الملخص
Estradiol (E2) accelerates oviductal egg transport through nongenomic pathways involving oviductal protein phosphorylation in non-mated rats, and through genomic pathways in mated rats. Here we investigated the ability of cervico-vaginal stimulation (CVS) to switch the mode of action of E2 in the absence of other male-associated components. Pro-estrous rats were subjected to CVS with a glass rod and 12 hours later were injected subcutaneously with E2 and intrabursally with the RNA synthesis inhibitor Actinomycin D or the protein phosphorylation inhibitor H-89. The number of eggs in the oviduct, assessed 24 h later, showed that Actinomycin D, but not H-89 blocked the E2-induced egg transport acceleration. This clearly indicates that CVS alone, without other mating-associated signals, is able to shift E2 signaling from nongenomic to genomic pathways. Since mating and CVS activate a neuroendocrine reflex that causes iterative prolactin (PRL) surges, the involvement of PRL pathway in this phenomenon was evaluated. Prolactin receptor mRNA and protein expression in the rat oviduct was demonstrated by RT-PCR and Western blot, but their levels were not different on day 2 of the cycle (C2) or pregnancy (P2). Activated ST AT 5a/b (phosphorylated) was detected by Western blot on P2 in the ovary, but not in the oviduct, showing that mating does not stimulate this PRL signalling pathway in the oviduct. Other rats subjected to CVS in the evening of pro-estrus were treated with bromoergocriptine to suppress PRL surges. In these rats, H-89 did not block the E2-induced acceleration of egg transport suggesting that PRL surges are not essential to shift E2 signaling pathways in the oviduct. We conclude that CVS is one of the components of mating that shifts E2 signaling in the oviduct from nongenomic to genomic pathways, and this effect is independent of PRL surges elicited by mating.
الموضوعات
Animals , Female , Rats , Estradiol/pharmacology , Estrogens/pharmacology , Fallopian Tubes/drug effects , Ovum Transport/drug effects , Signal Transduction/drug effects , Dactinomycin/pharmacology , Estrous Cycle , Estradiol/administration & dosage , Estrogens/administration & dosage , Fallopian Tubes/physiology , Isoquinolines/pharmacology , Protein Kinase Inhibitors/pharmacology , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Sulfonamides/pharmacologyالملخص
Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.
الموضوعات
Animals , Humans , Mice , Alpha-Amanitin/pharmacology , Antibodies, Monoclonal/analysis , Antibodies, Protozoan/analysis , Dactinomycin/pharmacology , Fluorescent Antibody Technique, Direct , Gene Expression/physiology , Green Fluorescent Proteins/genetics , HeLa Cells , Mice, Inbred BALB C , Nucleic Acid Synthesis Inhibitors/pharmacology , Nucleolus Organizer Region/drug effects , Pol1 Transcription Initiation Complex Proteins/metabolism , Protein Sorting Signals/physiology , Protozoan Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Toxoplasma/physiology , Transfectionالملخص
The present study evaluated the effects of hyperthyroid state on lipid peroxidation and antioxidant enzymes in the crude (CF), post nuclear (PNF) and mitochondrial fractions (MF) of the fish liver. The in vivo injection of T3 (200ng) did not change the lipid peroxidation products, malondialdehyde (MDA) and conjugated dienes (CD), while actinomycin D (10microg), a potent mRNA inhibitor when administered with T3 increased them. The antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) had an increased activity in CF and MF of hyperthyroid group to compete the increased oxidative stress, but actinomycin D partially inhibited the T3-induced activity. SOD and CAT activities in PNF of hyperthyroid group had no change, the glutathione concentration varied depending on the GPx and GR activity. Hyperthyroidism decreased the protein content, while simultaneous administration of actinomycin D inhibited the T3 action of elevating the protein content. The results suggest that the antioxidant defense status in A. testudineus is modulated by thyroid hormone, through an action sensitive to actinomycin D.
