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Biomedical subjects

F Morel

Publications and source records attributed to F Morel.

At least 91 records · Page 5Linked to original sources
PubMed1997

Oltipraz stimulates the transcription of the manganese superoxide dismutase gene in rat hepatocytes.

Oltipraz (4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione) (OPZ) is recognized as a potent chemoprotective agent against chemical-induced carcinogenesis in several animal models and is thought to act mainly by inducing phase II conjugating together with inhibiting phase I detoxication enzymes. The present study was undertaken to determine whether oltipraz can also influence expression of genes encoding antioxidant enzymes. In rat hepatocytes in primary culture, this compound was found to selectively induce the transcription of the manganese superoxide dismutase (Mn-SOD) gene while it had no effect on copper/zinc-SOD and glutathione peroxidase genes. Oltipraz increased Mn-SOD gene expression in a time- and dose-dependent manner by 2- to 3-fold and enhanced the binding activity of the nuclear factor kappa B within 30 min. Moreover, the increase in Mn-SOD gene transcription was associated with a 2- to 3-fold increase of free malondialdehyde and conjugated dienes, two markers of lipid peroxidation, an index of oxidative stress. These results suggest that in rat hepatocytes, oltipraz induced a production of reactive oxygen species that probably acted as second messengers in order to trigger the transcription of many genes. Such a mechanism of action of OPZ and other dithiolethiones would account for the broad spectrum of action of these anticarcinogenic compounds.

Animals↗
PubMed1997

Effects of administration of the chemoprotective agent oltipraz on CYP1A and CYP2B in rat liver and rat hepatocytes in culture.

The success of oltipraz (OPZ) [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] as a chemoprotective agent against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat is thought to depend principally on its ability to enhance detoxication by inducing phase II enzymes, especially glutathione transferases. However, in primary cultures of human hepatocytes, we recently demonstrated that OPZ also has an important inhibitory effect on the major cytochromes P450 (CYPs) of human hepatic AFB1 metabolism. This has prompted a detailed study of the effect of OPZ on some CYPs involved in metabolism of AFB1 in the rat. Primary cultures of rat hepatocytes behaved similarly to human hepatocytes and responded to OPZ by inhibition of ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD) activities mainly associated, respectively, with CYP1A and CYP2B. A time-course shows that this inhibition is largely reversible, with EROD and PROD activities reaching a minimum at 12 h and tending towards control values within 24 h. As is to be expected, the incubation of isolated microsomes with OPZ also inhibits CYP1A and 2B. The effect of OPZ on CYP1A is not a phenomenon limited to cells in culture, but also occurs in vivo. Using the whole animal, we were able to demonstrate that OPZ also transiently inhibited CYP1A activity in a rat given caffeine, by measuring the amounts of methylxanthines found in the serum. However, microsomes isolated from rats, that had been treated with OPZ in vivo, show no such inhibition, presumably because, since OPZ is a reversible inhibitor, it dissociates and is lost during the course of conventional procedures of microsomal preparation. This explains some earlier failures in studies of isolated microsomes to observe the inhibition of CYPs by OPZ. In addition to inhibiting their enzymatic activity, OPZ is also an inducer of CYP1A and 2B as shown by the increased levels of their mRNAs and of caffeine metabolism in vivo after 24 h or more. It is concluded that the mechanism of chemoprotection by OPZ, of toxic chemical metabolism in the rat, is complex and involves competitive inhibition of activation succeeded by induction of the enzymes of both activation and detoxication.

Aflatoxin B1↗
PubMed1997

Precipitation of Arsenic Trisulfide by Desulfotomaculum auripigmentum.

A newly discovered bacterium, Desulfotomaculum auripigmentum, precipitates arsenic trisulfide (As(inf2)S(inf3)). Precipitation of As(inf2)S(inf3) by this organism results from its reduction of As(V) to As(III) and S(VI) to S(-II). At the As(III) concentration range of interest (0.1 to 1 mM), the stability of As(inf2)S(inf3) is highly sensitive to pH and [S(-II)]. Thus, the relative rates at which D. auripigmentum reduces As(V) and S(VI) are critical to its formation of As(inf2)S(inf3). Other As(V)- or S(VI)-reducing bacteria are unable to precipitate As(inf2)S(inf3) either due to their inability to reduce both As(V) and S(VI) or because they reduce S(VI) too rapidly. Electron microscopy of thin sections showed that the precipitate forms both intra- and extracellularly. Microbial As(inf2)S(inf3) formation nucleates precipitation of the mineral in the bulk milieu, whereas heat-killed cells alone do not serve as templates for its formation. Precipitation of As(inf2)S(inf3) by D. auripigmentum suggests that As(inf2)S(inf3) formation may be important in the biogeochemical cycle of arsenic.

