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P Maurel

Publications and source records attributed to P Maurel.

At least 55 records · Page 3Linked to original sources
PubMed1996

The fetal specific gene CYP3A7 is inducible by rifampicin in adult human hepatocytes in primary culture.

We investigated by RNase protection the differential expression of CYP3A4 and CYP3A7 mRNAs in fetal and adult human livers and in adult hepatocytes in primary culture. Our results show that CYP3A7 is expressed in the liver of 8 of 9 adult Caucasians examined, at an average level of 1.7% of the level found in a fetal liver. CYP3A4 mRNA appeared to be expressed in this fetal liver. In adult hepatocytes in primary culture, the constitutive level of CYP3A4 mRNA was low but detectable after 96 hours in untreated cells, but CYP3A7 mRNA remained undetectable. However, when the cells were treated for 48 hours with 25 microM rifampicin, both CYP3A4 and CYP3A7 mRNAs were strongly induced in the 3 and in 2 of 3 cultures examined, respectively. Using an isoelectric focusing immunoblotting the two proteins were resolved. Protein CYP3A4 was detectable in induced cells but CYP3A7 was not. These results show for the first time that CYP3A7 and CYP3A4 mRNAs, but not the proteins, are co-inducible by rifampicin in adult human hepatocytes in culture.

Adult↗
PubMed1996

TAG-1/axonin-1 is a high-affinity ligand of neurocan, phosphacan/protein-tyrosine phosphatase-zeta/beta, and N-CAM.

Proteoglycans appear to play an important role in modulating cell-cell and cell-matrix interactions during nervous tissue histogenesis. The nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta were found to be high-affinity ligands of the neural cell adhesion molecule TAG-1/axonin-1, with dissociation constants of 0.3 nM and 0.04 nM, respectively. Phosphacan binding was decreased by approximately 70% following chondroitinase treatment, whereas binding of neurocan was not affected. The contribution of chondroitin sulfate chains to the binding of neurocan and phosphacan to TAG-1/axonin-1 is therefore the opposite of that previously observed for their binding to two other Ig-superfamily neural cell adhesion molecules, Ng-CAM/L1 and N-CAM. Moreover, whereas phosphacan interactions with certain proteins are mediated at least in part by N-linked oligosaccharides on the proteoglycan, N-deglycosylation of phosphacan had no effect on its binding to TAG-1/axonin-1. In addition to the chondroitin sulfate proteoglycans described above, we have demonstrated that N-CAM is a high-affinity ligand of TAG-1/axonin-1 (Kd approximately 1 nM), and specific binding of TAG-1/axonin-1 to tenascin-C was also observed (Kd approximately 9 nM). Immunocytochemical studies of embryonic and early postnatal nervous tissue showed an overlapping localization of TAG-1/axonin-1 with all four of these ligands, further supporting the biological significance of their ability to interact in vitro.

Animals↗
PubMed1996

Induction of CYP1A1 gene by benzimidazole derivatives during Caco-2 cell differentiation. Evidence for an aryl-hydrocarbon receptor-mediated mechanism.