الموضوعات
Animals , Antioxidants/metabolism , Dactinomycin/pharmacology , Glutathione/metabolism , Lipid Peroxidation/drug effects , Perciformes/metabolism , Triiodothyronine/pharmacologyالملخص
15-deoxy-delta12,14-PGJ2(15d-PGJ2) is a natural ligand that activates the peroxisome proliferators-activated receptor (PPAR) gamma, a member of nuclear receptor family implicated in regulation of lipid metabolism and adipocyte differentiation. Recent studies have shown that 15d-PGJ2 is the potent anti-inflammatory agent functioning via PPARgamma-dependent and -independent mechanisms. Most postulated mechanisms for anti-inflammatory action of PPARgamma agonists are involved in inhibiting NF-kappaB signaling pathway. We examined the possibility that IL-6 signaling via the Jak-Stat pathway is modulated by 15d-PGJ2 in lymphocytes and also examined whether the inhibition of IL-6 signaling is dependent of PPARgamma. 15d-PGJ2 blocked IL-6 induced Stat1 and Stat3 activation in primary human lymphocytes, Jurkat cells and immortalized rheumatoid arthritis B cells. Inhibition of IL-6 signaling was induced rapidly within 15 min after treatment of 15d-PGJ2. Other PPARgamma-agonists, such as troglitazone and ciglitazone, did not inhibit IL-6 signaling, indicating that 15d-PGJ2 affect the IL-6-induced Jak-Stat signaling pathway via PPARgamma-independent mechanism. Although cycloheximide reversed 15d-PGJ2-mediated inhibition of Stat3 activation, actinomycin D had no effect on 15d-PGJ2-mediated inhibition of IL-6 signaling, indicating that inhibition of IL-6 signaling occur independent of de novo gene expression. These results show that 15d-PGJ2 specifically inhibit Jak-Stat signaling pathway in lymphocytes, and suggest that 15d-PGJ2 may regulate inflammatory reactions through the modulation of different signaling pathway other than NF-kappaB in lymphocytes.
الموضوعات
Humans , Arthritis, Rheumatoid/metabolism , Chromans/pharmacology , Cycloheximide/pharmacology , DNA-Binding Proteins/metabolism , Dactinomycin/pharmacology , Gene Expression Regulation , Hypoglycemic Agents/pharmacology , Interleukin-6/pharmacology , Jurkat Cells/metabolism , Lymphocytes/cytology , NF-kappa B/metabolism , PPAR gamma/metabolism , Phosphorylation , Prostaglandin D2/analogs & derivatives , Protein Synthesis Inhibitors/pharmacology , Signal Transduction , Thiazolidinediones/pharmacology , Trans-Activators/metabolismالملخص
The effects of administration of cortisol, corticosterone, testosterone, progesterone and a synthetic estrogen. diethylstilbestrol (DES) on total brain Na(+)-K+- ATPase were investigated in tilapia, O. mossambicus. Exogenous administration of 0.125 and 0.25 microg/g body weight of glucocorticoids and 0.125, 0.25 and 0.5 microg/g body weight of DES for 5 days significantly stimulated Na+(-) K+ ATPase activity by 14-41% in the brain, while 0.5 microg/g body weight of glucocorticoids did not evoke any response on the activity of the enzyme. Progesterone (0.125 and 0.25 microg/g body weight) administration significantly decreased the enzyme activity by 21-36% and high dose (0.5 microg/g body weight) was ineffective. Testosterone exhibited a biphasic effect on Na(+)-K+ ATPase activity--a low dose stimulated by 14% while middle and high doses inhibited it by 19-24%. The results seem to be the first report on the effect of steroids on brain ATPase activity in a teleost. When 0.25microg/g body weight of actinomycin D or puromycin was administered prior to the treatment of similar doses of hormones, the inhibitors significantly inhibited the effect of the hormones by 24-52%. This clearly shows that the effect of the hormones was sensitive to the action of inhibitors suggesting a possible genomic mode of action under long-term treatment. The results suggest that cortisol, corticosterone and DES may possibly stimulate the co-transport of glucose and excitation of membrane potential while progesterone and testosterone inhibit them in the brain of O. mossambicus by regulating the activity of Na(+)-K+ ATPase.
الموضوعات
Animals , Body Weight , Brain/drug effects , Corticosterone/pharmacology , Dactinomycin/pharmacology , Diethylstilbestrol/pharmacology , Dose-Response Relationship, Drug , Fishes , Hydrocortisone/pharmacology , Progesterone/pharmacology , Puromycin/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Steroids/pharmacology , Testosterone/pharmacology , Tilapiaالملخص
Meiotic arrest of oocyte in an Indian carp, Labeo rohita Ham. has been found for the first time to be withdrawn by insulin only. Addition of insulin to oocytes in vitro caused germinal vesicle breakdown (GVBD), one of the first visual markers to determine initiation of the final maturational process. Under the influence of insulin the germinal vesicle (GV) of the oocyte migrated towards the animal pole, reached the micropyle and then dissolved (GVBD). By using different concentrations of insulin i.e., 0.063, 0.63, 6.3 and 12.6 mM, optimum amount required was found to be 6.3 mM. Induction of GVBD by insulin could be blocked by cycloheximide (Chx), a translation inhibitor, while actinomycin D (AcD) had no effect suggesting non-involvement of transcriptional activity in this process. Addition of the maturation-inducing steroid 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) stimulated (P<0.01) GVBD of carp oocytes and its combination with insulin showed an additive effect. Gonadotropin (GtH) caused GVBD but its effect was greatly augmented by insulin. Our results demonstrate that not only can insulin alone induce GVBD in carp oocytes, but it also augments the stimulatory effect of DHP or IGF-I or GtH on GVBD. This information will be important in hormonal manipulation during induced breeding of carp.