Journal Article↗
PubMed1997

Proteolytic potential of human neutrophil membranes.

A synergistic role for proteases in the degradation of extracellular matrix proteins has been proposed. Plasma membrane was isolated from a neutrophil homogenate, on a sucrose gradient, and shown to activate gelatinolysis when purified 92 kDa gelatinase was added to the medium. This stimulatory activity was enhanced by the addition of phorbol 12-myristate 13-acetate (PMA), in a dose-dependent manner, and was partially sensitive to phenylmethylsulfonyl fluoride treatment. The effect was abolished by the addition of 1 M KCl or 0.05% Brij 35 extraction. Both elastase and urinary type plasminogen activator were shown to be involved in the process. Moreover, upon neutrophil stimulation by PMA, 92 kDa gelatinase, as elastase, became associated with the plasma membrane, as shown by a subcellular fractionation experiment. These in vitro observations suggest that human neutrophils may be able, in vivo, to recruit endogenous or exogenous proteinases to mediate proteolysis associated with diapedesis and chemotactism during the inflammation process.

Alkaline Phosphatase↗
PubMed1997

A study of metalloproteinases in fifty joint fluid specimens.

OBJECTIVES: To study the activity and concentrations in joint fluid of gelatinases (A or matrix metalloproteinase (MMP)-2 and B or MMP-9), tissue inhibitor of metalloproteinases 1 (TIMP-1), and stromelysin-1 (MMP-3). METHODS: Synovial fluid specimens obtained as part of a diagnostic or therapeutic procedure were studied. Protein levels were determined, cells counted, crystals looked for and microbiological studies done. Gelatinolytic activity was determined quantitatively using computerized zymography. Proteins were identified by electrotransfer and immunorevelation. TIMP-1 and stromelysin-1 were assayed using an ELISA. Results were confronted with laboratory test and clinical findings. RESULTS: Of the 50 specimens studied, 25 were from joints with mechanical disorders and 25 from joints with inflammatory disorders. Activated MMP-2 was found in all the specimens, with no differences between the two groups. MMP-9 was found only in its inactive form. MMP-9, TIMP-1 and MMP-3 were found more often in inflammatory than in mechanical fluids and the levels of MMP-9 and TIMP-1 were correlated with neutrophil counts. In the 16 fluids from rheumatoid arthritis patients, levels of MMP-9 and TIMP-1 were closely correlated with serum C-reactive protein levels. CONCLUSIONS: MMP-9, TIMP-1, and MMP-3 levels show striking differences between inflammatory and mechanical joint fluids.

Adolescent↗
PubMed1996

Primitive hematopoietic cells in murine bone marrow express the CD34 antigen.

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin-/10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.

Animals↗
PubMed1996

Differential expression and secretion of gelatinases and tissue inhibitor of metalloproteinase-1 during neutrophil adhesion.

Transmigrating neutrophils secrete a 92 kDa gelatinase (MMP-9) in order to degrade type IV endothelial basement membrane collagen. A model system for neutrophil adhesion combining a short pre-adhesion time (30 min) in plastic or endothelium-coated wells, medium removal and addition of soluble stimuli (fMLP, TNF alpha), enabled us to induce the release of a basal level of gelatinase activity (> 12% total cell content) from tertiary granules, while the release of vitamin B12 binding protein from specific granules was limited to 4% total cell content. Neutrophil gelatinase activity in unfractionated supernatants from endothelium-coated wells was significantly reduced (P < 0.01) compared to levels obtained on plastic supports, even after TNF alpha treatment or when cell populations were physically separated by trans-well inserts. In contrast, gelatin zymograms of supernatants from plastic and endothelium-coated wells remained similar. These findings suggest that MMP-9 is equally secreted but differentially inhibited by the tissue inhibitor of metalloproteinase-1 originating from the neutrophils. MMP-9 RT-PCR from neutrophils, assessed after up to one hour adhesion on plastic, yielded a single 270 bp fragment which was almost undetectable in the endothelial RT-PCR counterpart, whereas the TIMP-1 PCR product was apparent in both cell types. Furthermore, neutrophil adhesion on endothelial cells and TNF alpha activation for one hour induced the disappearance of MMP-9 cDNA without changes in TIMP-1 and beta-actin PCR products. These results suggest the existence of a dual down-regulation during neutrophil-endothelial interaction, both at the level of secreted MMP-9 activity and of MMP-9 gene transcription.

Cell Adhesion↗
PubMed1996

A comparison of the effect of inducers on the expression of glutathione-S-transferases in the liver of the intact rat and in hepatocytes in primary culture.

