ABSTRACT
The transcription factor, nuclear factor kappa B (NF-kappa B), is activated by various stimuli including cytokines, radiation, viruses and oxidative stress. Here we show that, although induction with H(2)O(2) gives rise to NF-kappa B nuclear translocation in both lymphocyte (CEM) and monocyte (U937) cells, it leads only to the production of mRNA species encoding interleukin-8 (IL-8) and macrophage inflammatory protein 1 alpha in U937 cells. Under similar conditions these mRNA species are not observed in CEM cells. With the use of a transient transfection assay of U937 cells transfected with reporter constructs of the IL-8 promoter and subsequently treated with H(2)O(2), we show that (1) IL-8-promoter-driven transcription is stimulated in both U937 and CEM cells and (2) the NF-kappa B site is crucial for activation because its deletion abolishes activation by H(2)O(2). The production of IL-8 mRNA in U937 cells is inhibited by the NF-kappa B inhibitors clasto-lactacystin-beta-lactone and E-64D (l-3-trans-ethoxycarbonyloxirane-2-carbonyl-L-leucine-3-methyl amide) but requires protein synthesis de novo. Moreover, inhibition of the p38 mitogen-activated protein kinase also decreases the IL-8 mRNA up-regulation mediated by H(2)O(2). Taken together, these results show the importance of post-transcriptional events controlled by a p38-dependent pathway in the production of IL-8 mRNA in U937. The much lower activation of p38 in CEM cells in response to H(2)O(2) could explain the lack of stabilization of IL-8 mRNA in these cells.
Subject(s)
Chemokines/genetics , Chemokines/metabolism , Oxidative Stress/genetics , RNA Processing, Post-Transcriptional , Cell-Free System/drug effects , Cell-Free System/metabolism , Chemokine CCL2/genetics , Chemokine CCL2/metabolism , Chemokines/biosynthesis , Enzyme Activation/genetics , Gene Expression Regulation/drug effects , Humans , Hydrogen Peroxide/pharmacology , Interleukin-8/genetics , Interleukin-8/metabolism , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Oxidative Stress/drug effects , Promoter Regions, Genetic , Protein Binding/genetics , RNA Processing, Post-Transcriptional/drug effects , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , Transcription, Genetic/drug effects , Tumor Cells, Cultured , U937 Cells/drug effects , U937 Cells/metabolism , p38 Mitogen-Activated Protein KinasesABSTRACT
The present review focuses on the potential physiological regulations involving different isoforms of adenylyl cyclase (AC), the enzymatic activity responsible for the synthesis of cAMP from ATP. Depending on the properties and the relative level of the isoforms expressed in a tissue or a cell type at a specific time, extracellular signals received by the G protein-coupled receptors can be differently integrated. We report here on various aspects of such regulations, emphasizing the role of Ca(2+)/calmodulin in activating AC1 and AC8 in the central nervous system, the potential inhibitory effect of Ca(2+) on AC5 and AC6, and the changes in the expression pattern of the isoforms during development. A particular emphasis is given to the role of cAMP during drug dependence. Present experimental limitations are also underlined (pitfalls in the interpretation of cellular transfection, scarcity of the invalidation models, and so on).
Subject(s)
Adenylyl Cyclases/metabolism , Brain/enzymology , Isoenzymes/metabolism , Kidney/enzymology , Myocardium/enzymology , Adenylyl Cyclases/genetics , Animals , Gene Expression Regulation, Enzymologic , Isoenzymes/geneticsABSTRACT
Activation of transcription factor NF-kappa B involves the signal-dependent degradation of basally phosphorylated inhibitors such as I kappa B alpha. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-kappa B activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of I kappa B alpha. Indeed, mutation of Ser32 and Ser36 blocked IL-1 beta- or PMA-induced NF-kappa B activation, but had no effect on its activation by H2O2. Although I kappa B alpha was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-kappa B activation by H2O2, while it had no effect on activation by IL-1 beta or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on I kappa B kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole) blocks NF-kappa B activation. H2O2-induced I kappa B alpha phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-kappa B activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.
