Effects of propofol on endothelial cells subjected to a peroxynitrite donor (SIN-1).
Mathy, Marianne ; Mouithys-Mickalad, Ange ; et al
in Anaesthesia (2000), 55(11), 1066-71
We investigated the effect of propofol on endothelial cells subjected to the peroxynitrite (ONOO-) donor 3-morpholino sydnonimine (SIN-1). Cells were incubated overnight with 0.5, 1.0 or 2.0 mM SIN-1 ... [more ▼]
We investigated the effect of propofol on endothelial cells subjected to the peroxynitrite (ONOO-) donor 3-morpholino sydnonimine (SIN-1). Cells were incubated overnight with 0.5, 1.0 or 2.0 mM SIN-1, with or without 10-3 M propofol (Diprivan). Cytotoxicity, assessed by measuring the release of pre-incorporated 51Cr, increased when the concentration of SIN-1 increased, and was significantly decreased by 10-3 M propofol (90%, 78% and 28% of protection against 0.5, 1.0 and 2.0 mM SIN-1, respectively). Cell protection against 1 mM SIN-1 was tested with 0.03-1.0 mM propofol and this was compared to tyrosine, a target molecule for peroxynitrite. Propofol protected cells in a dose-dependent manner (r = 0.98; p < 0.001) and was as effective as tyrosine. Finally, using high-performance liquid chromatography, we demonstrated that propofol reacted with ONOO- more rapidly than did tyrosine, inhibiting nitrotyrosine formation. In the absence of propofol, 3.5 mM ONOO- with 1 mM tyrosine yielded 39.6% nitrotyrosine, but nitrotyrosine was not produced when 5 mM propofol was added. We conclude that propofol protects endothelial cells against the toxicity of ONOO-. The anti-oxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite, a property that might be relevant in pathological situations involving a significant contribution of peroxynitrite to tissue damage. [less ▲]Detailed reference viewed: 15 (0 ULg)
Plasma myeloperoxidase level and polymorphonuclear leukocyte activation in horses suffering from large intestinal obstruction requiring surgery: preliminary results.
Grulke, Sigrid ; ; et al
in Canadian Journal of Veterinary Research = Revue Canadienne de Recherche Vétérinaire (1999), 63(2), 142-7
Myeloperoxidase (MPO) is a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Thanks to a radioimmunoassay of equine myeloperoxidase, the authors have observed a ... [more ▼]
Myeloperoxidase (MPO) is a specific enzyme of neutrophil azurophilic granules with a strong oxidative activity. Thanks to a radioimmunoassay of equine myeloperoxidase, the authors have observed a significantly higher plasma level of MPO in horses operated for strangulation obstruction of the large intestine (n = 6) than in horses suffering from a non-strangulating displacement of the large intestine (n = 9). For the 2 groups, 3 phases were distinguished: reception (P1), intensive care (P2) and terminal phase (P3). The mean peak values of MPO for these phases were 121.6 ng/mL (P1), 168.6 ng/mL (P2), and 107.0 ng/mL (P3) for the non-strangulating group, and 242.6 ng/mL (P1); 426.0 ng/mL (P2), and 379.5 ng/mL (P3) for the strangulation group. The variations of the mean peak values of plasma MPO were significantly different between the 2 groups and between the different phases. A significant increase of the least square means of MPO was observed between P1 and P2. A significant decrease of the least square means of the number of circulating leukocytes was observed between P1 and P3. Polymorphonuclear neutrophil activation could play a major role in the pathogenesis of acute abdominal disease and endotoxic shock. [less ▲]Detailed reference viewed: 27 (1 ULg)
Oxidant-Scavenging Activities of Beta-Lactam Agents
; ; et al
in European Journal of Clinical Microbiology & Infectious Diseases : Official Publication of the European Society of Clinical Microbiology (1998), 17(1), 43-6
The relative antioxidant effect of ampicillin, ceftazidime, ceftriaxone, and cefuroxime on oxygen-reactive species was examined in vitro using stimulated human polymorphonuclear neutrophils. There was no ... [more ▼]
The relative antioxidant effect of ampicillin, ceftazidime, ceftriaxone, and cefuroxime on oxygen-reactive species was examined in vitro using stimulated human polymorphonuclear neutrophils. There was no evidence that any of the beta-lactam agents tested had an effect on superoxide or H2O2 generation. In contrast, all of the beta-lactam agents prevented hypochlorous acid (HOCI) chlorination of 1,1-dimethyl-4-chloro-3,5-cyclo-hexanedione in a cell-free system at concentrations of < 10 microg/ml. Furthermore, all antibiotics provided dose-dependent protection against HOCI cytotoxicity to 16HBE140 bronchial epithelial cells. Taken together, these data indicate a possible therapeutic role for beta-lactam agents in protecting host tissues from HOCI-induced oxidative damage. [less ▲]Detailed reference viewed: 13 (0 ULg)
Equine neutrophil myeloperoxidase in plasma: design of a radio-immunoassay and first results in septic pathologies.
