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M Denis

Publications and source records attributed to M Denis.

At least 181 records · Page 10Linked to original sources
PubMed1989

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol.

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.

Animals↗
PubMed1989

The Mr approximately 90,000 heat shock protein: an important modulator of ligand and DNA-binding properties of the glucocorticoid receptor.

This brief report deals with some recent observations relating to the assoclation of the Mr approximately 90,000 heat shock protein (hsp90) with the glucocorticoid receptor. In its nonactivated state, stabilized by sodium molybdate, the glucocorticoid receptor exists as a 9S heteromeric complex containing a single Mr approximately 94,000 steroid-binding unit and a dimer of hsp90. Monospecific antibodies raised against the purified rat glucocorticoid receptor-associated hsp90 interact with the molybdate-stabilized receptor. They also immunoprecipitate the Mr approximately 27,000 steroid-binding fragment of the receptor generated by trypsin treatment. Thus, hsp90 interacts with the ligand-binding domain of the glucocorticoid receptor. Furthermore, dissociation of the glucocorticoid receptor-hsp90 complex results in a major reduction of the affinity of the Mr approximately 94,000 receptor entity for its ligand. The heteromeric 9S complex does not bind to DNA. When it is activated to a DNA-binding state, the hsp90 dissociates from the ligand-binding protein. In vitro, activation of the cytosolic rat glucocorticoid receptor to a DNA-binding state is inducible by the binding of ligand. Taken together, our observations indicate the existence of important connections between the association of hsp90 and the functions of ligand- and DNA-binding of the glucocorticoid receptor.

Animals↗
PubMed1989

Translation of glucocorticoid receptor mRNA in vitro yields a nonactivated protein.

The glucocorticoid receptor is present in cytosol prepared from cell extracts of nonhormone-treated cells as a large nonactivated (i.e. non-DNA binding) 9 S heteromeric complex which contains the Mr approximately 90,000 heat shock protein, hsp90. hsp90 is expressed under physiological conditions in mammalian cells and is also present in reticulocyte lysate, as assessed by Western immunoblotting using specific anti-hsp90 antibodies. We have translated glucocorticoid receptor mRNA in reticulocyte lysates. The receptor synthesized under cell-free conditions also interacts with hsp90 both in the presence and absence of ligand, as determined by sucrose gradient centrifugation. The in vitro synthesized glucocorticoid receptor does not bind to DNA-cellulose but can be converted to a DNA binding form following labeling with dexamethasone and heat treatment. Thus, the glucocorticoid receptor is synthesized in a nonactivated form under cell-free conditions. These data indicate that the 9 S glucocorticoid receptor complex found in cytosol does not represent an artifact due to cell homogenization and supports the existence in vivo of the glucocorticoid receptor-hsp90 complex.

Animals↗
PubMed1989

Human neutrophils activated by interferon-gamma and tumour necrosis factor-alpha kill Entamoeba histolytica trophozoites in vitro.

The interaction between cytokine-activated human neutrophils and Entamoeba histolytica trophozoites was studied as well as the mechanism(s) involved. Treatment of neutrophils with rIFN-gamma alone allowed them to kill 30% of E. histolytica trophozoites; however, rIFN-gamma and rTNF-alpha pretreatments in combination increased neutrophil killing to 70%. In the absence of direct contact between neutrophils and amebae, rIFN-gamma-treated neutrophils were shown to kill 70% of amebae, and rIFN-gamma- and rTNF-alpha-treated neutrophils killed 97% of amebae. Neutrophil enhancement of amebicidal activity following cytokine treatments was correlated with increased neutrophil resistance to amebic contact-dependent killing and was shown to be 73% H2O2 dependent.

Animals↗
PubMed1989

The non-activated glucocorticoid receptor: structure and activation.

Glucocorticoid hormone receptors are present in the soluble fraction of target cell homogenates as large entities (Mr approximately 300,000) that are unable to interact with DNA. These large complexes contain an Mr approximately 94,000 steroid- and DNA-binding polypeptide, in association with an Mr approximately 90,000 non-ligand-binding entity, which has been identified as a heat shock protein, hsp90. This protein has been purified to near homogeneity as a component of the non-activated receptor complex. Characterization of the purified protein revealed its presence as a dimer in the large receptor form. Dissociation of the receptor-hsp90 complex can be induced by heat treatment only when ligand is bound to the receptor, as demonstrated by specific DNA-binding assay and sucrose gradient ultracentrifugation, hsp90 represents ca 1% of total proteins in rat liver cytosol, and milligram amounts were purified using a combination of high performance ion exchange and gel permeation chromatography. Monospecific antibodies were raised in rabbits. They were found to precipitate the intact non-activated glucocorticoid receptor, as well as the Mr approximately 27,000 steroid-binding fragment of the receptor generated by trypsin treatment, indicating that hsp90 interacts with the steroid-binding domain of the glucocorticoid receptor. Finally, translation of glucocorticoid receptor mRNA in reticulocyte lysate yields a protein which also interacts with hsp90 and binds to DNA only after ligand-binding and heat treatment. Thus, the glucocorticoid receptor is synthesized in a non-activated form also in vitro.

