182 results on '"Lieberman, M W"'
Search Results
2. Physiology: The ventilatory response to hypoxia
- Author
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Gozal, D., Gaston, B. M., Lipton, A. J., Johnson, M. A., Macdonald, T., and Lieberman, M. W.
- Published
- 2002
3. Low molecular weight silicones are widely distributed after a single subcutaneous injection in mice
- Author
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Kala, S. V., Lykissa, E. D., Neely, M. W., and Lieberman, M. W.
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Molecular Weight ,Mice ,Siloxanes ,Breast Implants ,Injections, Subcutaneous ,Animals ,Female ,Tissue Distribution ,Gas Chromatography-Mass Spectrometry ,Research Article - Abstract
To examine the distribution of low molecular weight silicones in body organs, separate groups of female CD-1 mice were injected with either breast implant distillate composed primarily of hexamethylcyclotrisiloxane, octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, dodecamethylcyclohexasiloxane, and tetradecamethylcycloheptasiloxane or a polydimethylsiloxane oil containing low molecular weight linear siloxanes. Mice were injected subcutaneously in the suprascapular area and killed at different times. Levels of individual low molecular weight silicones were measured in 10 different organs (brain, heart, kidney, liver, lung, mesenteric lymph nodes, ovaries, spleen, skeletal muscle, and uterus). In mice treated with the cyclosiloxane mixture and killed at 3, 6, or 9 weeks, highest levels of cyclosiloxanes were found in the mesenteric lymph nodes, ovaries, and uterus, but all organs examined contained cyclosiloxanes. In a cohort killed at 1 year, most organs contained measurable cyclosiloxanes with highest levels in mesenteric lymph nodes, abdominal fat, and ovaries. Of the individual cyclosiloxanes measured, selective retention of decamethylcyclopentasiloxane and dodecamethylcyclohexasiloxane relative to octamethylcyclotetrasiloxane was seen in all organs at all time points studied. Organs from animals receiving the linear siloxane mixture were harvested at 9, 12, and 15 weeks. We found maximum levels in the brain, lungs, and mesenteric lymph nodes, but all other organs contained measurable levels. These data are, to the best of our knowledge, the first demonstration that after a single subcutaneous injection silicones are widely distributed throughout the body and can persist over an extended period.
- Published
- 1998
4. gamma-Glutamyl transpeptidase. What does the organization and expression of a multipromoter gene tell us about its functions?
- Author
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Lieberman, M. W., Barrios, R., Carter, B. Z., Habib, G. M., Lebovitz, R. M., Rajagopalan, S., Sepulveda, A. R., Shi, Z. Z., and Wan, D. F.
- Subjects
Gene Expression Regulation ,Animals ,Humans ,gamma-Glutamyltransferase ,Promoter Regions, Genetic ,Research Article - Abstract
gamma-Glutamyl transpeptidase is a key enzyme in glutathione (GSH) salvage, metabolism of endogenous mediators such as leukotrienes and prostaglandins, detoxification of xenobiotics including environmentally important compounds and carcinogens, and cellular processes dependent on the oxidation/reduction of glutathione. The enzyme is widely distributed, and these functions often occur in separate tissues and in response to different stimuli. Evidence indicates that gamma-glutamyl transpeptidase plays a direct role in some hepatic and renal responses to injury. In the mouse gamma-glutamyl transpeptidase is a single copy gene expressed from at least seven promoters, and many of the transcribed gamma-glutamyl transpeptidase RNAs are restricted in their expression. Studies that combine analyses of cellular processes with a knowledge of gene structure and expression hold promise for unravelling how these two different levels of function are integrated.
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- 1995
5. Expression of the rasT24 oncogene in the ciliary body pigment epithelium and retinal pigment epithelium results in hyperplasia, adenoma, and adenocarcinoma
- Author
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Chévez-Barrios, P., Schaffner, D. L., Barrios, R., Overbeek, P. A., Lebovitz, R. M., and Lieberman, M. W.
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Adenoma ,Hyperplasia ,Oncogene Proteins, Fusion ,Histocytochemistry ,Ciliary Body ,Mice, Transgenic ,gamma-Glutamyltransferase ,Adenocarcinoma ,Eye ,Polymerase Chain Reaction ,Mice ,Genes, ras ,Animals, Newborn ,Animals ,RNA, Messenger ,Pigment Epithelium of Eye ,Promoter Regions, Genetic ,Research Article - Abstract
We examined eye lesions in five lines of transgenic mice carrying the human rasT24 oncogene driven by the type I gamma glutamyl transferase (gamma GT) promoter. In three lines, hyperplasia developed as early as 11.5 days postconception in the outer neuroectodermal layer, which gives rise to ciliary body and retinal pigment epithelium. At birth, the eyes from many animals contained adenomas, and by day 27, mice developed invasive adenocarcinomas originating in the region of the ciliary body. Microphthalmia, cataracts, and chronic nongranulomatous inflammation involving the anterior and/or posterior segments of the eye were also found. gamma GT is detectable histochemically as early as 11.5 gestational days in the outer neuroectodermal layer and after birth is more abundant in the ciliary body than in the retinal pigment epithelium. Using a reverse transcriptase-polymerase chain reaction, we found that type I (but not types II or III) gamma GT RNA is made by the mouse eye; the gamma GT(I)rasT24 transgene transcription product was detected in the eyes of all five transgenic lines. The sequential progression of hyperplasia to invasive neoplasms in the ciliary body in response to gamma GT(I)rasT24 expression differs from the process in the kidney of these animals in which tubular hyperplasia and microadenomas with little evidence of progression are the major lesions.