Deby, Ginette ; Grulke, Sigrid ; et al
in Veterinary Immunology and Immunopathology (1998), 66(3-4), 257-71
The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme ... [more ▼]
The strangulated intestinal pathologies of horses are accompanied by a local activation of the neutrophils, that can be revealed by measuring the tissular enzymatic activity of the granulocytic enzyme myeloperoxidase (MPO). To estimate the possible spreading of this neutrophil activation to the systemic circulation, we designed a radioimmunoassay (RIA) for equine neutrophil myeloperoxidase (MPO) (EC 18.104.22.168) using a specific rabbit antiserum. MPO was labeled with 1 mCi 125I by a technique of self-labeling in the presence of 10(-4) M hydrogen peroxide. The RIA was performed by incubation of 100 microl diluted antiserum, 100 microl labeled MPO (+/-30,000 cpm) and 100 microl of the reference molecule (unlabeled MPO) solution or the unknown sample, at room temperature for 18 h. The antibody-antigen complexes were isolated by double antibody precipitation. The sensitivity of the RIA was 2 ng/ml. The RIA showed good precision and accuracy with intra- and inter-assay coefficients of variation 6% and 8%, respectively, for MPO concentrations ranging from 2 ng/ml to 60 ng/ml. The best sampling technique for MPO measurement in plasma was to collect blood into EDTA, which allowed us to get a plasmatic value stable with time. The mean MPO value in normal horses was 69.5 +/- 19.4 ng/ml in EDTA anticoagulated plasma (n = 48). The stress of transport and anaesthesia did not modify the mean plasmatic value of MPO. No significant increase of plasma MPO was observed in 17 horses submitted to surgery for pathologies without systemic impact. But, in 25 horses with obstructive intestinal pathologies, persistent abnormal MPO concentrations were measured (until 740 ng/ml). [less ▲]Detailed reference viewed: 68 (2 ULg)
The antibiotic ceftazidime is a singlet oxygen quencher as demonstrated by ultra-weak chemiluminescence and by inhibition of AAP consumption.
Deby, Ginette ; ; Mouithys-Mickalad, Ange et al
in Biochimica et Biophysica Acta (1998), 1379(1), 61-8
We demonstrated that the cephalosporin antibiotic ceftazidime (CAZ) deactivated singlet oxygen (1O2). We then studied the mechanisms of the CAZ effects on the ultra weak chemiluminescence (uwCL ... [more ▼]
We demonstrated that the cephalosporin antibiotic ceftazidime (CAZ) deactivated singlet oxygen (1O2). We then studied the mechanisms of the CAZ effects on the ultra weak chemiluminescence (uwCL) associated with the energy decay of 1O2 generated by the Mallet reaction (H2O2 + HOCl --> HCl + H2O + 1O2), and on the anthracene-9,10-dipropionic acid (AAP) consumption by 1O2 generated by irradiation of Rose Bengal (RB). The uwCL generated by the Mallet reaction was amplified (6.2 times) by CAZ. The use of red and blue filters, which absorb radiation below 610 nm and between 470 and 700 nm respectively, demonstrated that CAZ increased the uwCL by a radiation emission at wavelengths shorter than the 633 and 704 nm wavelength emissions of 1O2. CAZ was excited by scavenging the energy excess of 1O2, which so returned to its fundamental state, while CAZ deactivated with light emission between 430-480 nm. CAZ also inhibited in a dose-dependent manner the consumption of AAP by 1O2 generated by the irradiation of RB. The protection of AAP by 5 x 10(-3) M CAZ was equivalent to that of 10(-3) M histidine and 3 X 10(-6) M sodium azide. This process of 1O2 deactivation will be useful in diseases characterized by an excessive PMN activation with a release of activated oxygen species. [less ▲]Detailed reference viewed: 83 (18 ULg)
Purification of myeloperoxidase from equine polymorphonuclear leucocytes.