Animals↗
PubMed1989

Occurrence of glucocorticoid binding sites in solubilized microsomes from rat liver.

Recent studies suggested the presence of specific glucocorticoid binding sites on rat liver microsomal membranes. We report here a new solubilization procedure which allows the physicochemical characterization of the microsomal glucocorticoid binding sites. Solubilization was achieved with 2 mM CHAPS in the presence of 5 mM benzamidine. Binding of [3H]cortisol showed a high affinity (Kd = 5.1 x 10(-9) M) and a limited capacity (0.72 pmol/mg of protein). The binding activity was abolished by elevated temperature and pronase. Competition experiments revealed that natural glucocorticoids and progesterone were highly potent competitors whereas dexamethasone and triamcinolone acetonide did not compete. Chromatography on DEAE Trisacryl and heparin Ultrogel confirmed that the solubilized protein is different from corticosteroid binding globulin and the cytosolic glucocorticoid receptor. Treatment of microsomal fractions with phosphatidyl inositol phospholipase C promoted the release of specific binding activity suggesting a putative glycosylphosphatidyl anchor for this protein. This finding may have interesting implications concerning the mechanism of glucocorticoid hormone action.

Animals↗
PubMed1989

Ligand-dependent interaction of the dioxin receptor with target DNA.

Wild type and nuclear transfer deficient mouse hepatoma cell lines were used to study the specific DNA binding of a dioxin inducible factor. This factor interacts with XRE only after dioxin treatment and is absent in receptor mutant containing cells even after treatment. Thus, evidence is provided to substantiate the claim that the dioxin receptor is involved in the specific DNA interaction with dioxin response enhancer elements. It is also shown that the molybdate stabilised dioxin-receptor interacts with hsp90 suggesting that, in similarity to the glucocorticoid receptor, the dioxin receptor is kept in a non-transformed state in the absence of ligand.

Animals↗
PubMed1989

Macrophage killing of human papillomavirus type 16-transformed cells.

Papillomaviruses (HPV) are involved in proliferation of epithelial cells and are implicated in several human malignancies. We determined the capability of activated macrophages to exert cytostatic and/or cytolytic activities against HPV type 16 DNA-transformed cells. Both NIH-3T3 and A31-3T3 cells transformed with HPV DNA were susceptible to killing by activated macrophages; however, transformed and nontransformed cells were not susceptible to killing by soluble mediators. The HPV-transformed cells were as resistant as their nontransformed counterparts to recombinant TNF-alpha. These data suggest that the killing of the HPV-16-transformed 3T3 cells by macrophages occurred by a mechanism independent of TNF-alpha.

Animals↗
PubMed1989

Salt requirements in the denitrifying bacterium Pseudomonas nautica 617.

Pseudomonas nautica 617, which was isolated from superficial marine sediment, was found to require sodium for growth. Growth also appeared to be sensitive to the divalent cation, Mg2+, the presence of which, together with that of Na+, was necessary for achieving maximal growth. We investigated cell capacity to resist lysis after washing with either 0.05 M MgCl2 or 0.5 M NaCl, by monitoring suspension optical density changes as well as the release of ultraviolet absorbing material. Mg2+ turned out to play a significant role in stabilizing the structure of the cell envelope. Respiratory activity was also sensitive to ionic environment. With cells washed with 0.05 M MgCl2 and suspended in 0.05 M Tris buffer, the respiration rate, assessed by N2O evolution, was 15% of that measured in artificial sea water. Upon addition of 0.5 M Na+, nitrous oxide production rose to 32% of the reference level. The dinitrification rate was fully restored by further addition of 0.05 M Mg2+. K+ alone had almost no effect, but when added with Na+, the rate of denitrification increased to 45%.

Bacteriolysis↗
PubMed1989

Specific protein-DNA interactions at a xenobiotic-responsive element: copurification of dioxin receptor and DNA-binding activity.