Recently, we used human hepatocytes in primary culture to study the effects of inducers of glutathione-S-transferases (GSTs) in the expectation that information obtained can be used to predict the value of particular inducers for use in the chemoprevention of cancer and other toxicities. However, in vitro human studies cannot readily be confirmed by studies in vivo. This problem does not arise in experimental animals. In the current studies, the response of male rat hepatocytes in primary culture to the following inducers of GST isoenzymes has been determined: 3-methylcholanthrene (MC); phenobarbital (PB); 1,2-dithiole-3-thione and its 5-(2-pyrazinyl)-4-methyl derivative, oltipraz (OPZ), and the results have been compared with induction obtained in livers of MC- and OPZ-treated rats. Each type of inducer was found to elicit a different response. In vitro, phenobarbital increased messenger RNA (mRNA) levels of subunits 1b and 3 after 12 and 72 hours, respectively; MC had a rapid effect on GST alpha class mRNAs (bringing about increase after only 2 hours of treatment), increased subunit 7 mRNA slightly, and had no effect on mu class mRNAs; dithiolethiones induced both subunit lb and 7 mRNAs after 4 hours and, to a much lower extent, subunit 3 mRNA after 72 hours. In vivo, MC induced significantly both subunit lb and 7 mRNAs whereas OPZ increased significantly subunits lb, 3 and 7 mRNA levels, and to a lower extent those of subunit 2, after 3 days and beyond to at least 5 days of treatment. Results obtained in mRNA studies were confirmed by high-pressure liquid chromatography (HPLC) analysis of GST subunits. HPLC also showed an induction of subunit 10 at the protein level of which the mRNA was not analyzed. Our results show that rat hepatocytes in primary culture prove to be a good model for the effect of inducers on both the expression of GST mRNA and protein levels in the rat liver in vivo. The demonstration of this good correlation in the rat with respect to increases gives support for the use of human hepatocytes for predictive studies of chemoprotection in human pharmacology.

Animals↗
PubMed1996

Serum soluble CD23 levels in giant cell arteritis.

Lymphocytes and monocytes express various levels of membrane-bound CD23, the low affinity receptor for IgE (Fc epsilon RII), and in some cases release it as a soluble form. Soluble CD23 (sCD23) has been implicated in the regulation of many immunological functions of T and B lymphocytes, macrophages and myeloid cells in humans. To study serum sCD23 levels in inflammatory conditions, we selected a systemic disease sensitive to corticotherapy, the giant cell arteritis, which is characterized by an inflammation of the temporal artery. Serum sCD23 levels, as measured by a radioimmunoassay, were increased in these patients, and returned to normal values within the 24 h following initiation of corticotherapy. The data suggest that the increase in sCD23 levels in giant cell arteritis results from an overproduction.

Giant Cell Arteritis↗
PubMed1996

Biochemical heterogeneity of arginine metabolism along kidney proximal tubules.

By using an in vitro single tubule micromethod of high specificity, in four different species of mammals it has been observed that (a) arginine synthesis from citrulline (arginine synthase activity, E.C. 6.3.4.5 and E.C. 4.3.2.1) is restricted to the early portions of proximal convoluted tubules, whereas (b) urea production from arginine (arginase activity, E.C. 3.5.3.1.) is present mainly in the cortical (CPST) and even more in the outer medullary (OSPST) portions of straight proximal tubules. The data suggest that (a) in early PCT cells, the citrulline reabsorbed from glomerular filtrate is converted into arginine, which in turn crosses peritubular cell membranes together with reabsorbed arginine, and (b) the urea formed in CPST and OSPST cells might passively diffuse into the luminal fluid entering Henle's loops. Such urea secretion might contribute to sustain the process of urea recycling in kidney medulla and thereby participate in the mechanism of urine concentration.

Animals↗
PubMed1996

Analysis of the sex chromosomal equipment in spermatozoa of a 47,XYY male using two-colour fluorescence in-situ hybridization.

The sex chromosomes in spermatozoa of a 47,XYY fertile male were analysed simultaneously by dual fluorescence in-situ hybridization (FISH), with two probes (pHY2.1 and pXBR). Of the 100000 cells analysed, 95179 spermatozoa (95.18%) exhibited one or more hybridization signals. Of the hybridized nuclei, 85.37% showed a normal sex chromosome constitution (37.37% X-bearing cells and 48.00% Y-bearing cells), with an X:Y ratio of 0.78:1. A total of 14.63% of the hybridized nuclei exhibited sex chromosome aneuploidy with a majority of XY- and YY-bearing spermatozoa (9.37 and 4.65% respectively). Even if the majority of spermatozoa have chromosomal haploidy, a large proportion of them exhibits numerical errors for the sex chromosomes. These observations raise questions about the commonly-admitted notions concerning the absence of chromosomal risk for XYY male offspring.