Subject(s)
DNA-Binding Proteins/metabolism , I-kappa B Proteins , NF-kappa B/metabolism , Oxidative Stress , Peptide Fragments/physiology , Protein Tyrosine Phosphatases/physiology , Tyrosine/physiology , Animals , Calpain/physiology , Casein Kinase II , Cysteine Endopeptidases/physiology , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Enzyme Activation/drug effects , Humans , Hydrogen Peroxide/pharmacology , I-kappa B Kinase , Mice , Multienzyme Complexes/physiology , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , Peptide Fragments/genetics , Phosphorylation/drug effects , Point Mutation/drug effects , Proteasome Endopeptidase Complex , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Protein Structure, Tertiary/physiology , Protein Tyrosine Phosphatase, Non-Receptor Type 12 , Protein Tyrosine Phosphatases/genetics , Serine/genetics , Tumor Cells, Cultured , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
The structure of the DNA binding site of the Nuclear single-stranded Binding Factor (NssBF), located in the long terminal repeat of the Drosophila 1731 retrotransposon, was investigated by melting temperature experiments, chemical probing and fluorescence measurements using a macrocyclic bis-acridine. The most probable structure of this element, named Bc, mainly involves two hairpins in equilibrium at pH 6.0 at low concentration. The hairpins differ in their apical loop size; 4 and 8 nt. The structural flexibility of Bc probably derives from the three consecutive CATA repeats complementary to the GTAT nucleotides of the palindrome. In contrast, the Bc complementary strand adopts a single hairpin. Since Bc is implicated in repression of transcription via binding of two specific factors, its structural flexibility could be associated with this process.
Subject(s)
DNA/chemistry , DNA/genetics , Drosophila/genetics , Retroelements/genetics , Acridines , Animals , Base Sequence , Fluorescent Dyes , Hydrogen-Ion Concentration , Nucleic Acid Conformation , Oligonucleotide Probes/genetics , Terminal Repeat Sequences , ThermodynamicsABSTRACT
Changes in the activity and in the expression of adenylyl cyclase (AC) were examined in mouse skeletal muscle after denervation and during development. Four isoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blot analysis in gastrocnemius muscle, AC9 being the most abundant. After denervation, the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. AC activity in response to several neurotransmitters was increased after denervation. During development, AC activity was high in fetus and neonate and declined in the adult; the sensitivity of AC activity to various neurotransmitters was the highest on the third postnatal day. The levels of AC6 and AC7 mRNAs were high on the third postnatal day and then decreased in adult, paralleling the decline in AC activity. All the characteristics of AC expression and activity in fetus and neonate resembled those observed in denervated adult muscle. These results indicate that changes in AC activity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscle atrophy.
Subject(s)
Adenylyl Cyclases/genetics , Gene Expression , Muscle Denervation , Muscle Development , Muscle, Skeletal/growth & development , Muscle, Skeletal/innervation , RNA, Messenger/metabolism , Animals , Blotting, Northern , GTP-Binding Proteins/genetics , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/enzymology , Myogenin/geneticsABSTRACT
The expression of adenylyl cyclases (ACs) in the adult rat adrenal gland was examined. In situ hybridization revealed specific patterns of AC messenger RNA (mRNA) distribution. AC1 was limited exclusively to the adrenal medulla. AC5 and AC6 were mainly expressed in the adrenal medulla, with a weak expression in the zona glomerulosa. AC9 was found in all the three regions of the adrenal cortex but not in the adrenal medulla. All these ACs were detected on postnatal day 1 (PN1), and their pattern of expression was unchanged on PN7, PN21, and PN90 (adult). We analyzed the response of these ACs to various physiological conditions known to affect the synthesis of aldosterone and corticosterone in the adrenal cortex. Our study demonstrates a specific increase of AC6 but not AC5 mRNA in the zona glomerulosa of rats given a low sodium diet. AC9 mRNA was increased in all the three cortical zones of rats treated with ACTH. We suggest that AC6 and AC9 play important roles in different pathways associated with the regulation of aldosterone and corticosteroid production.