The Caco-2 cell line, derived from a human colon adenocarcinoma, is unique in its property of spontaneously differentiating into a mature enterocyte cell type during its growth in culture. In this work, we compared the response of the CYP1A1 gene with the benzimidazole derivatives omeprazole and lansoprazole, and with the classical inducer beta-naphthoflavone in the Caco-2 cells at various culture stages. In addition, we characterized the Caco-2 aryl-hydrocarbon receptor. The protein-synthesis inhibitor cycloheximide led to a derepression of the CYP1A1 gene transcription, and to a superinduction when combined with either beta-naphthoflavone or benzimidazoles. Taking advantage of the spontaneous differentiation of Caco-2 cells in long-term cultures, we observed a difference in behavior between the classical inducer beta-naphthoflavone and the atypical inducer omeprazole. In the poorly differentiated cells, both compounds elicited comparable dose/response and rate of induction of CYP1A1 gene expression. In the fully differentiated cells, in contrast, the induction by omeprazole was only transient, whereas the response to beta-naphthoflavone was long lasting. The Caco-2 aryl-hydrocarbon receptor exhibited binding characteristics similar to those determined for human liver and other tissues. The induction of CYP1A1 transcription by benzimidazole derivatives in Caco-2 cells occurred with no direct binding of benzimidazole derivatives to the aryl-hydrocarbon receptor, as in human hepatocytes. However, transient transfection experiments clearly showed that the xenobiotic-responsive element enhancer, with which the activated aryl-hydrocarbon receptor interacts, could drive the induction of a heterologous promoter in the presence of benzimidazoles. Finally the presence of the activated aryl-hydrocarbon receptor in the nuclei of the Caco-2 cells exposed to these molecules was clearly demonstrated by gel-retardation experiments. These results question about the mechanism of ligand-independent activation of the aryl-hydrocarbon receptor and intracellular signaling, initiated by benzimidazole derivatives.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1996

Detection of CYP3A5 allelic variant: a candidate for the polymorphic expression of the protein?

In liver samples from 19 Caucasian subjects, the CYP3A5 protein was detected in 74% of individuals (14/19), while the messenger was shown to be expressed in 100% of individuals, assessed by the RT-PCR method. In order to characterise the putative mutation(s) in the messenger, accounting for the absence of protein accumulation, the full coding region of the CYP3A5 cDNA was sequenced for two unrelated individuals, one expressing the protein at a high level and one being defective. A point mutation in exon 11 at position 1280 (C-->A) was found to cosegregate with the absence of protein accumulation in 2 of the 5 defective individuals. This mutation produces a change in the amino acid at position 398 (Thr-->Asn). Whether this mutation affects the stability of the protein or whether it is in linkage desequilibrium with other mutation(s) in the non-coding region of the messenger is not known. The C-->A change at 1280 generates a Tsp509 I site which can be used for routine evaluation of the frequency of this mutation in population studies.

Adult↗
PubMed1996

Metabolism of the new immunosuppressor cyclosporin G by human liver cytochromes P450.

Cyclosporin G is a new immunosuppressor structurally similar to cyclosporin A. Although this drug is pharmacologically as active as cyclosporin A, it is less toxic, in particular at the kidney level. The aim of this work was to identify the enzyme system(s) involved in the oxidative metabolism of cyclosporin G in man: (1) in a bank of human liver microsomes (n = 22), cyclosporin G oxidase activity correlated significantly with cyclosporin A oxidase activity (P < 0.0001) and with the level of CYP3A4 (P < 0.002), determined by immunoblot; (2) specific inhibitors of CYP3A4, troleandomycin, and ketoconazole, inhibited cyclosporin G oxidase activity by more than 80%; (3) antiCYP3A4 antibodies specifically inhibited this activity by nearly 90%; (4) cyclosporin A was a competitive inhibitor of cyclosporin G oxidase and vice versa; (5). Among a battery of cDNA-expressed CYPs, only CYP3A4 was able to generate detectable amounts of metabolites of cyclosporin G and cyclosporin A with a turnover number close to that calculated from experiments with liver microsomes; (6) in human hepatocytes in culture, pretreatment of cells with rifampicin and phenobarbital, 2 inducers of CYP3A4, produced a great increase in cyclosporin G oxidase activity, while beta-naphthoflavone, an inducer of CYP1As, did not. We conclude that CYP3A4 is the major enzyme involved in the oxidative metabolism of cyclosporin G in human liver.

Adult↗
PubMed1996

Chondroitin sulfate proteoglycans in the developing central nervous system. I. cellular sites of synthesis of neurocan and phosphacan.