Mathy, Marianne ; ; Grulke, Sigrid et al
in Canadian Journal of Veterinary Research = Revue Canadienne de Recherche Vétérinaire (1998), 62(2), 127-32
Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute ... [more ▼]
Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute inflammation). To develop an assay for measurement of plasma MPO in horses during the above-mentioned infectious and inflammatory conditions, MPO was purified from equine PMN isolated from blood anticoagulated with citrate. PMN were extracted in a saline milieu (0.2 M Na acetate, 1 M NaCl, pH 4.7) to eliminate most of cellular proteins. Pellets were then extracted in the same buffer containing cationic detergent (1% cetyltrimethyl ammonium bromide). The supernatant was further purified by ion exchange chromatography (Hiload S Sepharose HP column 0.5 x 26 cm, equilibrated with 25 mM Na acetate, 0.2 M NaCl, pH 4.7) with a NaCl gradient (until 1 M). Most of the peroxidase activity of MPO (spectrophotometrically measured by the oxidation of orthodianisidine by hydrogen peroxide) was eluted at 0.65 M NaCl. MPO was further purified by gel filtration chromatography (Sephacryl S 200 column 2.6 x 42 cm with 25 mM Na acetate, 0.2 M NaCl, pH 4.7). MPO (specific activity: 74.3 U/mg) was obtained with a yield of 30% from the detergent extraction supernatant. Electrophoresis (non-reducing conditions) showed 3 bands identified, by comparison with human MPO, (i) the mature tetrameric enzyme (150 kDa) with 2 light and 2 heavy subunits, (ii) the precursor form (88 kDa) and (iii) a form of the heavy subunit without the prosthetic heme group (40 kDa). The mature enzyme and its precursor were glycosylated and possessed peroxidase activity. Equine MPO showed strong similarities with human and bovine MPO, with an absorption peak at 430 nm (Soret peak) characteristic of ferrimyeloperoxidase. Enzymatic activity was pH dependent (optimal value at pH 5.5). [less ▲]Detailed reference viewed: 43 (1 ULg)
Protective activity of propofol, Diprivan and intralipid against active oxygen species.
Mathy, Marianne ; Deby, Ginette ; Hans, Pol et al
in Mediators of Inflammation (1998), 7(5), 327-33
We separately studied the antioxidant properties of propofol (PPF), Diprivan (the commercial form of PPF) and intralipid (IL) (the vehicle solution of PPF in Diprivan) on active oxygen species produced by ... [more ▼]
We separately studied the antioxidant properties of propofol (PPF), Diprivan (the commercial form of PPF) and intralipid (IL) (the vehicle solution of PPF in Diprivan) on active oxygen species produced by phorbol myristate acetate (10(-6) M)-stimulated human polymorphonuclear leukocytes (PMN: 5 x 10(5) cells/assay), human endothelial cells (5 x 10(5) cells/assay) or cell-free systems (NaOCl or H2O2/peroxidase systems), using luminol (10(-4) M)-enhanced chemiluminescence (CL). We also studied the protective effects of Diprivan on endothelial cells submitted to an oxidant stress induced by H2O2/MPO system: cytotoxicity was assessed by the release of preincorporated 51Cr. Propofol inhibited the CL produced by stimulated PMN in a dose dependent manner (until 5 x 10(-5) M, a clinically relevant concentration), while Diprivan and IL were not dose-dependent inhibitors. The CL produced by endothelial cells was dose-dependently inhibited by Diprivan and PPF, and weakly by IL (not dose-dependent). In cell free systems, dose-dependent inhibitions were obtained for the three products with a lower effect for IL. Diprivan efficaciously protected endothelial cells submitted to an oxidant stress, while IL was ineffective. By HPLC, we demonstrated that PPF was not incorporated into the cells. The drug thus acted by scavenging the active oxygen species released in the extracellular medium. IL acted in the same manner, but was a less powerful antioxidant. [less ▲]Detailed reference viewed: 33 (1 ULg)
Effects of sphingosine and sphingosine analogues on the free radical production by stimulated neutrophils: ESR and chemiluminescence studies.
