The Faculty of Biological Science and Technology at University of Isfahan, established in 1992, is a leading institution for life sciences research with 5 departments covering microbiology, biochemistry, biotechnology, plant biology, and zoology, and housing advanced facilities including biosafety level-2 labs and a DNA sequencing center.
1. The effects of the calcium channel blocking agent, nitrendipine, were studied on seizures in mice produced during withdrawal from chronic benzodiazepine treatment and on the development of tolerance to benzodiazepines. 2. Nitrendipine produced a dose-dependent decrease in seizure incidence, when seizures were produced by the partial inverse agonist FG7142 during withdrawal from seven days treatment with flurazepam. 3. Nitrendipine did not raise the seizure thresholds in naive mice to the full inverse agonist methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), or to the γ-aminobutyric acid (GABA) antagonist, bicuculline. 4. When given concurrently with flurazepam for seven days, nitrendipine did not affect the incidence of seizures during flurazepam withdrawal. 5. When given concurrently with the benzodiazepines, nitrendipine did not prevent the development of tolerance to midazolam general anaesthesia or tolerance to the ataxic actions of flurazepam or midazolam. 6. Chronic treatment with flurazepam for seven days did not affect the K(d) or B(max) of [3H]-nimodipine binding in mouse whole brain or cerebral cortex. 7. These results with benzodiazepines are partially in contrast with those for ethanol, where nitrendipine not only decreased ethanol withdrawal seizures when given acutely, but also prevented the development of tolerance and withdrawal signs when given concurrently with ethanol. However, they do confirm the selectivity of nitrendipine for withdrawal-induced seizures.
The competitive antagonists at the N‐methyl‐d‐aspartate (NMDA) receptor, CGP39551 and CGP37849, protected against the barbiturate withdrawal syndrome in mice, as measured by ratings of convulsive behaviour on handling. The effective doses of these compounds were lower than those required to prevent seizures due to NMDA in naive animals; these were in turn lower than those needed to prevent the convulsive effects of the α‐aminobutyric acid (GABA) antagonist, bicuculline. The NMDA‐receptor antagonists did not alter the increase in the incidence of convulsions due to the GABAA antagonist, bicuculline, that is seen during barbiturate withdrawal, although the latencies to these convulsions during barbital withdrawal were significantly increased after CGP39551. Barbiturate withdrawal did not affect the convulsive actions of NMDA, whether measured by the incidence of convulsions or by intravenous infusion. The Bmax for [3H]‐dizocilpine ([3H]‐MK801) binding was significantly increased by chronic barbital treatment in cerebrocortical but not in hippocampal tissues, while the Kd remained unaltered in either case. At 1 h and 24 h after administration of a single dose of barbitone, the Bmax for [3H]‐dizocilpine binding was unaltered in cerebrocortical tissue. Acute addition of barbitone in vitro did not alter [3H]‐dizocilpine binding or the displacement of binding of thienylcyclohexylpyridine. 1994 British Pharmacological Society
Gäken, J.A., Tavassoli, M., Gan, S., Vallian borujeni, S., Giddings, I., Darling, D.C., Galea-lauri, J., Thomas, M.G., Abedi, H., Schreiber, V.
Journal of Virology (10985514)70(6)pp. 3992-4000
Integration of proviral DNA into the host cell genome is a characteristic feature of the retroviral life cycle. This process involves coordinate DNA strand break formation and rejoining reactions. The full details of the integration process are not yet fully understood. However, the endonuclease and DNA strand-joining activities of the virus-encoded integrase protein (IN) are thought to act in concert with other, as-yet-unidentified, endogenous nuclear components which are involved in the DNA repair process. The nuclear enzyme poly(ADP-ribose) polymerase (PARP), which is dependent on DNA strand breaks for its activity, is involved in the efficient repair of DNA strand breaks, and maintenance of genomic integrity, in nucleated eukaryotic cells. In the present work, we examine the possible involvement of PARP in the retroviral life cycle and demonstrate that inhibition of PARP activity, by any one of three independent mechanisms, blocks the infection of mammalian cells by recombinant retroviral vectors. This requirement for PARP activity appears to be restricted to processes involved in the integration of provirus into the host cell DNA. PARP inhibition does not affect viral entry into the host cell, reverse transcription of the viral RNA genome, postintegration synthesis of viral gene products, synthesis of the viral RNA genome, or the generation of infective virions. Therefore, efficient retroviral infection of mammalian cells is blocked by inhibition of PARP activity.