Subject(s)
Adenylyl Cyclases/genetics , Adrenal Glands/metabolism , RNA, Messenger/metabolism , Adrenal Glands/growth & development , Adrenocorticotropic Hormone/pharmacology , Aging/metabolism , Aldosterone/biosynthesis , Animals , Corticosterone/biosynthesis , In Situ Hybridization , Isoenzymes/genetics , Rats , Rats, Sprague-Dawley , Tissue DistributionABSTRACT
Extensive analyses of Drosophila melanogaster retrotransposon transcriptions in cultured cells or during development have been reported, but little is known about their translation during the development of the fly. Analysis of the translational products of the 1731 Drosophila melanogaster retrotransposon in Kc Drosophila cultured cells has been reported, showing the existence of primary products (Gag and Pol) and of processed polypeptides of various sizes. Study of 1731 retrotransposon expression at both levels of transcription and translation during the development of Drosophila melanogaster, is presented. 1731 transcripts were detected by in situ hybridization and 1731 proteins were detected by immunostaining and immunoblotting in embryos and in adult gonads. 1731 transcripts and proteins were detected in the mesoderm and central nervous system during embryonic development, in nurse cells and follicle cells in adult ovaries and in primary spermatocytes in adult testes. Moreover, Western blot analysis of the 1731 proteins with anti-Gag or anti-Pol antibodies in gonads revealed that the 1731 mRNA could be translated differentially according to the expressing tissue: essentially, ovarian translation and/or processing of 1731 products is different from that operating in testes, where the Gag-Pol fusion polyprotein is the most prominent product. Our results indicate that expression of the 1731 mobile element is regulated not only at the transcriptional level but also at the translational level, and that this regulation is different in the two sexes.
Subject(s)
Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental , Gene Products, gag/genetics , Gene Products, pol/genetics , Insect Proteins/genetics , Retroelements , Animals , Blotting, Western , Drosophila melanogaster/embryology , Drosophila melanogaster/growth & development , Embryo, Nonmammalian/physiology , Female , Immunoblotting , In Situ Hybridization/methods , Insect Proteins/immunology , Insect Proteins/metabolism , Male , Ovary/physiology , Protein Biosynthesis , Rabbits , Testis/physiology , Tissue Distribution , Transcription, GeneticABSTRACT
Nucleic acids can undergo dynamic conformational changes associated with the regulation of biological processes. A molecule presenting larger affinities for alternative structures relative to a duplex is expected to modify such conformational equilibria. We have previously reported that macrocyclic bis-acridine binds preferentially to single-stranded regions, especially DNA hairpins, due to steric effects. Here, we show, using gel electrophoresis, fluorescence and melting temperature experiments, that the macrocycle bis-acridine shifts an equilibrium from a duplex towards the corresponding hairpins. Competition experiments enlighten the higher affinity of the macrocycle for hairpins compared with double-stranded DNA. The macrocycle bis-acridine destabilizes a synthetic polynucleotide, by the formation of premelted areas. By extrapolation, the macrocycle bis-acridine should be able to disrupt, at least locally, genomic DNA duplexes and to stabilize unpaired areas, especially palindromic ones forming hairpins. Such macrocyclic compounds may have potential applications in the therapy of diseases involving hairpins.
Subject(s)
Acridines/pharmacology , DNA/chemistry , Nucleic Acid Conformation/drug effects , Binding, Competitive , DNA/metabolism , DNA, Single-Stranded/metabolism , Electrophoresis, Agar Gel , Macromolecular Substances , Osmolar Concentration , Poly dA-dT/chemistry , Poly dA-dT/metabolism , Spectrometry, FluorescenceABSTRACT
We present here a real-time and single cell study of an oxidative stress mechanism in human fibroblasts. Hydrogen peroxide released by a single normal or SV40-transformed human fibroblast was detected at the surface of an ultramicroelectrode while puncturing the cell membrane with the ultramicroelectrode tip itself or with a micropipette. This mechanical intrusion induced the emission of large quantities (10(-15)-10(-14) mol) of H2O2 by the cell with a very short time delay (<0.5 s). We show that this H2O2 production was an active neo-production by fibroblasts when the membrane was stressed by the cellular puncture and is a model which could mimic similar effects as particle (virus, bacteria, etc.) intrusion into the cell. Cell incubations in the presence of some inhibitors of the different NADPH oxidase enzymes, using ultramicroelectrode measurements of the short time effects (<20 min) let us believe that an NADPH oxidase-like enzyme may be implicated in this induced-H2O2 generation. Phenylarsine oxide (PAO), a specific NADPH oxidase inhibitor, at concentrations between 0.5-50 microM seemed to quickly kill the transformed cells preferentially to the normal cells, pointing out for the future a possible anti-cancerous chemotherapic use.
