cancer gene therapy. In this study, the role of Dendrosome and Lipofectin mediated normal cDNA of TP53 into MOLT-4, CCRF-CEM(T-Iymphoma) and K562 cell lines was assessed. Material and Methods: At first, CCRF-CEM, MOL T-4 and K562 (erythroleukemic) cell lines were transfetted by Dendrosome and Lipofectin separately. Then, viability study was carried out by Trypan blue exclusion. The rate of apoptosis and necrosis in transfected cell lines was evaluated by Flow Cytometry. Results: Trypan blue exclusion assay in K562 and CCRF-CEM revealed 55.8% and 17.97 % viability reduction in the Dend+p53 in comparison to Dend+pcDNA3 (control), respectively. Flow Cytometry studies confirmed a significant enhancement of apoptosis and necrosis in TP53 transrected K562 and CCRF-CEM cells. Flow Cytometry and viability studies Qntransfected MOLT-4 cells showed no significant changes. Conclusion: The results showed that expression of normal cDNA of TP53 in K562 and CCRF-CEM cell lines could induce apoptosis in these cell lines with different levels. Transfection of MOLT-4 cell Line by Dend+p53 and Lipo+53 could not result in apoptosis.

Introduction: The aim of this study was to evaluate the relation between protamine deficiency assessed by CMA3 and protamine 1/ protamine 2 (P1/P2) ratio. Material and Methods: This study was carried out on 71 patients referring to Isfahan Fertility and Infertility center. Semen analysis was assessed according to WHO criteria. CMA3 staining was use to determine protamine deficiency. P1/P2 ratio was evaluated by nuclear protein extraction, acetic acid urea polyacrylamide gel-electrophoresis and analysis of protein bands with related software. Western blot was carried out with primary anti P1 and anti P2 antibody to determine protamine 1 and protamine 2. Of the 71 patients 45 patients underwent Intra cytoplasmic Sperm Injection (ICSI). Results: A negative significant correlation was observed between fertilization rate with protamine deficiency and P1/P2 ratio. However, no significant correlation was observed between protamine deficiencies with P1/P2 ratio.Conclusion: The results of this study showed that protamine deficiency can be assessed by CMA3; however this procedure does not indicate type of protamine deficiency.

Introduction: Human T cell Lymphotropic l.irus type I is responsible for Adult T cell Leukemia/Lymphoma and HTLV-I associated myelopathy. The aim of this study was constructing two reporter plasmids to enable us to evaluate the effects of HTLV-I Tax protein upon intra cellular signalling pathways which recruit CREB and NFkB proteins. Material and Methods: A complete coding region of bacterial betagalactosidase gene was subcloned into pUC18 , followed by inserting a poly adenylation signal downstream to it. Promoter regions of HTLV-I long terminal repeat and Interlukin 2 receptor alpha (which were stimulated by CREB and NFkB respectively) were amplified by PCR and separately inserted upstream to betagalactosidase gene, leading to construction of two reporter plasmids. The effect of cotransfection of a Tax expressing plasmid with each of these plasmids was evaluated by X-gal staining, beta galactosidase ELlSA or beta galactosidase activity assay with CPRG substrate. Results: Results clearly showed that both reporter plasmids responded well to stimulation of their promoters by Tax and the produced beta galactosidase could successfully be detected by all three methods. Results of ELlSA and assay tests for betagalactosidase showed a high correlation (r=0.949). Conclusion: Both reporter plasmids constructed in this study are able to produce considerably more betagalactosidase after stimulation by HTLV-I Tax. Advantages and disadvantages of three evaluating method for detection of betagalactosidase are studied and discussed.

Introduction: The value of embryonic stem-like cells in cloning is obvious. Production of cloned animals can be achieved by introduction of these cells into a tetraploid embryo. Tetraploid embryo is used as a feeder for development of embryonic stem-like cells and can be produced in vitro by electro-fusion of 2-cell embryos. The aim of this study was to ass,essthe effect of voltage alteration and duration on fusion and cleavage rates of bovine tetraploid embryo produced by electrofusion. Material and Methods: After in vitro maturation and fertilization of cumulus oocyte complexes, two-cell embryos were categorized into three groups: 1- Fused group (FG): include two-cell embryos fused by exposure to different voltages (0.5, 0.75, 1, 1.25 1.:& kV/cm) and durations (20, 40, 60, 80 & 100 I-Is).2- Exposed control group (ECG): two-cell embryos that remained unfused after electrofusion. 3- Unexposed control group (UCG): two-cell embryos cultured without exposure to any voltage. The embryos from each groupWere cultured in SOF1. The fusion and cleavage rates were compared in each group. Results: Increase in voltage resulted in significantly higher fusion and lower cleavage rate. The increased duration had no significant effect on fusion rate. The increased duration of high voltages caused decreased cleavage rate significantly, and in low voltage resulted in increased cleavage rate. The cleavage rate in ECG group followed the same as FG group, and they were lower when compared to UCG. Conclusion: Best fusion and cleavage !:ate was obtained at 0.75-1 kV/cm for 60l-ls duration.

Introduction: The aim of this study was to evaluate the effect of sperm protamine deficiency on fertilization and embryo development post ICSI. Material and Methods: Semen samples from 27 patients candidate for ICSI were assessed for semen parameters. Sperm processing was carried out using pure sperm and percentage of protamine deficient sperm was assessed by Chromomycin A3 post processing. Correlation between fertilization rate, embryo quality and cleavage score with protamine deficiency was assessed using SPSS statistical programme. Results: A significant negative correlation was observed between fertilization rate and embryo quality score on day 3 with peroentage of protamine deficient sperms. Conclusion: Semen samples with high percentage of protamine deficient sperms have lower fertilization rate and lower potential to develop good quality embryos