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- 1993
6. Cell survival and changes in gene expression in cells unable to synthesize glutathione
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Rojas, E., primary, Shi, Z.-Z., additional, Valverde, M., additional, Paules, R. S., additional, Habib, G. M., additional, and Lieberman, M. W., additional
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- 2003
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7. Physiology (communication arising): The ventilatory response to hypoxia
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Gozal, D., Gaston, B. M., Lipton, A. J., Johnson, M. A., Macdonald, T., and Lieberman, M. W.
- Subjects
Environmental issues ,Science and technology ,Zoology and wildlife conservation - Abstract
Author(s): D. Gozal; B. M. Gaston [1]; A. J. Lipton; M. A. Johnson; T. Macdonald; M. W. Lieberman Gozal et al . reply We do not challenge the 'classic' theory [...]
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- 2002
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8. The ventilatory response to hypoxia
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Gozal, D., primary, Gaston, B. M., additional, Lipton, A. J., additional, Johnson, M. A., additional, Macdonald, T., additional, and Lieberman, M. W., additional
- Published
- 2002
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9. Cyclosiloxanes produce fatal liver and lung damage in mice.
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Lieberman, M W, primary, Lykissa, E D, additional, Barrios, R, additional, Ou, C N, additional, Kala, G, additional, and Kala, S V, additional
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- 1999
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10. Suramin inhibits wound healing following filtering procedures for glaucoma
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Mietz, H., primary, Chevez-Barrios, P., additional, Feldman, R. M, additional, and Lieberman, M. W, additional
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- 1998
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11. N-acetylcysteine as an antidote in methylmercury poisoning.
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Ballatori, N, primary, Lieberman, M W, additional, and Wang, W, additional
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- 1998
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12. Growth retardation and cysteine deficiency in gamma-glutamyl transpeptidase-deficient mice.
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Lieberman, M W, primary, Wiseman, A L, additional, Shi, Z Z, additional, Carter, B Z, additional, Barrios, R, additional, Ou, C N, additional, Chévez-Barrios, P, additional, Wang, Y, additional, Habib, G M, additional, Goodman, J C, additional, Huang, S L, additional, Lebovitz, R M, additional, and Matzuk, M M, additional
- Published
- 1996
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13. Six mRNAs with different 5' ends are encoded by a single gamma-glutamyltransferase gene in mouse.
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Rajagopalan, S, primary, Wan, D F, additional, Habib, G M, additional, Sepulveda, A R, additional, McLeod, M R, additional, Lebovitz, R M, additional, and Lieberman, M W, additional
- Published
- 1993
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14. Cloning and analysis of the rat gamma-glutamyltransferase gene.
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Rajagopalan, S, primary, Park, J H, additional, Patel, P D, additional, Lebovitz, R M, additional, and Lieberman, M W, additional
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- 1990
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15. Elimination of the differential chemoresistance between the murine B-cell lymphoma LY-ar and LY-as cell lines after arsenic (As2O3) exposure via the overexpression of gsto1 (p28).
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Giri, U., Terry, N. H. A., Kala, S. V., Lieberman, M. W., and Story, M. D.
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ARSENIC ,APOPTOSIS ,CELL proliferation ,NEOVASCULARIZATION ,CELL lines ,GLUTATHIONE - Abstract
Purpose: Arsenic, in the form of As
2 O3 , has gained therapeutic importance because it has been shown to be very effective clinically in the treatment of acute promyelocytic leukemia (APL). Via numerous pathways arsenic induces cellular alterations such as induction of apoptosis, inhibition of cellular proliferation, stimulation of differentiation, and inhibition of angiogenesis. Responses vary depending on cell type, dose and the form of arsenic. GSTO1, a member of the glutathione S-transferase superfamily omega, has recently been shown to be identical to the rate-limiting enzyme, monomethyl arsenous (MMAV ) reductase which catalyzes methylarsonate (MMAV ) to methylarsenous acid (MMAIII ) during arsenic biotransformation. In this study, we investigated whether arsenic trioxide (As2 O3 ) induces apoptosis in both chemosensitive and chemoresistant cell lines that varied in their expression of p28 (gsto1), the mouse homolog of GSTO1. Methods: The cytotoxicity of arsenic in the gsto1- and bcl-2- expressing chemoresistant and radioresistant LY-ar mouse lymphoma cell line, was compared with that of the LY-ar's parental cell line, LY-as. LY-as cells are radiosensitive, apoptotically permissive, and do not express gsto1 or bcl-2. Cell survival, glutathione (GSH) levels, mitochondrial membrane potential, and stressactivated kinase status after arsenic treatment were examined in these cell lines. Results: As2 O3 induced an equivalent dose- and time-dependent increase in apoptosis in these cell lines. Cellular survival, as measured after a 24-h exposure, was also the same in each cell line. Reduced GSH was modulated in a similar time- and dose-dependent manner. Apoptosis was preceded by loss of mitochondrial membrane potential that triggered caspase-mediated pathways associated with apoptosis. With a prolonged exposure of As2 O3 , both cell lines showed decreased activation of ERK family members, ERK1, ERK2 and ERK5. As2 O3 enhanced the death signals in LY-ar cells through a decrease in GSH, loss of mitochondrial membrane potential, and abatement of survival signals. This effect is similar to that seen when LY-ar cells are treated with thiol-depleting agents or by the removal of methionine and cysteine (GSH precursor) from the growth medium. This response is also completely contrary to that seen for radiation, actinomycin D, VP-16 and other agents, where LY-ar cells do not succumb to apoptosis. Conclusions: The overexpression of gsto1 in normally chemoresistant and radioresistant LY-ar cells renders them vulnerable to the cytotoxic effects of As2 O3 , despite the 30-fold overexpression of the survival factor bcl-2. Gsto1 and its human homolog, GSTO1, may serve as a marker for arsenic sensitivity, particularly in cells that are resistant to other chemotherapeutic agents. [ABSTRACT FROM AUTHOR]- Published
- 2005
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16. The Influence of Ras Oncogene Expression on Radiation Response in the Rat-1 Cell.