Arif, S., Vallian borujeni, S., Farzaneh, F., Zanone, M.M., James, S.L., Pietropaolo, M., Hettiarachchi, S., Vergani, D., Conway, G.S., Peakman, M.
Journal of Clinical Endocrinology and Metabolism (0021972X)81(12)pp. 4439-4445
Autoantibodies directed against steroid hormone-producing cells (SCA) detectable by immunofluorescence are typically found in a small proportion of patients with premature ovarian failure (POF) as well as in other endocrine autoimmune diseases. The SCA pattern stains cells in the outer zones of the adrenal cortex, ovary, and testis. To identify the molecular target of SCA, an adrenal complementary DNA expression library was screened using SCA- positive serum, and the steroid enzyme 3β-hydroxysteroid dehydrogenase (3βHSD) was identified. Only 1 of 48 (2%) patients with idiopathic POF, not preselected for the presence of other autoimmune diseases, had SCA by immunofluorescence, whereas 10 of 48 (21%) had anti-3βHSD autoantibodies detectable by immunoblot using recombinant human enzyme compared with 6 of 115 (5%) control subjects (P = 0.002). Absorption of SCA-positive serum with recombinant human 3βHSD abolished the immunofluorescence pattern. We also examined the prevalence of anti-3βHSD autoantibodies in other endocrine autoimmune diseases. Two of 112 (2%) diabetic patients, but none of the thyroid or Addisonian patients, had SCA by immunofluorescence. Twenty-six (23%) diabetic subjects (P < 0.001 vs. controls), 3 of 18 thyroid patients (P > 0.05 vs. controls), and none of 4 Addisonian patients had anti-3βHSD autoantibodies. 3βHSD is the first steroid cell autoantigen defined at the molecular level to be associated with idiopathic POF occurring in the absence of other polyglandular diseases. Autoantibodies to 3βHSD in patients with other organ-specific autoimmune diseases indicate that the enzyme behaves as a typical target of polyendocrine autoimmunity. Anti-3βHSD autoantibodies in patients with POF may provide a marker of those subjects whose ovarian failure is autoimmune in origin and, as recent studies suggest, may be salvageable with glucocorticoid treatment.
Our previous studies demonstrated that PML is a growth suppressor that suppresses oncogenic transformation of NIH/3T3 cells and rat embryo fibroblasts. PML is a nuclear matrix-associated phosphoprotein whose expression is regulated during the cell cycle. Disruption of PML function by t(15;17) in acute promyelocytic leukemia (APL) plays a critical role in leukemogenesis. To further study the role of PML in the control of cell growth, we have stably overexpressed PML protein in the HeLa cell line. This overexpression of PML significantly reduced the growth rate of HeLa cells and suppressed anchorage-independent growth in soft agar. We consequently investigated several parameters correlated with cell growth and cell cycle progression. We found that, in comparison with the parental HeLa cells, HeLa/PML stable clones showed proportionally more cells in G1 phase, fewer cells in S phase and about the same number in G2/M phase. The HeLa/PML clones showed a significantly longer doubling time as a result of a lengthening of the G1 phase. No effect on apoptosis was found in HeLa cells overexpressing PML. This observation indicates that PML suppresses cell growth by increasing cell cycle duration as a result of G1 elongation. To further understand the mechanism of the effect of PML on HeLa cells, expression of cell cycle-related proteins in HeLa/PML and parental HeLa cells was analyzed. We found that Rb phosphorylation was significantly reduced in PML stable clones. Expression of cyclin E, Cdk2 and p27 proteins was also significantly reduced. These studies indicate that PML affects cell cycle progression by mediating expression of several key proteins that normally control cell cycle progression. These results further extend our current understanding of PML function in human cells and its important role in cell cycle regulation.
Vallian borujeni, S., Gäken, J.A., Trayner, I.D., Gingold, E.B., Kouzarides, T., Chang k.-s., K., Farzaneh, F.