الموضوعات
Animals , Carps/physiology , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Gonadotropins/pharmacology , Hydroxyprogesterones/pharmacology , Insulin/pharmacology , Insulin-Like Growth Factor I/pharmacology , Meiosis/physiology , Oocytes/cytology , Protein Synthesis Inhibitors/pharmacologyالملخص
The present study was designed to investigate the effect of actinomycin D, a transcription inhibitor, and cycloheximide, a translation inhibitor, on the delayed cardioprotective effect of ischemic preconditioning. Left thoracotomy was performed in anaesthetized rats at 4th/5th intercostal space and polypropylene suture (5-0) was employed to occlude left common coronary artery. Ischemic preconditioning was produced by four episodes of 5 min of coronary artery occlusion followed by 5 min of reperfusion and thoracic cavity was sutured. Left thoracotomy was performed again after 24 hr of ischemic preconditioning and left coronary artery was occluded for 30 min followed by reperfusion for 120 min. Area at risk and infarct size was estimated by patent blue and TTC staining respectively. Total left ventricular RNA was isolated and estimated quantitatively. Ischemic preconditioning, 24 hr after its induction, produced significant decrease in myocardial infarct size occurred as a result of sustained ischemia and reperfusion but produced no marked effect on ventricular RNA content. Actinomycin D and cycloheximide only, in high dose, markedly attenuated ischemic preconditioning induced decrease in myocardial infarct size. However, no such effect was noted with low dose of cycloheximide. The results suggest that delayed cardioprotective effect of ischemic preconditioning may be mediated through denovo synthesis of protein(s) which is regulated both at transcriptional and translational level.
الموضوعات
Animals , Cardiotonic Agents/pharmacology , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Ischemic Preconditioning , Rats , Rats, Wistarالملخص
There was a linear increase in poly (A+) polymerase activity in the C. arietinum epicotyls during germination. Six-day-old auxin treated seedlings showed about 3-4 fold stimulation in enzyme activity, accompanied with 3- fold rise in the relative abundance of poly (A+) RNA levels. Actinomycin D, cycloheximide, cordycepin and amino acid analogues caused dramatic decline in poly (A+) polymerase as well as poly (A+) RNA levels. It seems that auxin induced a de novo synthesis of this enzyme.
الموضوعات
Amino Acids/metabolism , Antineoplastic Agents/pharmacology , Cicer/drug effects , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Deoxyadenosines/pharmacology , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Plant , Germination , Indoleacetic Acids/pharmacology , Poly A/metabolism , Polynucleotide Adenylyltransferase/isolation & purification , Protein Synthesis Inhibitors/pharmacology , RNA/metabolism , Seeds/drug effectsالملخص
Rat gene 33 (g33) mRNA has a widespread tissue distribution. Insulin and various agents such as glucocorticoids, phorbol esters and plant lectins regulate G33 expression in rat hepatoma cells. The regulation of g33 by insulin and a phorbol ester was examined in two Chinese Hamster ovary (CHO) cell lines, CHO-T cells (which overexpress human insulin receptors (hIR)) and wild type CHOwt cells. These cell lines were used to determine how expression of the hIR influences the capacity of g33 to respond to insulin and phorbol myristate acetate (PMA). Treatment of CHOwt and CHO-T cells with insulin increased mRNAg33 levels three to four-fold, with a maximum effect reached after three hours of treatment. PMA treatment of CHOwt and CHO-T cells caused a similar elevation of mRNAg33 levels after three hours. Insulin had no effect on mRNAg33 stability in both CHO cell lines. Additionally, the effects of insulin and PMA on mRNAg33 levels were additive only in CHO-T cells. Insulin or PMA-pretreated CHO-T cells were able to respond to both agents, but elevation ofmRNAg33 levels was maximal. In contrast, when insulin and/or PMA-pretreated CHOwt cells were exposed to insulin or PMA, g33 was able to respond maximally. These results suggest that insulin and phorbol esters act through different signaling mechanisms in CHOwt cells. Additionally, insulin's ability to stimulate g33 expression in CHOwt cells suggests that this insulin effect may be independent of the insulin eceptor. There are differences in the regulation pattern of g33 by insulin and PMA in rat hepatoma and among the two CHO cell lines used in this study
الموضوعات
Humans , Animals , Cricetinae , Rats , CHO Cells , Gene Expression Regulation , Insulin/pharmacology , RNA, Messenger/analysis , Tetradecanoylphorbol Acetate/pharmacology , Antibiotics, Antineoplastic/pharmacology , Blotting, Northern , CHO Cells/metabolism , Dactinomycin/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Insulin/physiology , Receptor, Insulin/physiology , Gene Expression Regulation , RNA, Messenger/adverse effects , RNA, Messenger/isolation & purificationالملخص