We have used in situ hybridization histochemistry to examine the cellular sites of synthesis of two major nervous tissue proteoglycans, neurocan and phosphacan, in embryonic and postnatal rat brain and spinal cord. Both proteoglycans were detected only in nervous tissue. Neurocan mRNA was evident in neurons, including cerebellar granule cells and Purkinje cells, and in neurons of the hippocampal formation and cerebellar nuclei. In contrast, phosphacan message was detected only in astroglia, such as the Golgi epithelial cells of the cerebellum. At embryonic day 13-16, phosphacan mRNA is largely confined to areas of active cell proliferation (e.g., the ventricular zone of the ganglionic eminence and septal area of the brain and the ependymal layer surrounding the central canal of the spinal cord) as well as being present in the roof plate. The distribution of neurocan message is more widespread, extending to the cortex, hippocampal formation, caudate putamen, and basal telencephalic neuroepithelium, and neurocan mRNA is present in both the ependymal and mantle layers of the spinal cord but not in the roof plate. The presence of neurocan mRNA in areas where the proteoglycan is not expressed suggests that the short open reading frame in the 5'-leader of neurocan may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing central nervous system.

Animals↗
PubMed1996

Contribution of primary cultures of adult human hepatocytes to the pathophysiology of hepatocellular carcinoma.

BACKGROUND/AIMS: The mechanisms of hepatocarcinogenesis are still poorly understood. The development of hepatocellular carcinoma has recently been shown to be associated with increased DNA synthesis in cirrhosis. The aim of this work was to determine whether the high rate of hepatocyte regeneration observed in cirrhotic liver with hepatocellular carcinoma is associated with the presence of a growth factor that could be detectable in the serum. METHODS: Adult human hepatocytes in primary culture, allowing the evaluation of the release of circulating hepatotrophic factors, were used. These cultures were treated for 48 h with serum from patients with cirrhosis with and without hepatocellular carcinoma, from patients with liver metastasis, and from healthy subjects. The rate of DNA synthesis in these cultures was assessed by measuring the amount of [3H]-thymidine incorporation into genomic DNA. RESULTS: On average, the synthesis of DNA was increased 2.5-, 2.2-, 2.1-, and 2.3-fold, respectively, in response to serum from patients with cirrhosis with hepatocellular carcinoma, from patients with cirrhosis without hepatocellular carcinoma, from patients with liver metastasis, and from healthy subjects. CONCLUSIONS: We conclude that the hepatotrophic activity of the serum is not significantly different in patients with cirrhosis with or without hepatocellular carcinoma. These results suggest that the increased DNA synthesis in hepatocytes of cirrhotic liver with hepatocellular carcinoma might be due to proliferative factor(s) acting by paracrine or autocrine pathways.

Adult↗
PubMed1996

Neurocan and phosphacan: two major nervous tissue-specific chondroitin sulfate proteoglycans.

Neurocan is a multidomain hyaluronan-binding chondroitin sulfate proteoglycan that is synthesized by neurons, whereas the astroglial proteoglycan phosphacan is an mRNA splice variant representing the entire extracellular portion of a receptor-type protein tyrosine phosphatase. A glycoform of phosphocan (phosphocan-KS) that contains both chondroitin sulfate and keratan sulfate is present in the postnatal rat central nervous system (CNS). The concentration of neurocan in brain increases during late embryonic development but then declines steeply during the early postnatal period together with hyaluronan, and neurocan also undergoes extensive proteolytic processing during the course of brain development. In contrast, the concentrations of both phosphocan and phosphocan-KS rise steadily after embryonic day 20 to reach a plateau at about 2 weeks postnatally. In the embryonic CNS the distribution of neurocan mRNA is more widespread than that of phosphocan, which is primarily present in regions of active cell proliferation. Neurocan mRNA is also present in areas where the proteoglycan is not expressed, and there is evidence that the short open reading frame in its 5'-leader may function as a cis-acting regulatory signal for the modulation of neurocan expression in the developing CNS. Neurocan and phosphocan bind saturably, reversibly, and with high affinity to neural cell adhesion molecules (Ng-CAM/L1, NCAM, TAG-1/axonin-1) and to tenascin-C. The proteoglycans and their ligands have overlapping localizations in the CNS, and binding of phosphocan to Ng-CAM/L1, NCAM, and tenascin-C is mediated by complex-type N-linked oligosaccharides on the proteoglycan. Neurocan and phosphocan also bind to neurons and are potent inhibitors of neuronal and glial adhesion and neurite outgrowth. Through their interactions with neural cell adhesion and extracellular matrix molecules, these proteoglycans may play a major role in modulating cell adhesion, neurite growth, and signal transduction across the plasma membrane during the development of the CNS.