Experimental Cell Research (10902422)237(2)pp. 371-382
Acute promyelocytic leukemia is characterized by the presence of a t(15; 17) chromosomal translocation which results in the expression of a chimeric gene product, PMLRARα, consisting of an N-terminal-truncated retinoic acid receptor-α fused to a C-terminal-truncated PML. Several structural features, and regions of homology to known transcription factors, suggest that PML may be involved in the regulation of gene expression. In this study we have analyzed the transcriptional regulatory activity of PML using chimeric GAL4/PML constructs and GAL4-responsive reporter plasmids. The data presented demonstrate that PML, when fused to the DNA-binding domain of GAL4 (GAL4/PML), inhibits transcription from GAL4-responsive promoters. The magnitude of this repression is cell type and promoter dependent, and deletion studies show that the putative coiled-coil and part of the serine- rich regions of PML are required for this activity. These regions are also shown to be responsible for the repression of transcription activity from the EGFR promoter. The data presented also demonstrate that GAL4/PML can recruit PMLRARα resulting in the retinoid-inducible transcriptional activation of a GAL4-responsive promoter, a function dependent on the presence of the coiled- coil region of PMLRARα.
Obstetrics and Gynecology (00297844)91(3)pp. 319-323
Objective: To determine whether the mechanism for the retention of interstitial fluid in trisomy 21 fetuses presenting with nuchal translucency at 10-14 weeks' gestation is an alteration in the composition of collagen type VI, which is normally a triple helix formed of three single chains, α1, α2, and α3. The genes responsible for the α1 and α2 chains, COL6A1 and COL6A2, are located on chromosome 21 and therefore may be overexpressed in trisomy 21, whereas COL6A3 is located in chromosome 2. Methods: Skin tissue was obtained after termination of pregnancy at 11-16 weeks' gestation in five fetuses with trisomy 21 and five normal controls. Total RNA was extracted and the steady-state levels of COL6A1 and COL6A3 mRNA expression of the gene transcripts were determined. Additionally, the distribution of collagen type VI in the skin of trisomy 21 and normal fetuses was analyzed using an immunohistochemical method. Results: The ratio of the normalized densitometric scores for the mRNA expression of COL6A1 to COL6A3 in the skin of trisomy 21 fetuses was twice as high as in normal fetuses. Immunohistochemistry demonstrated that in trisomy 21 fetuses collagen type VI formed a dense network extending from the epidermal basement membrane to the subcutis, whereas in normal fetuses dense staining was confined to the upper region of the dermis. Conclusion: The distribution for collagen type VI is different from normal in the skin of trisomy 21 fetuses, and there is overexpression of COL6A1 compared with COL6A3.
Vallian borujeni, S., Gäken, J.A., Gingold, E.B., Kouzarides, T., Chang k.-s., K., Farzaneh, F.
Oncogene (14765594)16(22)pp. 2843-2853
The growth and transformation suppressor function of promyelocytic leukemia (PML) protein are disrupted in acute promyelocytic leukemia (APL) as a result of its fusion to the RARα gene by t(15;17) translocation. There is significant sequence homology between the dimerization domain of PML and the Fos family of proteins, which imply that PML may be involved in AP-1 activity. Here we show that PML can cooperate with Fos to stimulate its AP-1-mediated transcriptional activity. Cotransfection of PML, with GAL4/Fos strongly induced Fos-mediated activation of GAL4-responsive reporters, indicating a functional interaction between Fos and PML in vivo. Deletion analysis of Fos and PML demonstrated that the intact C-terminal domain of Fos (containing the dimerization domain), and the RING-finger, B1 box and nuclear localization domains of PML are involved in the cooperative activity of Fos and PML. Immunoprecipitation and electrophoretic mobility shift assay showed that PML is associated with the AP-1 complex. PMLRARα was also found to enhance the transcriptional activity of GAL4/Fos. The addition of retinoic acid abrogated the PMLRARα, but not PML-induced stimulation of GAL4/Fos activity in a dose-dependent manner. This study demonstrated that PML is involved in the AP-1 complex and can modulate Fos-mediated transcriptional activity, which may contribute to its growth suppressor function.