Upon binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (called dioxin or TCDD), the dioxin receptor exhibits increased affinity for the cell nucleus in vivo and for DNA in vitro. To define the recognition sequence of the dioxin receptor and its relationship with that of the glucocorticoid receptor, oligonucleotides derived from dioxin-responsive elements of the rat cytochrome P-450c gene were tested for their ability to form specific protein-DNA complexes in a gel retardation assay. We found that a previously defined sequence motif that is similar to the glucocorticoid-responsive element and exhibits strong enhancer activity in response to dioxin receptor ligands bound a dioxin-inducible factor with high specificity but was not recognized by the DNA-binding domain of the glucocorticoid receptor. Binding to this element was only observed in nuclear extracts of wild-type mouse hepatoma cells in a time- and dose-dependent manner and not in nuclear extracts from a nonresponsive mutant cell line deficient in DNA binding of the dioxin receptor. The specific DNA-binding activity in wild-type nuclear extracts comigrated in a Superose size-exclusion column and cosedimented on sucrose gradients with the in vivo labeled dioxin receptor. These experiments strongly suggest that the dioxin receptor is a sequence-specific DNA-binding protein and is not only biochemically but also functionally similar to the steroid receptor family.

Animals↗
PubMed1989

Anti-interleukin 2 receptor monoclonal antibody in the treatment of ongoing acute rejection episodes of human kidney graft--a pilot study.

Monoclonal antibodies (MoAbs) against human interleukin 2 receptor (IL-2-R) have been shown to prevent early kidney rejection in animals and humans. We report here the effect of an anti-IL-2-R MoAb (33B3.1) inhibiting IL-2 binding high-affinity sites on activated lymphocytes in 10 declared acute rejection episodes of first cadaveric kidney grafts. Six patients were under cyclosporine treatment only at the time of diagnosis of the rejection. All rejection episodes but one were biopsy-proved cellular rejections. Treatment consisted of intravenous infusions of 33B3.1 at 20 mg/day x 2 days, followed by 10 mg/day for 8 additional days. In case of MoAb ineffectiveness at day 5, anti-IL-2-R MoAb was discontinued and a rescue treatment of corticosteroid boluses (CSb) was given. If not, in all cases corticosteroids (CS) were given (1 mg/kg) at the end of MoAb treatment (day 10) and tapered off thereafter. Two rejection episodes immediately responded to 33B3.1 treatment. During 33B3.1 treatment four other patients had only a stabilization of their blood creatinine concentration, which nevertheless returned to prerejection levels after day 10 when anti-IL-2-R was discontinued and CS administered at 1 mg/kg (no rescue treatment). The four remaining patients had an increase of their blood creatinin levels at day 5 despite 33B3.1 treatment, and their renal function only improved with CSb rescue treatment. One of these patients lost the graft despite rescue treatment, as well as a 9-day course of antithymocyte globulin. Trough levels of MoAb reached a plateau as early as day 2 (approximately 6 micrograms/ml). All patients developed antibodies (IgM and IgG) after day 14. In no instance could unresponsiveness be related to low circulating 33B3.1 trough levels or to early host anti-MoAb immune response (IgM or IgG). We conclude that 33B3.1, known to be effective in preventing early rejection, has only inconsistent and/or incomplete effects on the ongoing rejection process. Our data suggest that once IL-2-dependent clones are expanded in the rejected graft, interference with IL-2/IL-2-R signals does not block the effector mechanisms sustaining acute rejection.

Antibodies, Monoclonal↗
PubMed1989

Cytokine activation of murine macrophages for in vitro killing of Entamoeba histolytica trophozoites.

Macrophage-mediated effector mechanisms against the protozoan parasite Entamoeba histolytica were studied. Unstimulated macrophages were inefficient at killing E. histolytica trophozoites in vitro and were killed by the trophozoites. Conversely, immature cells of the mononuclear phagocyte lineage (promonocytes) were shown to display a strong spontaneous amebicidal activity. The acquisition of macrophage amebicidal activity following cytokine treatment was investigated. Gamma interferon, tumor necrosis factor alpha, and macrophage colony-stimulating factor 1, or combinations thereof, were shown to endow murine bone marrow-derived macrophages with significant amebicidal activity. Low doses of gamma interferon and tumor necrosis factor alpha and of gamma interferon and colony-stimulating factor 1 were shown to act synergistically in this phenomenon. This enhancement of amebicidal activity was shown to operate on bone marrow-derived macrophages, elicited peritoneal macrophages, and, to a much lesser extent, spleen macrophages. Although acquisition of amebicidal activity was associated with a strong respiratory burst, the addition of oxygen-free radical scavengers showed that the killing activity was approximately 45% H2O2 dependent. In addition, amebicidal activity by macrophages was shown to be contact dependent and was inhibited by 61% with the protease inhibitor tosyl lysyl chloromethyl ketone. Our results indicate that immunologic production of gamma interferon, tumor necrosis factor alpha, and colony-stimulating factor 1 could be important in the activation of macrophages for host defense against amebiasis and that promonocytes are strong effector cells against virulent amebae.