Chemokine KC has been considered to be a murine homologue of human GRO/MGSA and was identified as chemoattractant for monocytes and neutrophils. This study examined the expression of KC mRNA in thioglycollate-elicited mouse peritoneal macrophages that were stimulated in vitro with Candida albicans (CA). Also examined were the inhibitory effects of IL-10 on the CA-induced expression of KC gene by Northern blot analysis. CA was found to induce chemokine gene expression in a gene-specific manner, CXC chemokine IP-10 mRNA expression was not detected in CA-stimulated macrophages. Maximum KC mRNA expression was observed approximately 2 hr after adding CA. The inhibitory action of IL-10 to CA-induced KC mRNA expression on mouse peritoneal macrophages was independent on concentration and stimulation time of IL-10 and was observed approximately one hour after adding IL-10 and CA simultaneously. IL-10 produced a decrease in the stability of KC mRNA, and CA-stimulated macrophages with cycloheximide blocked the suppressive effect of IL-10. These results suggest that CA also induces chemokine KC from macrophages, and IL-10 acts to destabilize CA-induced KC mRNA and de novo synthesis of an intermediate protein is a part of the IL-10 suppressive mechanism.
الموضوعات
Mice , Animals , Blotting, Northern , Candida albicans/metabolism , Cells, Cultured , Chemotactic Factors/genetics , Dactinomycin/pharmacology , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Growth Substances/genetics , Interleukin-10/pharmacology , Interleukin-10/metabolism , Macrophages/physiology , Mice, Inbred BALB C , Nucleic Acid Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , RNA, Messenger/drug effectsالملخص
In this study the programmed cell death [apoptosis] of human neutrophilic granulocytes was investigated in the presence and absence of fetal calf serum [FCS], cycloheximide and actinomycin-D. The results show that when FCS is omitted from cultures, apart from a decrease in viability, the percentage of apoptotic cells and DNA fragmentation increases. Apoptosis is accelerated in serum withdrawal cultures at 6 hours of incubation time. The use of fluorescent dyes and diphenylamine reaction procedures confirm the above results. Treatment of cells with protein synthesis inhibitors, actinomycin-D and cycloheximide promotes apoptosis and produces a typical ladder of internucleosomal cleavage in the cellular chromatin; the extent of fragmentation however, differs
الموضوعات
Humans , Neutrophils/drug effects , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Protein Synthesis Inhibitors/drug effectsالملخص
MP 84 is a novel protein synthesized in response to all cytokines. This antigen is expressed only in stimulated mesangial cells and diseased kidney sections but not in the normal kidney sections [Mukhtar A., 1992 and 1994]. This is the first study of the mechanism of MP 84 expression by stimulated mesangial cells by incubating cells with cyclohexamide [inhibitor of portein synthesis], tunicamycin [inhibitor of transcription] and actinomycin D [inhibitor of N-linked glycosylation]. Anti phosphatase anti alkaline phosphatase [APAAP] was used. This study shows that both transcription and translation are required for expression since both cyclohexamide [inhibitor of protein translation] and actinomycin D [inhibitor of transcription] inhibited the traget antigen
الموضوعات
Cytokines/drug effects , Protein Synthesis Inhibitors , Dactinomycin/pharmacologyالملخص
Os autores estudaram a nível ultra-estrutural, a resposta da epiderme na reparaçao de feridas cutâneas sob efeito de dactinomicina. Foram observadas alteraçoes celulares envolvendo a organizaçao citoplasmática e nuclear.
الموضوعات
Animals , Male , Rats , Wound Healing , Dactinomycin/pharmacology , Epidermis/ultrastructure , Epidermis/drug effects , Rats, Inbred Strainsالملخص
The change in activity of cinnamic acid 4-hydroxylase (CA4H) in potato parenchyma tissue exposed to various conditions has been examined. Maximum induction of CA4H activity was obtained at 18 hr of incubation. Though CA4H induction can occur in dark, over 100% increase in enzyme activity was obtained on exposure of the tissue to light. Actinomycin D and cycloheximide inhibited the induction process. Mn2+, though known to cause an induction of CA4H in Jerusalem Artichoke, strongly inhibited potato CA4H induction. Dithiothreitol enhanced the CA4H activity due to either activation or protection of the enzyme. CA4H induction was significantly regulated at very low concentrations of trans-cinnamate and paracoumarate.