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Garden, A. S., Meyn, R. E., Weil, M. M., Lebovitz, R. M., and Lieberman, M. W.
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- 1992
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17. Formation and Urinary Excretion of Arsenic Triglutathione and Methylarsenic Diglutathione
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Kala, S. V., Kala, G., Prater, C. I., Sartorelli, A. C., and Lieberman, M. W.
- Abstract
Taking advantage of mice deficient in γ-glutamyl transpeptidase that are unable to metabolize glutathione (GSH), we have identified two previously unrecognized urinary metabolites of arsenite: arsenic triglutathione and methylarsenic diglutathione. Following administration of sodium arsenite to these mice, ~60−70% of urinary arsenic is present as one of these GSH conjugates. We did not detect the dimethyl derivative, dimethyl arsenic GSH; however, dimethyl arsenic (DMAV) represented approximately 30% of urinary arsenic. Administration of buthionine sulfoximine, an inhibitor of GSH synthesis, to wild-type mice reduced urinary arsenic excretion by more than 50%, indicating the GSH dependence of arsenic metabolism, transport, or both. Rodents deficient in three known ABC family transporters (MRP1, MRP2, and MDR1a/1b) exhibited urinary arsenic levels similar or greater than those in wild-type rodents; however, administration of MK571, an MRP inhibitor, reduced urinary arsenic excretion by almost 50%. MK571-treated mice showed ~50% reduction of AsIII, MMAV, and AsV as compared to untreated wild-type controls, while DMAV levels were unchanged. These findings suggest that arsenic excretion is in part dependent on GSH and on an MRP transporter other than MRP1 or 2.
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- 2004
18. Accumulation of DNA damage in the organs of mice deficient in g-glutamyltranspeptidase
- Author
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Rojas, E., Valverde, M., Kala, S. V., Kala, G., and Lieberman, M. W.
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- 2000
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19. Genomic hypomethylation and far-5' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene
- Author
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Barr, F G, Rajagopalan, S, MacArthur, C A, and Lieberman, M W
- Abstract
We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.
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- 1986
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20. MTrasT24, a metallothionein-ras fusion gene, modulates expression in cultured rat liver cells of two genes associated with in vivo liver cancer.
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Li, Y C, Seyama, T, Godwin, A K, Winokur, T S, Lebovitz, R M, and Lieberman, M W
- Abstract
We studied the effects of a zinc-inducible metallothionein-ras fusion gene (MTrasT24) in cultured rat liver epithelial (RLE) cells on expression of two genes induced during liver carcinogenesis in vivo: gamma-glutamyltransferase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] and glutathione S-transferase-P (RX:glutathione R-transferase, EC 2.5.1.18). Expression of MTrasT24 increased steady-state RNA levels of gamma-glutamyltransferase and glutathione transferase-P 6- to 100-fold and 1.6- to 6-fold, respectively; in contrast, levels of alpha-tubulin RNA fell slightly or were unchanged. RNA gel blots verified that gamma-glutamyltransferase and glutathione transferase-P RNAs were of the appropriate size, and results from immunocytochemistry on transfected cells demonstrated that RLE cells carrying MTrasT24 synthesized immunoreactive, appropriately localized gamma-glutamyltransferase and glutathione transferase-P. Zinc induction studies indicated that gamma-glutamyltransferase and glutathione transferase-P RNA levels were directly dependent on MTrasT24 RNA levels. These data suggest that expression of gamma-glutamyltransferase and glutathione transferase-P expression are part of a reorientation of cellular gene expression during carcinogenesis and that activated ras expression, like chemical carcinogens, can bring about this change.
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- 1988
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21. A 346-base pair region of the mouse gamma-glutamyl transpeptidase type II promoter contains sufficient cis-acting elements for kidney-restricted expression in transgenic mice.
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Sepulveda, A R, Huang, S L, Lebovitz, R M, and Lieberman, M W
- Abstract
The mouse gamma-glutamyl transpeptidase (GGT) gene encodes seven distinct mRNAs that are transcribed from seven separate promoters. Type II mRNA is the most abundant in kidney. We have developed a cell line with features of renal proximal tubular cells which expresses GGT mRNA types with a pattern similar to that of mouse kidney. Because a 346-bp sequence from the type II promoter directed the highest level of CAT activity in these cells, this region was used to drive the expression of a beta-galactosidase reporter gene in transgenic mice. Two transgenic mouse lines expressed beta-galactosidase limited to the renal proximal tubules. Site-directed deletions within this 346-bp promoter region demonstrated that cis-elements containing the consensus binding sites for AP2, a glucocorticoid response element (GRE)-like element, and the initiator region were required for transcriptional activity and were not additive. Purified AP2 bound and footprinted the AP2 consensus region, making it likely that transcription from the GGT type II promoter is regulated in part by AP2. These data suggest that transcription of the type II promoter requires multiple protein DNA interactions involving at least an AP2 element, and probably a GRE-like element and the initiator region.
- Published
- 1997
22. Differences in removal of acetylaminofluorene and pyrimidine dimers from the DNA of cultured mammalian cells.
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Amacher, D E, Elliott, J A, and Lieberman, M W
- Abstract
The rate and extent of disappearance of two DNA lesions (pyrimidine dimers and covalently bound acetylaminofluorene), both thought to be removed by the so-called wide-patch (approximately 100 nucleotides) repair process, were studied in a variety of cultured mammalian cells. With the exception of mouse cells, dimers were removed more rapidly and extensively than covalently bound acetylaminofluorene. In human cells, for example, about 50% of the dimers were excised from DNA in 1 hr while only 25-50% of the chemically induced lesions were excised from DNA after 48 hr. Surprisingly mouse cells, which remove few dimers, were about as competent as control human fibroblasts at removing acetylaminofluorene lesions; however, xeroderma pigmentosum cells (group D) removed fewer N-acetoxy-2-acetylaminofluorene-induced lesions than control human cells. Our data raise the possibility of separate repair processes for these two types of lesions and suggest that their expression may be under similar genetic control in human cells.