Subject(s)
Fibroblasts/enzymology , NADPH Oxidases/metabolism , Stress, Mechanical , Antioxidants/pharmacology , Arsenicals/pharmacology , Cell Line, Transformed/drug effects , Cell Membrane/pathology , Cell Transformation, Viral , Cells, Cultured/drug effects , Child, Preschool , Enzyme Activation , Enzyme Inhibitors/pharmacology , Fetus , Fibroblasts/drug effects , Humans , Hydrogen Peroxide/metabolism , Microelectrodes , NADPH Oxidases/antagonists & inhibitors , Oxidative Stress , Oxygen Consumption , Reactive Oxygen Species , Simian virus 40/physiologyABSTRACT
Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release.
Subject(s)
Hypochlorous Acid/pharmacology , I-kappa B Proteins , NF-kappa B/metabolism , T-Lymphocytes/metabolism , Transcriptional Activation/physiology , Antioxidants/pharmacology , Blotting, Western , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/pharmacology , Dimerization , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation/genetics , Hot Temperature , Humans , Hydrogen Peroxide/pharmacology , Kinetics , NF-KappaB Inhibitor alpha , Nuclear Proteins , Oxidation-Reduction , Oxidative Stress/physiology , Transcription Factors/metabolism , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Phenylarsine oxide (PAO), which is described as an inhibitor of tyrosine phosphatase activity, inhibits H2O2 release from human peripheral blood mononuclear cells (PBMCs) as measured by electrochemistry. Since human immunodeficiency virus type 1 (HIV-1) replication is known to be favored under oxidative stress conditions, ex vivo experiments using uninfected PBMCs, primary monocytes or a latently infected promonocytic U1 cell line show that HIV-1 replication and reactivation, monitored by p24 antigen measurement, are inhibited by PAO in a time- and concentration-dependent manner. These observations can be linked with the inhibition of NF-kappa B activation when uninfected monocytes are induced by either tumor necrosis factor alpha (TNF-alpha) phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS).
Subject(s)
Arsenicals/pharmacology , HIV-1/drug effects , Monocytes/physiology , Monocytes/virology , Virus Replication/drug effects , Cell Line , Enzyme Inhibitors/pharmacology , HIV Core Protein p24/biosynthesis , HIV-1/physiology , Humans , Hydrogen Peroxide/analysis , Lipopolysaccharides/pharmacology , Monocytes/drug effects , NF-kappa B/metabolism , Oxidative Stress , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/antagonists & inhibitors , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Phylogenetic analysis of the retrotransposon and retrovirus suggests an evolutionary relationship between them and indicates that transactivation of the long terminal repeat (LTR)-containing retroelements could be ubiquitous. Using constructs expressing a reporter gene under the control of the entire or deleted LTR of 1731, which is a retrotransposable element of Drosophila melanogaster, we were able to show that the UVB-irradiation activation of the 1731-LTR requires the same short sequence of U3 region in a human epithelial cell line as in Schneider's Drosophila cell line (S2). This sequence is similar to the binding sequence of the members of the nuclear factor-kappa B (NF-kappa B)/rel family. In addition, human colonic carcinoma cells (HT29), in response to UVB-irradiation, produce some extracellular factor(s) that activates the 1731-LTR in nonirradiated cells.
Subject(s)
Drosophila melanogaster/genetics , Drosophila melanogaster/radiation effects , HT29 Cells/physiology , HT29 Cells/radiation effects , Repetitive Sequences, Nucleic Acid , Retroelements/genetics , Retroelements/radiation effects , Ultraviolet Rays , Up-Regulation/radiation effects , Animals , DNA/genetics , Humans , Sequence Homology, Nucleic Acid , TransfectionABSTRACT
Using constructs expressing the reporter gene under the control of the entire or deleted long terminal repeats (LTRs) of 1731, a Drosophila melanogaster retrotransposable element, we show that 1731-LTR is activated by X-irradiation in a dose- and time-dependent manner, and that a sequence located in the U3 region of these LTRs is required. The cis-acting element conferring X-responsiveness shows similarities to kappa B (kappa B)-like binding sequence. In response to X-irradiation, S2 Drosophila cells produced an extracellular factor which activates the 1731-LTR in nonirradiated cells. This factor was detected both when transfected cells were cocultured with inducing cells and when a conditioned medium taken from irradiated cultures was added.
