Browsing by Author "Manzano, M"

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    Induction of the multispecific organic anion transporter (cMoat/mrp2) gene and biliary glutathione secretion by the herbicide 2,4,5-trichlorophenoxyacetic acid in the mouse liver
    (PORTLAND PRESS, 1999) Wielandt, AM; Vollrath, V; Manzano, M; Miranda, S; Accatino, L; Chianale, J
    The canalicular multispecific organic anion transporter. cMoat, is an ATP-binding-cassette protein expressed in the canalicular domain of hepatocytes. In addition to the transport of endo- and xenobiotics, cMoat has also been proposed to transport GSH into bile, the major driving force of bile-acid-independent bile flow. We have shown previously that the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a peroxisome-proliferator agent, significantly increases bile-acid-independent bile flow in mice. On this basis, the effect of the herbicide on cMoat gene expression was studied. A 3.6-fold increase in cMoat mRNA levels and a 2.5-fold increase in cMoat protein content were observed in the liver of mice fed on a diet supplemented with 0.125% 2,4,5-T. These effects were due to an increased rate of gene transcription (3.9-fold) and were not associated with peroxisome proliferation. Significant increases in bile flow (2.23 +/- 0.39 versus 1.13 +/- 0.15 mu l/min per g of liver: P < 0.05) and biliary GSH output (7.40 +/- 3.30 versus 2.65 +/- 0.34 nmol/min per g of liver; P < 0.05) were observed in treated animals. The hepatocellular concentration of total glutathione also increased in hepatocytes of treated mice (10.95 +/- 0.84 versus 5.12 +/- 0.47 mM; P < 0.05), because of the induction (2.4-fold) of the heavy subunit of the gamma-glutamylcysteine synthetase (GCS-HS) gene. This is the first model of co-induction of cMoat and GCS-HS genes in vivo in the mouse liver, associated with increased glutathione synthesis and biliary glutathione output. Our observations are consistent with the hypothesis that the cMoat transporter plays a crucial role in the secretion of biliary GSH.
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    Role of tfdCIDIEIFI and tfdDIICIIEIIFII gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4)
    (2000) Pérez-Pantoja, D; Guzmán, L; Manzano, M; Pieper, DH; González, B
    The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, a nd maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes, To assess the role of both ya modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene, These constructs were introduced into R. eurtropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,3-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I, The R, eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II) In the case of P, putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB, These results indicate that efficient degradation of 3-CB by R, eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.
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    The copy number of the catabolic plasmid pJP4 affects growth of Ralstonia eutropha JMP134 (pJP4) on 3-chlorobenzoate
    (2002) Trefault, N; Clément, P; Manzano, M; Pieper, DH; González, B
    Ralstonia eutropha JMP134 (pJP4) grows on 3-chlorobenzoate (3-CB) or 2,4-dichlorophenoxyacetate (2,4-D). The copy number of chlorocatechol genes has been observed to be important for allowing growth of bacterial strains on chloroaromatic compounds. Despite the fact that two functional chlorocatechol degradation tfd gene clusters are harbored on plasmid pJP4, a single copy of the region comprising all tfd genes in strain JMP134-F was insufficient to allow growth on 3-CB, whereas growth on 2,4-D was only slightly retarded compared to the wild-type strain. Using competitive PCR, approximately five copies of pJP4 per genome were observed to be present in the wild-type strain, whereas only one copy of pJP4 was present per chromosome in strain JMP134-F. Therefore, several copies of pJP4 per chromosome are required for full expression of the tfd-encoded growth abilities in the wild-type R. eutropha strain. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.