Aging↗
PubMed1995

Evidence for the ligand-independent activation of the AH receptor.

Benzimidazole derivatives are potent inducers of CYP1A1 in rabbit and human hepatocytes, but apparently do not bind the AH receptor. To resolve this paradoxical behaviour, studies have been concerned with the question of whether an alternative ligand-independent mechanism could explain the activation of the AH receptor. From experiments in cultured rabbit hepatocytes we show that benzimidazoles bind early and transiently to an unknown protein. Moreover, they are able to deplete the AHR in a time- and dose-dependent manner. In contrast, benzimidazoles are unable to induce CYP1A1 mRNA in mouse hepa-1 cells and to deplete the high-affinity AHR form from these cells. Taken together these data suggest that a signal transduction pathway, similar to that involved in the ligand-independent activation of steroid receptors, could only activate the low-affinity forms of AHR as those existing in rabbit and human cells.

Animals↗
PubMed1995

Nucleotide sequence and molecular variants of rat receptor-type protein tyrosine phosphatase-zeta/beta.

We have previously described the cloning of phosphacan, a chondroitin sulfate proteoglycan of nervous tissue which interacts with neurons, glia, neural cell adhesion molecules, and tenascin, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase. We now report the complete cDNA and deduced amino acid sequences of the rat transmembrane phosphatase, and demonstrate that the phosphatase and the extracellular proteoglycan have different 3'-untranslated regions. Northern analysis showed three probable splice variants, comprising the extracellular proteoglycan (phosphacan) and long and short forms of the transmembrane phosphatase. PCR studies of rat genomic DNA indicated that there are no introns at the putative 5' and 3' splice sites or in the 2.6 kb segment which is deleted in the short transmembrane protein. Using variant-specific riboprobes corresponding to sequences in the 3'-untranslated region of phosphacan and in the first or second phosphatase domains of the transmembrane protein, in situ hybridization histochemistry of embryonic rat brain and spinal cord and early postnatal cerebellum demonstrated identical localizations of phosphacan and phosphatase mRNAs.

Alternative Splicing↗
PubMed1995

Differential effects of cytokines on the inducible expression of CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture.

We have investigated the effect of cytokines, including interleukin-6 (Il-6), interleukin-1 alpha (Il-1 alpha), and tumor necrosis factor-alpha (TNF-alpha), on the inducible expression of cytochrome P450s (CYP) CYP1A1, CYP1A2, and CYP3A4 in human hepatocytes in primary culture. The ability of these cultures to mimic the acute phase response when stimulated with cytokines was evaluated using immunoblotting to measure the production of albumin, ferritin, fibrinogen, and ceruloplasmin. The cytokines exhibited specific patterns of action on the production of these proteins. Albumin was depressed by all the cytokines. In contrast to Il-6 and Il-1 alpha, TNF-alpha reduced the production of fibrinogen and ceruloplasmin but stimulated the production of ferritin. When cells were treated with the CYP inducer alone, large increases in the expression of CYP1A1 and CYP1A2 by beta-naphthoflavone and of CYP3A4 by rifampicin were observed at messenger RNA (mRNA) and protein levels, by ribonuclease protection and immunoblotting, respectively. When the cells were treated with the inducer plus cytokines, the induction of mRNA was greatly reduced. Again, specific patterns of action were revealed: Il-6 had the most potent effect on CYP3A4, whereas TNF-alpha was the most potent with CYP1A genes. In all cases, changes at the protein levels paralleled changes at the mRNA levels. In cells preinduced with beta-naphthoflavone or rifampicin, the decay with time of the levels of the CYP1A2 or CYP3A4 proteins, after the removal of the inducer, was not affected by cytokines. We conclude that cytokines strongly repress the inducibility of CYP1As and CYP3A4 genes at a transcriptional or a posttranscriptional level, but affect neither the rate of translation of CYP mRNAs nor the rate of degradation of the CYP proteins in these cultures.