Subject(s)
Drosophila melanogaster/genetics , Drosophila melanogaster/radiation effects , Repetitive Sequences, Nucleic Acid/radiation effects , Retroelements/radiation effects , Animals , Base Sequence , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Culture Media, Conditioned , DNA/genetics , NF-kappa B/genetics , Sequence Homology, Nucleic Acid , Transcription, Genetic/radiation effects , TransfectionABSTRACT
A 1731 is a Drosophila melanogaster retrotransposon, the transcripts of which decrease in Drosophila cells after treatment by the 20-hydroxyecdysone (20-OH), the steroid-molting hormone of insects. In order to analyze the regulation of the long terminal repeat (LTR) directed transcription by UV-B, S2 Drosophila cells were transfected with various chimeric constructs carrying the LTR of 1731 linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene and then subjected to UV-B irradiation. The results demonstrated that the 1731 LTR is activated by UV-B irradiation in a dose- and time-dependent manner. Using constructions expressing the reporter gene under the control of either the entire or deleted LTRs of 1731, we established that a sequence located in the U3 region was required for the retrotransposon to respond to UV-B. The cis-acting element is identical to the binding sequence of the dorsal transcription factors. In addition, the S2 Drosophila cell produced extracellular factor(s) in response to UV-B irradiation which activated the 1731 LTR in nonirradiated cells. This factor(s) was detected when responding cells were cocultured with inducing cells and when conditioned medium from irradiated cultures was added to the cell cultures.
Subject(s)
Drosophila melanogaster/genetics , Repetitive Sequences, Nucleic Acid/radiation effects , Retroelements/radiation effects , Animals , Base Sequence , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , DNA/genetics , DNA/radiation effects , Drosophila melanogaster/radiation effects , Drosophila melanogaster/virology , Genes, Reporter , Molecular Sequence Data , Signal Transduction , Transfection , Ultraviolet Rays , Up-RegulationABSTRACT
The NssBF element has been characterized as a 26 nt sequence in the long terminal repeat of Drosophila melanogaster retrotransposon 1731. This sequence has been shown to be implicated in transcriptional repression of the 1731 promoter. We here report the cloning of a cDNA encoding a nuclear DNA binding protein named p11 that binds specifically to the NssBF element. P11 is a 98 amino acid polypeptide. It exhibits similarities with the mouse p9 single-stranded DNA binding protein, raising the possibility of a very general family of protein factors. Co-transfection experiments in human U937 cells showed repression of the 1731 promoter by overexpression of p11.
Subject(s)
DNA, Complementary/isolation & purification , DNA-Binding Proteins/genetics , Drosophila melanogaster/genetics , Retroelements/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , DNA-Binding Proteins/metabolism , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolismABSTRACT
1731 is a Drosophila melanogaster retrotransposon whose nucleotide sequence shows a proviral architecture with two long terminal repeats (LTRs) framing two internal Open Reading Frames (ORFs). The pol ORF2 of this mobile genetic element was demonstrated to code for an active Reverse Transcriptase (RT) and the ORF1 is expected to code for the structural Gag proteins of the virus-like particles (VLP). Using specific anti-Gag antibodies, we have characterized the 1731 Gag polypeptides expressed either in vitro or in Kc Drosophila melanogaster cultured cells. Together with the 1731 RT, the largest, likely post-translationaly-modified Gag polypeptides are gathered into cytoplasmic virus-like particles. Moreover and consistent with the nuclear localization signal present in the Gag sequence, we observed that a short 1731 Gag polypeptide is associated to the cell nuclei.
Subject(s)
Cell Nucleus/chemistry , Drosophila melanogaster/genetics , Gene Products, gag/analysis , Retroelements , Virion/chemistry , Amino Acid Sequence , Animals , Blotting, Western , Cells, Cultured , Gene Products, gag/genetics , Immunosorbent Techniques , Molecular Sequence Data , Open Reading Frames , RNA-Directed DNA Polymerase/genetics , Repetitive Sequences, Nucleic Acid , Restriction MappingABSTRACT
Am important aspect of human immunodeficiency virus (HIV-1) infection is the regulation of its expression by nuclear factor kappa B (NF-kappa B) by redox-controlled signal transduction pathways. In this study, we demonstrate that selenium supplementation can effectively increase glutathione peroxidase (GPx) activity in latently infected T lymphocytes. The Se-supplemented cells exhibited an important protection against the cytotoxic and reactivating effects of hydrogen peroxide (H2O2). Concomitantly, NF-kappa B activation by H2O2 was also decreased in Se-supplemented cells. Selenium stimulation of GPx activity also induces a protective effect against cell activation by tumor necrosis factor alpha (TNF-alpha) but less significantly by phorbol esters such as PMA. These Se-mediated effects were specific because they were not found when AP-1 DNA-binding activity was studied after H2O2-induced stress. Hyperthermia was also studied because it could promote intracellular electron leakage in electron transport chains. Elevating the temperature to 42 degrees C did not induce NF-kappa B directly. Rather, it sensitized infected cells to subsequent oxidative stress by H2O2, demonstrating the importance of hyperthermia, often associated with opportunistic infections in the development of immunodeficiency. In this case, Se induced partial protection against the sensitizing effect of hyperthermia.