Amebicides↗
PubMed1989

Murine T-cell clones against Entamoeba histolytica: in vivo and in vitro characterization.

Eleven T-cell clones were raised from the spleens of BALB/c mice hyperimmunized against a crude soluble extract of Entamoeba histolytica trophozoites. Seven clones were of the Lyt-1+, and four of the Lyt-23+ phenotype. All clones proliferated in the presence of E. histolytica antigens but not to a purified protein derivative; five clones proliferated to a crude extract of the E. histolytica-like Laredo amoebae. Ten clones secreted T-cell growth factors in response to E. histolytica antigens. Two clones (Lyt-23+) mediated direct lymphocytotoxicity (73% and 86%) against amoebic trophozoites that was inhibited with rabbit anti-mouse TNF-alpha. Supernatants of five of the clones (all Lyt-1+) activated mouse peritoneal macrophages (Mphi) to kill E. histolytica trophozoites in vitro, seemingly independent of secreted reactive oxygen intermediates (O2- and H2O2) in the case of three clones supernatants. All of the clones that were activating Mphi to kill amoeba in vitro also mediated a local DTH reaction in mouse footpad. Our results demonstrate direct lymphocyte cytotoxicity via a cytolytic molecule antigenically related to TNF-alpha and lymphokines activating Mphi for amoebic killing by oxidative and non-oxidative mechanisms, the latter process mediated by a macrophage-activating factor (MAF) distinct from interferon-gamma (IFN-gamma).

Animals↗
PubMed1989

Identification and characterization of a BCG antigen expressed at the membrane surface of in vitro infected mouse peritoneal macrophages.

A murine monoclonal antibody to Mycobacterium bovis (BCG) (230A4A6) was used to identify an antigenic determinant expressed at the surface of murine peritoneal macrophages infected in vitro. This determinant was shown to be expressed with a delay in infected macrophages following in vitro infection. Isolation of the 230A4A6 reactive antigen showed that it was part of a 12-kD molecule. This protein was shown to be capable of eliciting delayed-type hypersensitivity in infected mice in a standard footpad assay as well as provide stimulus for mitogenesis of BCG-primed spleen cells.

Animals↗
PubMed1989

[Activity of 5 fluoroquinolones on hospital Gram-negative bacilli with different sensitivities to pefloxacin].

The minimum inhibitory concentrations (MIC) of 5 fluoroquinolones, fleroxacin (FLE), ciprofloxacin (CIP), ofloxacin (OFL), enoxacin (ENO) and norfloxacin (NOR) have been determined by the agar dilution method towards 140 strains of Pseudomonas aeruginosa (Pa) and 146 Enterobacteriaceae showing different sensitivities to pefloxacin (PEF). The strains were isolated in 1988 at the Bellevue Hospital. The modal MIC is 0.12 for CIP, 0.25 for NOR, 0.5 for OFL, and 1 for FLE and ENO when used on Pa strains which are sensitive to PEF (n = 35) (MIC less than or equal to 1mg/1). The modal MIC is 0.25 - 0.5 for CIP, 0.5 for NOR, 1 for OFL and ENO, and 2 for FLE when used on Pa strains which are of intermediate sensitivity to PEF (n = 70) (1 less than MIC less than or equal to 4). The modal MIC is 2 for CIP, 8 for NOR and OFL, 8 - 16 for ENO, and 32 for FLE when used on Pa strains which are resistant to PEF (n = 35) (MIC greater than 4). The modal MIC is 0.015 for CIP, 0.06 for OFL, 0.12 for FLE, NOR and ENO when used on Escherichia coli strains which are sensitive to PEF (n = 47). The modal MIC is 0.5 for CIP, 1 for OFL and NOR, and 2 FLE and ENO, when used on Escherichia coli strains which are of intermediate sensitivity to PEF (n = 38). The modal MIC is 1 for CIP, 4 for OFL and NOR, 16 for FLE, and 32 for ENO when used on E coli strains which are resistant to PEF (n = 15). The 26 Serratia marcescens and 20 Citrobacter with MIC greater than or equal to 8 for PEF all have MICs greater than 1 and modal MICs greater than or equal to 4 for all the fluoroquinolones studied. CIP always showed greater activity than the other quinolones whatever the sensitivity shown towards PEF.

Anti-Infective Agents↗