Acute-Phase Reaction↗
PubMed1995

Effect of anti-ulcer drugs on DNA synthesis in adult normal human hepatocytes in culture.

The aim of this work was to investigate the effect of four H2 receptor antagonist, cimetidine, ranitidine, famotidine, nizatidine, and of two proton pump inhibitors, omeprazole and lansoprazole, on the mitotic response of human hepatocytes in primary culture. After plating at subconfluent density, cells were exposed to 0.2 to 20 mumol/L of these drugs for 48 hours, either in the absence or in the presence of epidermal growth factor (EGF). The rate of DNA synthesis was evaluated by [3H]-thymidine incorporation into genomic DNA. Both the basal rate of DNA synthesis and the extent of stimulation by EGF exhibited a wide interindividual variability, and were not correlated with the viability of freshly prepared cells. In contrast, the effects of anti-ulcer drugs on the rate of DNA synthesis were clearly reproducible from one culture to another. H2 receptor antagonists had no significant effect (P > .2) over the entire range of concentration tested, whereas omeprazole and lansoprazole significantly inhibited the rate of DNA synthesis by 60% to 90% at 30 mumol/L (P = .016). This effect was concentration dependent between 2 and 20 mumol/L. Neither of the drugs tested was cytotoxic under the conditions used in this work, as assessed by measurements of the do nov protein synthesis. We conclude that, in contrast to H2 receptor antagonists, omeprazole and lansoprazole are able to interfere with the replicative synthesis of DNA in human hepatocytes in culture, at suprapharmacological concentrations. Whether or not this effect is clinically significant remains to be established.

Anti-Ulcer Agents↗
PubMed1995

Oxidative metabolism of lansoprazole by human liver cytochromes P450.

The aim of this work was to identify the form(s) of human cytochrome P450 (P450) involved in the hepatic biotransformation of lansoprazole to its two main metabolites, i.e., the sulfone and the hydroxy derivative. In liver microsomes, the production of the sulfone of lansoprazole correlated with the level of P450 3A4, cyclosporin oxidase, and the production of the hydroxy derivative, as well as of omeprazole sulfone. The production of hydroxylansoprazole moderately correlated with the level of P450 3A4, cyclosporin oxidase, and (S)-mephenytoin 4'-hydroxylase. The production of the sulfone and of the hydroxy derivative of lansoprazole was significantly inhibited by anti-P450 3A4 antibodies, by cyclosporin and ketoconazole, and by tolbutamide. Anti-P450 2C8 and 2C3 antibodies moderately inhibited the biotransformation of lansoprazole, whereas they completely inhibited (S)-mephenytoin 4'-hydroxylase activity under the same conditions. In primary cultures of human hepatocytes, the biotransformation of lansoprazole and the oxidation of cyclosporin were strongly increased by rifampicin and phenobarbital, whereas (S)-mephenytoin 4'-hydroxylation was not. beta-Naphthoflavone did not induce the formation of the sulfones but stimulated the production of hydroxylansoprazole. Among several forms of cDNA-expressed human P450s, 3A4 generated significant amounts of the sulfones of lansoprazole and omeprazole and 2C18 was active for the production of hydroxylansoprazole but inactive in the 4'-hydroxylation of (S)-mephenytoin. We conclude that P450 3A4 is the major enzyme involved in the production of the sulfone of lansoprazole and that this P450, as well as P450 2C18 and/or another 2C-related form, could contribute to the production of hydroxylansoprazole.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1995

Oxidative metabolism of zolpidem by human liver cytochrome P450S.