- Published
- 1977
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23. gamma-glutamyl leukotrienase, a gamma-glutamyl transpeptidase gene family member, is expressed primarily in spleen.
- Author
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Carter, B Z, Shi, Z Z, Barrios, R, and Lieberman, M W
- Abstract
We have recently identified a mouse enzyme termed gamma-glutamyl leukotrienase (GGL) that converts leukotriene C4 (LTC4) to leukotriene D4 (LTD4). It also cleaves some other glutathione (GSH) conjugates, but not GSH itself (Carter, B. Z., Wiseman, A. L., Orkiszewski, R., Ballard, K. D., Ou, C.-N., and Lieberman, M. W. (1997) J. Biol. Chem. 272, 12305-12310). We have now cloned a full-length mouse cDNA coding for GGL activity and the corresponding gene. GGL and gamma-glutamyl transpeptidase constitute a small gene family. The two cDNAs share a 57% nucleotide identity and 41% predicted amino acid sequence identity. Their corresponding genes have a similar intron-exon organization and are located 3 kilobases apart. A search of Genbank and reverse transcription-polymerase chain reaction analysis failed to identify additional family members. Mapping of the GGL transcription start site revealed that the GGL promoter is TATA-less but contains an initiator, a control element for transcription initiation. Northern blots for GGL expression were negative. As judged by ribonuclease protection, in situ hybridization, and measurement of enzyme activity, spleen had the highest level of GGL expression. GGL is also expressed in thymic lymphocytes, bronchiolar epithelial cells, pulmonary interstitial cells, renal proximal convoluted tubular cells, and crypt cells of the small intestine as well as in cerebral, cerebellar, and brain stem neurons but not in glial cells. GGL is widely distributed in mice, suggesting an important role for this enzyme.
- Published
- 1998
24. Metabolism of leukotriene C4 in gamma-glutamyl transpeptidase-deficient mice.
- Author
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Carter, B Z, Wiseman, A L, Orkiszewski, R, Ballard, K D, Ou, C N, and Lieberman, M W
- Abstract
We have investigated the metabolism of leukotriene C4 (LTC4) in gamma-glutamyl transpeptidase (GGT)-deficient mice (Lieberman, M. W., Wiseman, A. L., Shi, Z-Z., Carter, B. Z., Barrios, R., Ou, C-N., Chevez-Barrios, P., Wang, Y., Habib, G. M., Goodman, J. C., Huang, S. L., Lebovitz, R. M., and Matzuk, M. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7923-7926) and have found substantial conversion of LTC4 to leukotriene D4 by high performance liquid chromatography and continuous flow fast atom bombardment-tandem mass spectrometric analyses. LTC4-converting activity has a tissue distribution different from GGT with highest activity in spleen followed by small intestine, kidney, and pancreas and lower activity in liver and lung. The activity is membrane-bound and is inhibited by acivicin, a known inhibitor of GGT. The enzyme was partially purified from the small intestine of GGT-deficient mice by papain treatment and gel filtration chromatography. The partially purified fragment released by papain has an apparent molecular mass of 65-70 kDa and the same substrate specificity as the tissue homogenate. In addition to LTC4, S-decyl-GSH is also cleaved. GSH itself, oxidized GSH, and the synthetic substrates used to analyze GGT activity (gamma-glutamyl-p-nitroanilide and gamma-glutamyl-4-methoxy-2-naphthylamide) are not substrates for this newly discovered enzyme. These data demonstrate that in addition to GGT at least one other enzyme cleaves LTC4 in mice. To reflect this enzyme's preferred substrate, we suggest that it be named gamma-glutamyl leukotrienase.
- Published
- 1997
25. Identification of DNA polymerases involved in DNA excision repair in diploid human fibroblasts.
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Dresler, S L and Lieberman, M W
- Abstract
We have used inhibitors to identify the DNA polymerases which are involved in DNA excision repair induced in confluent diploid human fibroblasts by several DNA damaging agents: UV radiation, N-acetoxy-2-acetylaminofluorene, N-methyl-N-nitrosourea, and bleomycin. We find that DNA repair synthesis involves both DNA polymerase alpha and a non-alpha DNA polymerase, probably polymerase beta. The fraction of repair synthesis mediated by each of the two polymerases is dependent on which DNA-damaging agent is administered and on the dose of damaging agent. Low doses of DNA damage induce DNA repair synthesis which is mediated to a great extent by a non-alpha DNA polymerase, and with an increasing dose of damage there is increasing participation of DNA polymerase alpha in repair synthesis. At high doses of damage, the fraction of repair synthesis mediated by DNA polymerase alpha reaches a maximal level which is dependent on the damaging agent; the maximal level of polymerase alpha involvement is about 80% for UV radiation and N-acetoxy-2-acetylaminofluorene, about 70% for N-methyl-N-nitrosourea, and about 40% for bleomycin.
- Published
- 1983
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26. The distribution of DNA excision-repair sites in human diploid fibroblasts following ultraviolet irradiation.