Our previous studies demonstrated that the promyelocytic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing effect in breast cancer cells were examined. We showed that Ad-PML effectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post-infection and a detectable level remained at day 16. Ad-PML significantly suppressed the growth rate, clonogenicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude K mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the effect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a significant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphorylated 48 h post-infection. These findings indicate that PML exerts its growth suppressing effects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppressing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy.
Molecular and Cellular Biology (02707306)18(12)pp. 7147-7156
The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein with growth- and transformation-suppressing ability. Having previously shown it to be a transcriptional repressor of the epidermal growth factor receptor (EGFR) gene promoter, we have now shown that PML's repression of EGFR transcription is caused by inhibition of EGFR's Sp1-dependent activity. On functional analysis, the repressive effect of PML was mapped to a 150-hp element (the sequences between -150 and -16, relative to the ATG initiation site) of the promoter. Transient transfection assays with Sp1-negative Drosophila melanogaster SL2 cells showed that the transcription of this region was regulated by Sp1 and that the Sp1-dependent activity of the promoter was suppressed by PML in a dose-dependent manner. Coimmunoprecipitation and mammalian two-hybrid assays demonstrated that PML and Sp1 were associated in vivo. In vitro binding by means of the glutathione S-transferase (GST) pull-down assay, using the full-length and truncated GST- Sp1 proteins and in vitro-translated PML, showed that PML and Sp1 directly interacted and that the C-terminal (DNA-binding) region of Sp1 and the coiled-coil (dimerization) domain of PML were essential for this interaction. Analysis of the effects of PML on Sp1 DNA binding by electrophoretic mobility shift assay (EMSA) showed that PML could specifically disrupt the binding of Sp1 to DNA. Furthermore, cotransfection of PML specifically repressed Sp1, but not the E2F1-mediated activity of the dihydrofolate reductase promoter. Together, these data suggest that the association of PML and Sp1 represents a novel mechanism for negative regulation of EGFR and other Sp1 target promoters.
Iranian Journal Of Medical Sciences (02530716)(3-4)
Background: The cAMP signal transduction systems are known to play a critical role in the acute and chronic effects of ethanol and other drugs of abuse. Objective: To investigate the role of protein kinase C (PKC) in the action of ethanol on platelets and human erythroleukemia (HEL) cells. Methods: HEL, HEK 293 cells [transfected with AC type VII(AC7)] and platelets were used to study the effects of ethanol. cAMP accumulation assay was used to determine the percentage conversion of [3H]ATP into [3H]cAMP. Each fraction was separated through sequential chromatography and the radioactive material was then quantified using a liquid scintillation counter. Results: Ethanol enhanced the PGE1-stimulated AC activity in HEL cells. The percent enhancement of cAMP accumulation by 200 mM ethanol was similar in HEL cells, platelets and HEK 293 cells transfected with AC7. When combined with PDBu, 200 mM ethanol further enhanced the PDBu stimulation of AC activity suggesting a separate mechanism by which ethanol and PDBu exert their effects on the AC. Pretreatment of the cells with staurosporine and chelerythrine significantly blocked the PDBu-and/or ethanol-enhancement of cAMP accumulation. Conclusion: These results indicate a clear role for PKC in the action of ethanol on AC in platelets.
Chronic barbital treatment significant increased the net K+-stimulated uptake of 45Ca2+ in cerebrocortical synaptosomal preparations, 24 h after withdrawal from chronic barbital administration. Basal uptake was not significantly changed. Hippocampal synaptosomal preparations showed a similar pattern, but the increase was not significant. The synaptosomal Ca2+ uptake was not affected by incubation with the dihydropyridine Ca2+ channel antagonist, nitrendipine, in controls or after chronic barbital treatment. Acute administration of a single dose of barbital did not alter the basal or stimulated uptake of 45Ca2+ in cortical synaptosomes, when this was measured 36 h after the barbital administration. Hippocampal slices prepared 24 h after withdrawal from chronic barbital treatment showed a significant increase in K+-stimulated uptake of 45Ca2+, and the basal uptake was significantly decreased. Both changes were prevented by nitrendipine. An increase in the density of dihydropyridine-sensitive binding sites was found in the cerebral cortex. The results indicate that both dihydropyridine-sensitive and insensitive neuronal Ca2+ channels are altered by chronic barbiturate treatment. These changes may be involved in physical dependence on barbiturates. Copyright (C) 1999 Elsevier Science B.V.