Mouithys-Mickalad, Ange ; Deby, Ginette ; Hoebeke, Maryse et al
in Mediators of Inflammation (1997), 6(5-6), 327-33
Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of ... [more ▼]
Sphingolipids inhibit the activation of the neutrophil (PMN) NADPH oxidase by protein kinase C pathway. By electron spin resonance spectroscopy (ESR) and chemiluminescence (CL), we studied the effects of sphingosine (SPN) and ceramide analogues on phorbol 12-myristate 13-acetate (PMA, 5 x 10(-7)M) stimulated PMN (6 x 10(6) cells). By ESR with spin trapping (100 mM DMPO: 5,5-dimethyl-1-pyrroline-Noxide), we showed that SPN (5 to 8 x 10(-6)M), C(2)-ceramide (N-acetyl SPN) and C(6)-ceramide (N-hexanoyl SPN) at the final concentration of 2 x 10(-5) and 2 x 10(-4)M inhibit the production of free radicals by stimulated PMN. The ESR spectrum of stimulated PMN was that of DMPO-superoxide anion spin adduct. Inhibition by 5 x 10(-6)M SPN was equivalent to that of 30 U/ml SOD. SPN (5 to 8 x 10(-6)M) has no effect on in vitro systems generating superoxide anion (xanthine 50 mM/xanthine oxidase 110 mU/ml) or hydroxyl radical (Fenton reaction: 88 mM H(2)O(2), 0.01 mM Fe(2+) and 0.01 mM EDTA). SPN and N-acetyl SPN also inhibited the CL of PMA stimulated PMN in a dose dependent manner (from 2 x 10(-6) to 10(-5)M), but N-hexanoyl SPN was less active (from 2 x 10(-5) to 2 x 10(-4)M). These effects were compared with those of known PMN inhibitors, superoxide dismutase, catalase and azide. SPN was a better inhibitor compared with these agents. The complete inhibition by SPN of ESR signal and CL of stimulated PMN confirms that this compound or one of its metabolites act at the level of NADPH-oxidase, the key enzyme responsible for production of oxygen-derived free radicals. [less ▲]Detailed reference viewed: 41 (8 ULg)
Experimental model for the study by chemiluminescence of the activation of isolated equine leucocytes.
; Deby, Ginette ; et al
in Research in Veterinary Science (1996), 61(1), 59-64
The activation of human polymorphonuclear leucocytes (the respiratory burst) can be studied by measuring their chemiluminescent response. This technique was adapted to equine leucocytes to investigate the ... [more ▼]
The activation of human polymorphonuclear leucocytes (the respiratory burst) can be studied by measuring their chemiluminescent response. This technique was adapted to equine leucocytes to investigate the effects of cell number, activator concentration, enhancers of chemiluminescence, pH, temperature and inhibitors. Leucocytes were isolated from citrated blood from healthy horses and chemiluminescence was measured with a Bio-Orbit luminometer sensitive to 900 nm light. The optimal cell density for the maximal chemiluminescent response ranged from 10(6) to 10(7) leucocytes 600 microliters-1. Chemiluminescence increased as a function of temperature, and the concentrations of luminol, lucigenin and phorbol myristate acetate (PMA), and was pH related (optimal pH value = 8.0 for lucigenin and 8.5 for luminol). The inhibition of chemiluminescence by 5 x 10(-5) M azide was 88 per cent for luminol and 37 per cent for lucigenin. Superoxide dismutase (100 IU) totally inhibited the chemiluminescence response. Approximately 30 per cent variability in chemiluminescence was observed under the same assay conditions, depending on the origin of the leucocytes. Based on these results, the conditions selected for the measurement of equine leucocyte chemiluminescence were: 10(6) to 10(7) leucocytes 600 microliters-1, 1 x 10(-6)M PMA, 1 mM luminol or 0.4 mM lucigenin, physiological pH (7.4) and physiological temperature (37.8 degrees C). These conditions were similar to those used for measuring the chemiluminescent response of human leucocytes. [less ▲]Detailed reference viewed: 32 (1 ULg)
Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime.