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Cohn, S M and Lieberman, M W
- Abstract
Using the technique for separating DNA fragments containing excision-repair sites from total genomic DNA as described in the previous paper (Cohn, S. M., and Lieberman, M. W. (1984) J. Biol. Chem. 259, 12456-12462), we have developed a method for directly determining the distribution of excision-repair sites in the genome. DNA was prepared from confluent, diploid human fibroblasts which had been irradiated with ultraviolet light and incubated in the presence of 5-bromo-2'-deoxyuridine (BrdUrd), repaired fragments were isolated, and the dependence of the fraction of total DNA fragments containing excision-repair sites on DNA fragment length was determined by electrophoretic analysis. The observed dependence was compared to the relationship expected for a random distribution of repair sites. At 36 h following 3 J/m2 UV, the distribution of repair sites was indistinguishable from a random distribution; however, at doses of UV above 6 J/m2, the observed dependence indicated that the distribution of repair sites was nonrandom. A time course of the distribution of repair sites following 12 J/m2 UV was clearly nonrandom from 4 h after irradiation until at least 36 h following irradiation. By 72 h, however, the distribution had become random. In cells treated with hydroxyurea, a reduced number of excision-repair sites were present, but the distribution of repair sites was also nonrandom. Autoradiographic analysis of the amount of unscheduled DNA synthesis in individual nuclei suggested that the nonrandom distribution of repair sites did not result from variable extents of repair synthesis in different cell populations or from cell death.
- Published
- 1984
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27. The use of antibodies to 5-bromo-2'-deoxyuridine for the isolation of DNA sequences containing excision-repair sites.
- Author
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Cohn, S M and Lieberman, M W
- Abstract
We have developed an immunological method for isolation and identification of DNA sequences containing 5-bromo-2'-deoxyuridine (BrdUrd) incorporated during UV-induced excision-repair synthesis. DNA fragments containing BrdUrd incorporated during repair synthesis were incubated with goat anti-BrdUrd and rabbit anti-goat IgG, and the antibody-DNA complexes were separated from bulk DNA by nitrocellulose filter binding. With this method, 80% of DNA sequences containing BrdUrd-labeled excision-repair sites were recovered, contaminated with less than 1% of DNA fragments devoid of excision-repair sites. Recovery of DNA fragments containing repair sites was independent of size from 2 to 20 kilobases. We have used this method in conjunction with blot hybridization to demonstrate that repair synthesis occurs in human ribosomal gene sequences in cells treated with UV.
- Published
- 1984
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28. Nucleosome rearrangement in human chromatin during UV-induced DNA- reapir synthesis.
- Author
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Smerdon, M J and Lieberman, M W
- Abstract
The distribution of UV-induced DNA repair synthesis within chromatin was measured in confluent human fibroblasts that were pulse-labeled with [3H]dThd (10 or 90 min) immediately after irradiation and chased in nonradioactive medium for different time periods. Initially (i.e., at the end of the pulse period), most of the repair synthesis occurs in staphylococcal nuclease-sensitive regions. With increasing chase times the nucleotides inserted during repair synthesis become progressively more nuclease resistant. Gel electrophoresis data indicate that nuclease resistance is conferred on these nucleotides by their appearance in core DNA. The kinetics of this rearrangement process are biphasic: greater than 85% of the repair synthesis sites undergo rapid rearrangement (4--5 hr); the remaining sites ( less than 15%) rearrange much more slowly, if at all. The time courses of nucleosome rearrangement and repair synthesis are similar, suggesting that nucleosome rearrangement may be induced by the repair process or that the rate of repair synthesis may be regulated by nucleosome rearrangement.
- Published
- 1978
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29. Cloning of cDNA and genomic structure of the mouse gamma-glutamyl transpeptidase-encoding gene
- Author
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Shi, Z.-Z., Habib, G. M., Lebovitz, R. M., and Lieberman, M. W.
- Published
- 1995
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30. Synthesis of nuclear proteins during DNA repair synthesis in human diploid fibroblasts damaged with ultraviolet radiation of N-acetoxy-2-acetylaminofluroene.
- Author
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Stein, G S, Park, W D, Stein, J L, and Lieberman, M W
- Abstract
We have examined the accumulation of newly synthesized nuclear proteins into nuclei during DNA repair synthesis in confluent WI-38 human diploid fibroblasts damaged with ultraviolet radiation or N-acetoxy-2-acetylaminofluroene. In contrast to a marked stimulation of DNA repair synthesis, stimulation of amino acid incorporation into histone polypeptides or into the various molecular weight classes of nonhistone nuclear proteins was not observed. These results suggest that detectable stimulation of newly synthesized nuclear protein incorporation into nuclei does not accompany DNA repair synthesis induced by ultraviolet radiation or a direct acting chemical carcinogen. At least for the special case of repair, DNA synthesis may be uncoupled from histone synthesis.
- Published
- 1976
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31. Four distinct membrane-bound dipeptidase RNAs are differentially expressed and show discordant regulation with gamma-glutamyl transpeptidase.
- Author
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Habib, G M, Barrios, R, Shi, Z Z, and Lieberman, M W
- Abstract
Membrane-bound dipeptidase (MBD) participates in the degradation of glutathione by cleaving the cysteinyl-glycine bond of cystinyl bisglycine (oxidized cysteinyl-glycine) following removal of a gamma-glutamyl group by gamma-glutamyl transpeptidase (GGT). In the mouse, MBD RNA is most abundant in small intestine, kidney, and lung and is represented by four distinct RNA species. These are generated by transcription from two promoters located 6 kilobases apart in the 5' flanking region of the gene and by the use of two different poly(A) addition sites. Promoter I is used primarily in small intestine and kidney, whereas promoter II is most active in lung and kidney. We found a discordance in the expected co-expression of MBD and GGT; as expected, MBD and GGT are both expressed at high levels in the kidney and small intestine. However, in the lung, MBD is expressed at high levels, whereas GGT is almost undetectable. The reverse is true in the seminal vesicles and fetal liver. Thus, although both enzymes may function in concert to metabolize glutathione in kidney and small intestine, in other tissues they appear to act independently, suggesting that they have independent roles in other biological processes.