Alcoholism: Clinical and Experimental Research (01456008)(1)
Ethanol is known to enhance the activity of adenylyl cyclase (AC) in a number of cells and tissues. Recent work has suggested that the various isoforms of AC show differential sensitivity to ethanol, with Type VII AC being most sensitive. However, the mechanism of action of ethanol is unclear. In the present work, we investigated the effect of ethanol on AC activity in the human erythroleukemia (HEL) cell line, platelets, and AC VII-transfected HEK 293 cells. The HEL cells contain abundant amounts of mRNA for Type VII AC. We found that both ethanol and phorbol dibutyrate (PDBu) treatment enhanced agonist (prostaglandin E1; PGE1)-stimulated AC activity in HEL cells, as well as in platelets and HEK 293 cells transfected with AC VII. Inhibitors of protein kinase C (PKC) blocked the stimulatory effects of both ethanol and PDBu. However, the effects of ethanol and PDBu on AC activity were additive, suggesting that the mechanisms of action of ethanol and PDBu were not identical. Furthermore, a 30-min exposure of HEL cells to ethanol attenuated (desensitized) the ability of ethanol, but not PDBu, to enhance agonist-activated AC activity. On the other hand, a 30-min pretreatment with PDBu attenuated the AC response to the phorbol ester, but not to ethanol; but, after a 20 hr preincubation with phorbol ester, the ability of both PDBu and ethanol to enhance prostaglandin E1-stimulated AC activity was completely eliminated. Finally, pretreatment of HEL cells with pertussis toxin blocked the effect of PDBu, but not ethanol, on AC activity. The results support the involvement of phorbol ester-sensitive PKC(s) in ethanol's enhancement of agonist-activated activity of AC in HEL cells, but suggest that the mechanism of ethanol's action is different from that of PDBu. The findings with pertussis toxin suggest that PDBu activation of PKC(s) may affect AC activity through phosphorylation of a G(i) protein, whereas ethanol may act by promoting phosphorylation of a different substrate (e.g., AC VII).
Rahgozar, S., Poorfathollah, A.A., Moafi, A., Old, J.M.
American Journal of Hematology (03618609)65(3)pp. 192-195
Sickle cell anemia is not considered to be a significant disease in Iran, although the sickle cell trait is estimated to have a high incidence in the Southern provinces. Since 1977, when the presence of a mild sickle cell anemia was reported in this country, there have been no further investigations published giving precise data on the incidence and origins of the sickle cell mutation in Iran. We report here the finding of patients with the sickle cell trait, sickle cell anemia, and sickle-β thalassemia in Central Iran. A survey of 300 individuals from a village in Southeast Esfahan revealed an incidence of the sickle cell trait of 8.33%. 'Cascade screening' enabled 96 relatives in four surrounding villages to be tested, and the at-risk couples were offered counseling as a 'sickle cell control program.' The hematological indices and HbF levels of the affected patients were determined. The HbF levels were unusually high, ranging from 18% to 41.4%, and SS patients with the highest levels were asymptomatic. Linkage analysis revealed the β(s) gene haplotype in this population to be the Indian-Arab haplotype. (C) 2000 Wiley-Liss, Inc.
IRANIAN JOURNAL OF SCIENCE AND TECHNOLOGY (03601307)27(1)pp. 185-190
The effect of pH (ranging from 3 to 10), salinity (0.5, 2 and 4M NaCl) and light intensity (20, 60 and 300μmol quanta. m-2. s-1) on the growth curve of three species of D. salina, D.parva and D.psuedosalina were studied. Also, the effect of high temperature ranging from 35 to 60 °C on the motility and integrity of these three species at different salinity of 0.1, 0.5, 1, 1.5, 2, 3 and 4M NaCl in the medium, were studied. The results show that D.salina and D.parva could grow at pH ranges of 6 to 9 with optimum of 7 to 8 and D.psuedosalina at pH of 5 to 10 with optimum of 7 to 8. All three species had optimum growth rate at about 0.5 to 2.0M NaCl. The results also show that, in all three species cell motility and resistance to high temperature increased with increasing NaCl concentration in the medium.