Humans↗
PubMed1996

Positive and negative regulatory regions in promoters of human glutathione transferase alpha genes.

The human glutathione transferase (GST, EC 2.5.1.18) alpha class locus comprises several genes and pseudogenes. Genomic DNA encoding several human alpha-class-related genes and pseudogenes was cloned and characterized. Three distinct but highly similar 5'-flanking regions of GST alpha genes as well as a series of 5'-deletions were investigated for promoter activity by fusion to the luciferase reporter gene. Transient transfection of these luciferase constructs into human hepatoblastoma, kidney carcinoma, nephroblastoma or bladder carcinoma cells revealed that the promoters are active and contain both positive and negative regulatory regions that behave in a cell-type specific fashion. The 150 bp proximal promoter regions of the three sequences retained the same relative activities as the full length promoters. Two of them were equally active, whereas the third one showed only 20% of the activity of the two stronger promoters. Site-directed mutagenesis indicated that a conspicuous insertion of three nucleotides (TTT) in the weak promoter is not responsible for the different activities.

Cloning, Molecular↗
PubMed1996

[Hierarchy of cellular regulation in the kidney].

Among the numerous "signals" which may alter the functional state of renal epithelial cells, it appears convenient to distinguish two categories: under physiological conditions, on the one hand, "hormones" control the rate of specific transport across their respective kidney target cells. In order to under-stand how such cellular effects of hormones may contribute to maintain body fluid homeostasis, it is necessary to determine where and how each of them modifies the tubular fluid composition along the successive portions of the whole nephron; under pathological conditions or during kidney ontogenesis, on the other hand, a variety of "factors" present in the close environment induce, in kidney cells, functional programmes which favor either survival, growth, or division.

Epithelial Cells↗
PubMed1995

Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture.

Dithiolethiones are thought to act as potent chemoprotective agents against aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat by inducing glutathione S-transferases (GSTs). To determine whether these antioxidants can be similarly effective in human beings, we have investigated metabolism of AFB1, in primary human hepatocytes with or without pretreatment by oltipraz (OPZ), a synthetic derivative of the natural 1,2-dithiole-3-thione. Aflatoxin M1 (AFM1), glutathione conjugates of AFB1 oxides (AFBSGs), and unchanged AFB1 were quantitated in cultures derived from eight human liver donors. Parenchymal cells obtained from the three GST M1-positive livers metabolized AFB1 to AFM1 and to AFBSGs derived from the isomeric exo-and endo-8,9-oxides, whereas no AFBSGs were formed in the GST M1-null cells. Pretreatment of the cells with 3-methylcholanthrene or rifampicin, inducers of CYP1A2 and CYP3A4, respectively, caused a significant increase in AFB1 metabolism. Although OPZ induced GST A2, and to a lesser extent GST A1 and GST M1, it decreased formation of AFM1 and AFBSG, which involves CYP1A2 and CYP3A4. Inhibition by OPZ of AFB1 metabolism by reducing CYP1A2 and CYP3A4 was also demonstrated by decreased activity of their monooxygenase activities toward ethoxyresorufin and nifedipine, respectively. The significant inhibition by OPZ of human recombinant yeast CYP1A2 and CYP3A4 was also shown. These results demonstrate that AFBSG can be formed by GST M1-positive human hepatocytes only, and suggest that chemoprotection with OPZ is due to an inhibition of activation of AFB1, in addition to a GST-dependent inactivation of the carcinogenic exo-epoxide.

Aflatoxin B1↗
PubMed1995

Characterization of neutrophil NADPH oxidase activity reconstituted in a cell-free assay using specific monoclonal antibodies raised against cytochrome b558.

The immunochemical characterization of NADPH oxidase activity of cytochrome b558 purified from human neutrophils was determined after reconstitution in a cell-free assay using the native hemoprotein and recombinant purified cytosolic activating factors. The oxidase activity showed a strict dependence on the heme content at each step of the hemoprotein purification process. The immunochemical properties of the reconstituted oxidase made use of monoclonal antibodies raised against membrane-bound and octyl-glucoside-extracted cytochrome b. From nine specific monoclonal antibodies reacting with gp91-phox cytochrome b558, two were selected, both of which were found to bind to the beta subunit of cytochrome b558 and to inhibit superoxide formation in the oxidase reconstituted cell-free assay. The extent of inhibition was dependent on the phospholipid environment. Neutrophil membrane extracts from X-linked chronic granulomatous disease patients did not produce O2- in the reconstituted system and did not bind to the antibodies.

Animals↗