- Published
- 1996
32. Recognition of chemical carcinogen-modified DNA by a DNA-binding protein.
- Author
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Moranelli, F and Lieberman, M W
- Abstract
Using a filter binding assay, we have detected and partially purified a protein from human placenta that has a high affinity for N-acetoxy-2-acetylaminofluorene-modified double-stranded DNA (AAF-[3H]DNA) of bacteriophage T7. This protein has been partially purified from a 1 M NaCl extract of a crude nuclear fraction by a combination of ion-exchange and nucleic acid affinity chromatography. With AAF-[3H]DNA as the substrate, the binding reaction reached equlibrium within 1 hr at 4 degrees C, and the extent of binding ws proportional to the amount of protein added. Complex formation was dependent on both pH and salt concentration and was unaffected by the presence of sulfhydryl-blocking agents. The purest protein fraction also recognizes DNA modified with methylmethane-sulfonate or methylnitrosourea. It shows little or no recognition of single-stranded DNA, double-stranded DNA, supercoiled bacteriophage phiX174 DNA, partially depurinated DNA, glucosylated bacteriophage T4DNA, or UV-irradiated DNA. No endo- or exonuclease activity, DNA polymerase activity, or glucosylase activity for AAF-DNA was detectable in the preparation.
- Published
- 1980
- Full Text
- View/download PDF
33. Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.
- Author
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Cohn, S M, Krawisz, B R, Dresler, S L, and Lieberman, M W
- Abstract
A marked induction of DNA replication was observed in confluent human diploid fibroblast cultures treated with low relatively nontoxic doses of UV radiation, N-methyl-N-nitrosourea (MNU), and N-acetoxy-2-acetylaminofluorene (AAAF). Isopycnic CsCl density gradient analysis of newly synthesized DNA labeled with BrdUrd indicated that most of the synthesis was semiconservative. The rate of semiconservative DNA synthesis was maximal 24 hr after damage. This induction of DNA replication was greatest at approximately equal to 3 J/m2 UV, 0.5 mM MNU, or 1.0 microM AAAF; was inhibited by hydroxyurea and aphidicolin; and also occurred in repair-deficient xeroderma pigmentosum fibroblasts. Autoradiographic examination of both confluent cultures and serum-arrested cultures showed a large increase in the fraction of densely labeled (S phase) cells after UV treatment. These densely labeled cells retain the capacity for cell division and subsequent proliferation. We conclude that low doses of at least three different DNA damaging agents are capable of recruiting quiescent cells into a state of DNA replication similar to that observed in the normal cell cycle.
- Published
- 1984
- Full Text
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34. Identification of a sixth promoter that directs the transcription of gamma-glutamyl transpeptidase type III RNA in mouse.
- Author
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Habib, G M, Carter, B Z, Sepulveda, A R, Shi, Z Z, Wan, D F, Lebovitz, R M, and Lieberman, M W
- Abstract
We have previously identified five promoters in the 5'-flanking region of the mouse gamma-glutamyl transpeptidase (gamma GT) gene. We now report the localization of a sixth promoter that supports the transcription of type III RNA, the major gamma GT RNA in fetal liver. We made a fetal liver cDNA library enriched for gamma GT RNA and obtained 12 gamma GT type III-specific clones. The longest clone is consistent with a transcription start site for type III RNA at a position 5' to the type IV promoter and about 5 kilobase(s) (kb) 5' to the first coding exon. We estimated by ribonuclease protection assay that about 80% of the gamma GT mRNA in fetal liver was type III. Primer extension and nuclease protection analyses mapped the 5' end of type III mRNA in fetal liver and kidney to a single cluster of potential major and minor transcription start sites. Deletion analysis using transient expression of chloramphenicol acetyltransferase constructs of the type III promoter region revealed the greatest activity with a 1-kb 5'-flanking fragment in mouse kidney proximal tubular cells and no detectable activity in NIH-3T3 fibroblasts. These studies demonstrate that the type III 5' region of the mouse gamma GT gene is organized into two distinct exons (IIIa and IIIb) and that type III RNA is expressed under the control of its own promoter.
- Published
- 1995
35. Requirement of ATP for specific incision of ultraviolet-damaged DNA during excision repair in permeable human fibroblasts.
- Author
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Dresler, S L and Lieberman, M W
- Abstract
Studies from several laboratories have shown that ATP is required for DNA excision repair in UV-irradiated mammalian cells. Using permeable human fibroblasts, we have investigated this ATP requirement in detail. We find that ATP is required for specific incision of UV-damaged DNA in permeable cells. No ATP-dependent incision is seen in UV-irradiated permeable xeroderma pigmentosum (complementation group G) fibroblasts, indicating that the ATP-dependent incision observed in normal cells is part of the normal excision repair process. We conclude that, in mammalian cells, ATP is required for specific incision of UV-damaged DNA or for some obligatory step preceding incision in the excision repair pathway. ATP also protects the permeable cells from loss of the capacity to perform excision repair, probably in a nonspecific fashion. The actual synthesis of repair patches can proceed in the absence of ATP; however, our data do not exclude the possibility that ATP can also stimulate repair synthesis directly.