Subject(s)
Glutathione Peroxidase/metabolism , HIV-1/physiology , Antioxidants/metabolism , Cell Line , DNA, Viral/metabolism , HIV Infections/etiology , HIV Infections/prevention & control , HIV-1/drug effects , HIV-1/pathogenicity , Hot Temperature , Humans , Hydrogen Peroxide/pharmacology , NF-kappa B/metabolism , Oxidation-Reduction , Oxidative Stress/drug effects , Selenium/pharmacology , Signal Transduction , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , T-Lymphocytes/virology , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The entire 1731 retrotransposon of Drosophila melanogaster, tagged with the E. coli lac Z gene inserted in its gag sequence, was injected into oocytes and fertilized eggs of the urodele amphibian Pleurodeles waltl. Expression of the reporter gene indicated that the 1731 promoter (its 5'LTR) is active in the embryos and not in the oocytes. It appeared that this element is regulated as amphibian genes are at the beginning of the development, i.e. that expression was detected after the mid blastula stage and maintained up to four or five days after injection. Another construction associating the modified 1731 promoter with the CAT gene is also expressed in Pleurodeles embryos during the same period of development. This indicated that the 1731 promoter issued from a Drosophila species is activated as promoting sequences of amphibian zygotic genes are, suggesting that in the case of horizontal transfer, 1731 can be expressed into vertebrate organisms.
Subject(s)
Blastocyst/physiology , Drosophila melanogaster/genetics , Oocytes/physiology , Retroelements , Animals , Embryo, Nonmammalian/physiology , Escherichia coli/genetics , Female , Fertilization , Genes, Bacterial , Plasmids , Pleurodeles , Repetitive Sequences, Nucleic Acid , beta-Galactosidase/biosynthesisABSTRACT
The interaction of DNA topoisomerase II with the long terminal repeat (LTR) of the Drosophila melanogaster 1731 retrotransposon was studied. The covalent binding of topoisomerase II to the LTR was strongly stimulated by different inhibitors of the enzyme 4'-demethylepipodophyllotoxin-9-(4,6-O-2-ethylidene-beta-D-glucopy ranoside (VP-16), 4'-(9-acridinylamino)methanesulfon-m-anisidine) (m-AMSA) and an ellipticine derivative. Enzyme-mediated DNA cleavage could be observed in the absence of inhibitors and was stimulated in their presence. Cleavage occurred predominantly at sites located within or at the boundary of alternating purine/pyrimidine tracts in agreement with previous observations [Spitzner, J. R., Chung, I. K. & Muller, M. T. (1990) Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats, Nucleic Acids Res. 18, 1-11]. In addition, all of the cleavage sites observed in the absence of inhibitor were located in the U3 region of the LTR. The site specificity of drug-induced cleavage was studied and the conformity of the cleavage sites with previously established consensus sequences was examined. Our results suggest that DNA topoisomerase II, through its ability to alter the degree of DNA supercoiling, might be involved in the control of different functions of the LTR.
Subject(s)
DNA Topoisomerases, Type II/metabolism , DNA Transposable Elements , Repetitive Sequences, Nucleic Acid , Amsacrine/pharmacology , Animals , Base Sequence , DNA/genetics , DNA/metabolism , Drosophila , Ellipticines/chemistry , Ellipticines/pharmacology , Etoposide/pharmacology , Molecular Sequence DataABSTRACT
An entire copy of 1731, a Drosophila melanogaster retrotransposon, was tagged by fusing in frame its putative gag gene with the reporter LacZ sequence. The high transfection efficiency of Drosophila virilis cells added to the absence of 1731 in their genome allowed, by combining histochemical staining and immunological detections, the demonstration of the translation of the 1731 gag gene. The gag protein is gathered in virus-like particles. Its occurrence in nuclei is consistent with a nuclear localization signal. The expression of the sense construction was inhibited by cotransfections with its antisense homologue.