The aim of this study was to identify the form(s) of cytochrome P450 (CYP) responsible for the biotransformation of zolpidem to its alcohol derivatives which, after rapid conversion to carboxylic acids, represents the main way of metabolism in humans. In human liver microsomes, zolpidem was converted to alcohol derivatives. Production of these correlated with the level of CYP3A4 and with cyclosporin oxidation and erythromycin N-demethylation activities, but not with the level of CYP1A2 nor with ethoxyresorufin O-deethylation or S-mephenytoin 4'-hydroxylation activities. Liver microsomes from CYP2D6-deficient patients exhibited normal activity. Production of alcohol derivatives was significantly inhibited by anti-CYP3A antibodies and by ketoconazole. Antibodies directed against other CYP forms (including CYP1A1, CYP1A2, CYP2A6, CYP2B4, and CYP2C8), and CYP-specific substrates or inhibitors (including propranolol, coumarin, mephenytoin, sulfaphenazole, quinidine, aniline, and lauric acid) produced a moderate or no inhibitory effect. cDNA-expressed CYP3A4 and CYP1A2 generated significant amounts of one of the alcohol derivatives, whereas CYP2D6 generated both of them in similar amounts. In human hepatocytes in primary culture, zolpidem was extensively and almost exclusively converted to one of the carboxylic acid derivatives, the main species identified in vivo. Treatment of cells with inducers of CYP1A (beta-naphthoflavone) and CYP3A (rifampicin and phenobarbital) greatly increased the rate of production of this metabolite. We conclude that the formation of alcohol derivatives of zolpidem is rate-limiting and principally mediated by CYP3A4. Both CYP1A2 and CYP2D6 participate in alcohol formation; but, because of their low relative level of expression in the human liver, their contribution is minor.

Adult↗
PubMed1994

Sequence of the 5'-flanking region of CYP3A5: comparative analysis with CYP3A4 and CYP3A7.

Two genomic clones containing the 3'-end of CYP3A7, the whole 5'-flanking region plus the first exon of CYP3A5 were isolated from two different human genomic banks and sequenced. The 5'-flanking region of CYP3A5, from nucleotide -1 to -1434, was 60% and 59% similar with the corresponding regions of CYP3A4 and CYP3A7, respectively. However, the similarity increased to 74% when regions from nucleotide -1 to -700 were compared and dropped to less than 42% for regions upstream from -700. The CYP3A5 promoter contains a CATA box instead of the typical TATA box and a basic transcription element (BTE). Other consensus sequences previously found in CYP3A4 and CYP3A7 were also identified in the 5'-flanking of CYP3A5 from -1 to -700. Both CYP3A5 and CYP3A7 genes appear to be tandemly associated and transcribed in the same direction on chromosome 7.

Aryl Hydrocarbon Hydroxylases↗
PubMed1994

The pig as a model for studying AH receptor and other PAH-binding proteins in man.

In this report we compare the three major binding proteins existing in hepatic cytosols of pig and human. Many data suggest that for the AH receptor, which mediates the biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a structural as well as a functional variability exists across species. Similar conclusions can be drawn from the interspecies characterization of benzo(a)pyrene binding proteins, namely the 4S protein and the 8S protein. Using fractionation procedures such as sucrose gradient sedimentation and gel permeation chromatography we obtained enriched fractions containing each of these binding proteins. By saturation experiments and analysis of data by Scatchard and Woolf plots we determined binding characteristics and we conclude that pig and human AH receptors were closely related proteins since their Kd (18 +/- 0.4nM) were found quite similar. On the other hand, 4S proteins from pig (Kd = 16.7 nM; Bmax = 5.5 pmol/mg) and from human (Kd = 14 nM; Bmax = 4.5 pmol/mg) as well as 8S proteins (Kd approximately 300 nM) also exhibit remarkable similarities.

Animals↗