Mathy, Marianne ; Deby, Ginette ; et al
in Mediators of Inflammation (1995), 4(6), 437-43
We investigated the effects of the antibiotic ceftazidime (CAZ) on the cytolytic action of the neutrophil myeloperoxidase-hydrogen peroxide-chloride anion system (MPO/H(2)O(2)/Cl(-)). In this system ... [more ▼]
We investigated the effects of the antibiotic ceftazidime (CAZ) on the cytolytic action of the neutrophil myeloperoxidase-hydrogen peroxide-chloride anion system (MPO/H(2)O(2)/Cl(-)). In this system, myeloperoxidase catalyses the conversion of H(2)O(2) and CI(-) to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC) were capable of taking up active MPO. In presence of H(2)O(2) (10(-4) M), this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of (51)Cr from HUVEC and expressed as an index of cytotoxicity (IC). Dose dependent protection was obtained for CAZ concentrations ranging from 10(-5) to 10(-3) M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H(2)O(2), but when cytolysis was achieved with H(2)O(2) or O(2) (-) generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H(2)O(2)) was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon). So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis. [less ▲]Detailed reference viewed: 31 (4 ULg)
Multihormonal regulation of the human prolactin gene expression from 5000 bp of its upstream sequence
; ; Peers, Bernard et al
in Molecular & Cellular Endocrinology (1991), 80(1-3), 53-64
We have cloned DNA sequences extending up to 6000 bp upstream from the first exon of the human prolactin (hPRL) gene. 5000 bp of these upstream sequences were fused to a CAT reporter gene and shown to ... [more ▼]
We have cloned DNA sequences extending up to 6000 bp upstream from the first exon of the human prolactin (hPRL) gene. 5000 bp of these upstream sequences were fused to a CAT reporter gene and shown to provide tissue-specific transient expression in rat pituitary GH3 cells. Multihormonal response was found in this transient expression assay, leading to significant 2- to 5-fold induction by addition of 8-chlorophenylthio-cyclic AMP, thyrotropin-releasing hormone, epidermal growth factor, basic fibroblast growth factor, phorbol myristate acetate, a calcium channel agonist (Bay K-8644) and triiodothyronine. A 3-fold inhibition was observed in the presence of the glucocorticoid agonist dexamethasone. The sequence of the hPRL promoter was determined up to coordinate -3470. Computer similarity search between the rat and human sequences showed two highly conserved regions corresponding to the proximal and distal tissue specific enhancers described in both PRL promoters. [less ▲]Detailed reference viewed: 7 (1 ULg)
Regulatory elements controlling pituitary-specific expression of the human prolactin gene
Peers, Bernard ; Voz, Marianne ; et al
in Molecular & Cellular Biology (1990), 10(9), 4690-700
We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase ... [more ▼]
We have performed transfection and DNase I footprinting experiments to investigate pituitary-specific expression of the human prolactin (hPRL) gene. When fused to the chloramphenicol acetyltransferase (CAT) reporter gene, 5,000 base pairs of the 5'-flanking sequences of the hPRL gene were able to drive high cat gene expression in prolactin-expressing GH3B6 cells specifically. Deletion analysis indicated that this pituitary-specific expression was controlled by three main positive regulatory regions. The first was located just upstream from the TATA box between coordinates -40 and -250 (proximal region). We have previously shown that three motifs of this region bind the pituitary-specific Pit-1 factor. The second positive region was located in the vicinity of coordinates -1300 to -1750 (distal region). DNase I footprinting assays revealed that eight DNA motifs of this distal region bound protein Pit-1 and that two other motifs were recognized by ubiquitous factors, one of which seems to belong to the AP-1 (jun) family. The third positive region was located further upstream, between -3500 and -5000 (superdistal region). This region appears to enhance transcription only in the presence of the distal region. [less ▲]Detailed reference viewed: 15 (0 ULg)
Pituitary-specific factor binding to the human prolactin, growth hormone, and placental lactogen genes
; Peers, Bernard ; et al
in DNA (1989), 8(3), 149-59
The human genes coding for growth hormone (GH), chorionic somatomammotropin (placental lactogen, CS), and prolactin (Prl) are related evolutionarily but are expressed in phenotypically distinct cell types ... [more ▼]