- Published
- 1983
- Full Text
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36. Levels of DNA polymerases alpha, beta, and gamma in control and repair-deficient human diploid fibroblasts 1
- Author
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Parker, V P and Lieberman, M W
- Subjects
Xeroderma Pigmentosum ,DNA Repair ,Infant, Newborn ,Anemia, Aplastic ,DNA-Directed DNA Polymerase ,Syndrome ,Fibroblasts ,Cell Line ,Fanconi Anemia ,Caffeine ,Child, Preschool ,Humans ,Child ,Cells, Cultured - Abstract
The activities of DNA polymerases alpha, beta, and gamma were determined in control and repair-deficient human fibroblasts (xeroderma pigmentosum complementation groups A, C, and D; Fanconi's Anemia; and Bloom's syndrome). Assays were done on 103,000XG supernatants which had been chromatographed on DEAE cellulose to remove nucleic acids and on fractions containing polymerase activities which had been separated from one another on a second DEAE cellulose column. All repair-deficient cell types contained all three DNA polymerase activities. Caffeine, which has been observed to inhibit some DNA-repair processes in intact cells, had no effect on DNA polymerase activities from XP-A, XP-C, XP-D or XP-variant cells. These data indicate that all three polymerases are present in cells which have reduced or absent repair functions and that the caffeine effects observed in living cells are probably not due to the direct action of caffeine on DNA polymerases.
- Published
- 1977
37. Cell survival and changes in gene expression in cells unable to synthesize glutathione
- Author
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Emilio Rojas, Shi, Z. -Z, Valverde, M., Paules, R. S., Habib, G. M., and Lieberman, M. W.
38. Intragenomal Distribution of DNA Repair Synthesis: Repair in Satellite and Mainband DNA in Cultured Mouse Cells
- Author
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Lieberman, M. W., primary and Poirier, M. C., additional
- Published
- 1974
- Full Text
- View/download PDF
39. Gamma-glutamyl transpeptidase-deficient mice are resistant to the nephrotoxic effects of cisplatin.
- Author
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Hanigan MH, Lykissa ED, Townsend DM, Ou CN, Barrios R, and Lieberman MW
- Subjects
- Acetylcysteine pharmacology, Animals, Blood Urea Nitrogen, Body Weight, Creatinine blood, Drug Resistance, Female, Kidney metabolism, Kidney pathology, Mice, Mice, Inbred C57BL, Mice, Knockout genetics, Platinum metabolism, Platinum urine, Reference Values, gamma-Glutamyltransferase genetics, Antineoplastic Agents poisoning, Cisplatin poisoning, Kidney drug effects, gamma-Glutamyltransferase deficiency
- Abstract
We have proposed that the nephrotoxicity of cisplatin, a widely used chemotherapy drug, is the result of the binding of cisplatin to glutathione and the subsequent metabolism of the cisplatin-glutathione complex via a gamma-glutamyl transpeptidase (GGT)-dependent pathway in the proximal tubules. To test the hypothesis that GGT activity is essential for the nephrotoxicity of cisplatin, the effects of cisplatin were examined in wild-type and GGT-deficient mice. Mice were treated with 15 mg cisplatin/kg. Five days after treatment, renal histopathology, blood urea nitrogen levels, serum creatinine, platinum excretion, and platinum accumulation in the kidney were examined. Half the mice were supplemented with N-acetylcysteine, which has been shown to correct low levels of tissue glutathione in GGT-deficient mice. The data show that cisplatin was nephrotoxic in wild-type mice but not in GGT-deficient mice. The wild-type mice, with and without N-acetylcysteine supplementation, had significantly elevated levels of blood urea nitrogen, serum creatinine, and renal tubular necrosis. There was no evidence of nephrotoxicity in the GGT-deficient mice regardless of N-acetyl cysteine supplementation. No differences in platinum excretion were seen comparing wild-type and GGT-deficient mice, nor was there any significant difference in renal platinum accumulation. These data suggest that renal cisplatin toxicity is dependent on GGT activity, and is not correlated with uptake. The results support our hypothesis that the nephrotoxicity of cisplatin is the result of the metabolism of the drug through a GGT-dependent pathway.
- Published
- 2001
- Full Text
- View/download PDF
40. S-nitrosothiols signal the ventilatory response to hypoxia.
- Author
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Lipton AJ, Johnson MA, Macdonald T, Lieberman MW, Gozal D, and Gaston B
- Subjects
- Animals, Cell Hypoxia, Cysteine analogs & derivatives, Glutathione analogs & derivatives, Isoxazoles pharmacology, Mice, Oxygen blood, Rats, S-Nitrosoglutathione, gamma-Glutamyltransferase antagonists & inhibitors, Cysteine metabolism, Glutathione metabolism, Nitroso Compounds metabolism, Respiratory Physiological Phenomena, S-Nitrosothiols, Solitary Nucleus metabolism, Sulfhydryl Compounds metabolism, gamma-Glutamyltransferase metabolism
- Abstract
Increased ventilation in response to hypoxia has been appreciated for over a century, but the biochemistry underlying this response remains poorly understood. Here we define a pathway in which increased minute ventilation (&Vdot;E ) is signalled by deoxyhaemoglobin-derived S-nitrosothiols (SNOs). Specifically, we demonstrate that S-nitrosocysteinyl glycine (CGSNO) and S-nitroso-l-cysteine (l-CSNO)-but not S-nitroso-d-cysteine (d-CSNO)-reproduce the ventilatory effects of hypoxia at the level of the nucleus tractus solitarius (NTS). We show that plasma from deoxygenated, but not from oxygenated, blood produces the ventilatory effect of both SNOs and hypoxia. Further, this activity is mediated by S-nitrosoglutathione (GSNO), and GSNO activation by gamma-glutamyl transpeptidase (gamma-GT) is required. The normal response to hypoxia is impaired in a knockout mouse lacking gamma-GT. These observations suggest that S-nitrosothiol biochemistry is of central importance to the regulation of breathing.
- Published
- 2001
- Full Text
- View/download PDF
41. Disruption of gamma-glutamyl leukotrienase results in disruption of leukotriene D(4) synthesis in vivo and attenuation of the acute inflammatory response.