The human genes coding for growth hormone (GH), chorionic somatomammotropin (placental lactogen, CS), and prolactin (Prl) are related evolutionarily but are expressed in phenotypically distinct cell types despite their nucleotide sequence homology. We show here that the promoters of the human Prl and CS genes contain cis-acting sequences that confer pituitary-specific expression in a cell-free transcription assay. Similar data are obtained with the human GH gene, consistent with earlier work by others. Footprinting analysis shows that neighboring sequences in each of these three promoters are protected from deoxyribonuclease I digestion by rat pituitary cell extracts. Footprinting competition experiments and gel retardation assays with synthetic oligonucleotides suggest that a single factor is responsible for the pituitary-specific footprints seen on the human Prl, CS, and GH genes. They also suggest that this factor is identical or closely related to the trans-acting factor GHF-1/Pit-1. Similarities with and differences from the rat GH and Prl genes are discussed. [less ▲]Detailed reference viewed: 16 (0 ULg)
Approach to the molecular mechanisms of the modulation of growth hormone gene expression by glucocorticoid and thyroid hormones
; ; et al
in Journal of Steroid Biochemistry (1987), 27(1-3), 149-58Detailed reference viewed: 10 (2 ULg)
Localisation des séquences régulatrices de la transcription. Application aux gènes de la famille de la prolactine.
; ; et al
in Annales d'Endocrinologie (1986), 47
We are studying nucleotide sequences responsible for the regulation of eukaryotic gene expression. Our test system comprises the human genes coding for prolactin (hPRL), growth hormone (hGH-N) and ... [more ▼]
We are studying nucleotide sequences responsible for the regulation of eukaryotic gene expression. Our test system comprises the human genes coding for prolactin (hPRL), growth hormone (hGH-N) and placental lactogen (hCS-B). We have cloned these genes and are searching within their sequences for in vitro binding sites of the human glucocorticoid receptor on the hGH-N and hCS-B genes; the in vivo activity of such DNA sequences by assaying hybrid gene expression in transfected cells; in vivo "enhancer" activity of different hPRL gene fragments linked to a marker gene and transfected in cultured cells. [less ▲]Detailed reference viewed: 28 (7 ULg)
Location of transcriptional and regulatory sequences within the prolactin family genes
; ; et al
in Müller, E. E.; McLeod, R. M. (Eds.) Neuroendocrine Perspectives (1986)Detailed reference viewed: 9 (3 ULg)
Location of transcriptional regulatory sequences within the prolactin family genes.
; ; et al
in Neuroendocrine Perspectives. (1986)
Thyroid hormone receptors bind to defined regions of the growth hormone and placental lactogen genes
; Voz, Marianne ; et al
in Proceedings of the National Academy of Sciences of the United States of America (1986), 83(23), 9021-5
The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that ... [more ▼]
The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[125I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions. [less ▲]Detailed reference viewed: 11 (2 ULg)
Binding of the human glucocorticoid receptor to defined regions in the human growth hormone and placental lactogen genes
; ; et al
in DNA (1985), 4(6), 409-17
An in vitro competition assay was used to investigate whether binding sites for the human glucocorticoid receptor occur in the human genes for growth hormone (hGH) and placental lactogen (chorionic ... [more ▼]
An in vitro competition assay was used to investigate whether binding sites for the human glucocorticoid receptor occur in the human genes for growth hormone (hGH) and placental lactogen (chorionic somatomammotropin, hCS). These genes display 95% sequence homology. Two receptor-binding regions were found in the hGH gene, one of which is located within 290 bp upstream, and one within 251 bp downstream from the transcription initiation site. Two binding regions homologous to those in the hGH gene were found in the hCS gene. The receptor-binding DNA fragment from the structural part of the genes, but not that from their promoter area, contained a sequence homologous to a 15-bp consensus sequence proposed earlier for the glucocorticoid receptor binding site. It is unlikely that the putative difference in glucocorticoid sensitivity between the hGH and hCS genes is accounted for by major differences in glucocorticoid receptor binding pattern. [less ▲]Detailed reference viewed: 7 (1 ULg)