- Author
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Shi ZZ, Han B, Habib GM, Matzuk MM, and Lieberman MW
- Subjects
- Animals, Dipeptidases immunology, Gene Expression Regulation immunology, Gene Targeting, Leukotriene D4 biosynthesis, Leukotriene D4 immunology, Mice, Mutation, Dipeptidases genetics, Inflammation genetics, Leukotriene D4 genetics
- Abstract
To study the function of gamma-glutamyl leukotrienase (GGL), a newly identified member of the gamma-glutamyl transpeptidase (GGT) family, we generated null mutations in GGL (GGL(tm1)) and in both GGL and GGT (GGL(tm1)-GGT(tm1)) by a serial targeting strategy using embryonic stem cells. Mice homozygous for GGL(tm1) show no obvious phenotypic changes. Mice deficient in both GGT and GGL have a phenotype similar to the GGT-deficient mice, but approximately 70% of these mice die before 4 weeks of age, at least 2 months earlier than mice deficient only in GGT. These double-mutant mice are unable to cleave leukotriene C(4) (LTC(4)) to LTD(4), indicating that this conversion is completely dependent on the two enzymes, and in some organs (spleen and uterus) deletion of GGL alone abolished more than 90% of this activity. In an experimental model of peritonitis, GGL alone is responsible for the generation of peritoneal LTD(4). Further, during the development of peritonitis, GGL-deficient mice show an attenuation in neutrophil recruitment but not of plasma protein influx. These findings demonstrate an important role for GGL in the inflammatory response and suggest that LTC(4) and LTD(4) have distinctly different functions in the inflammatory process.
- Published
- 2001
- Full Text
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42. Oxygen-induced pulmonary injury in gamma-glutamyl transpeptidase-deficient mice.
- Author
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Barrios R, Shi ZZ, Kala SV, Wiseman AL, Welty SE, Kala G, Bahler AA, Ou CN, and Lieberman MW
- Subjects
- Animals, Cysteine metabolism, Glutamate-Cysteine Ligase metabolism, Hyperoxia etiology, Lung metabolism, Mice, RNA genetics, Acetylcysteine pharmacology, Glutathione physiology, Hyperoxia metabolism, Lung Injury, gamma-Glutamyltransferase deficiency
- Abstract
We used mice with a targeted disruption in g-glutamyl transpeptidase (GGT-deficient mice) to study the role of glutathione (GSH) in protection against oxygen-induced lung injury. These mice had reduced levels of lung GSH and restricted ability to synthesize GSH because of low levels of cysteine. When GGT-deficient mice were exposed to 80% oxygen, they developed diffuse pulmonary injury and died within eight days. Ten of 12 wild-type mice were alive after 18 days. Administration of N-acetylcysteine (NAC) to GGT-deficient mice corrected GSH values and prevented the development of severe pulmonary injury and death. Oxygen exposure induced an increase in lung GSH levels in both wild-type and GGT-deficient mice, but induced levels in the mutant mice were <50% of those in wild-type mice. Cysteine levels were approximately 50-fold lower than GSH levels the lungs of both wild-type and GGT-deficient mice. Levels of lung RNA coding for the heavy subunit of g-glutamyl cysteine synthetase rose three- to fourfold after oxygen exposure in both wild-type and GGT-deficient mice. In contrast, oxygen exposure failed to provoke increases in glutathione synthetase, glutathione peroxidase, glutaredoxin, or thioredoxin.
- Published
- 2001
- Full Text
- View/download PDF
43. Cataract development in gamma-glutamyl transpeptidase-deficient mice.
- Author
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Chévez-Barrios P, Wiseman AL, Rojas E, Ou CN, and Lieberman MW
- Subjects
- Acetylcysteine therapeutic use, Animals, Cataract drug therapy, Cataract etiology, Cysteine physiology, Electrophoresis, Free Radical Scavengers therapeutic use, Glutathione physiology, Lighting, Mice, Mice, Inbred C57BL, Cataract enzymology, gamma-Glutamyltransferase deficiency
- Abstract
The present study was undertaken to analyse the relationship of lens glutathione (GSH) and light to cataract development in mice deficient in gamma-glutamyl transpeptidase (GGT). These mice have reduced levels of cysteine and GSH in the eye and develop cataracts. GGT-deficient mice raised under normal vivarium conditions, showed no cataractous changes at birth, but by 1 week they had developed nuclear opacities. By 3 weeks more severe cataracts develop, and lens GSH levels are approximately 6-7% of wild type levels. By 6-11 weeks cataracts show nuclear and cortical involvement, liquefaction and calcification. Single cell DNA electrophoresis (comet assay) demonstrated mild DNA damage in the lens epithelium. GGT-deficient mice raised in the dark beginning the day after conception all developed cataracts, but these were less severe than those in GGT-deficient mice raised with normal vivarium lighting. Administration of N -acetyl cysteine (NAC) raises lens GSH and almost completely prevents cataract development. Our data indicate that cataract development in GGT-deficient mice is multifactorial and results from exogenous damage (exposure to light), reduced lens GSH levels, and nutritional effects secondary to low cysteine levels., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
44. Reproductive defects in gamma-glutamyl transpeptidase-deficient mice.
- Author
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Kumar TR, Wiseman AL, Kala G, Kala SV, Matzuk MM, and Lieberman MW
- Subjects
- Acetylcysteine pharmacology, Animals, Cysteine analysis, Female, Follicle Stimulating Hormone blood, Growth Disorders etiology, Growth Hormone blood, Infertility, Female pathology, Infertility, Female physiopathology, Infertility, Male pathology, Insulin-Like Growth Factor I analysis, Male, Mice, Mice, Knockout, Oligospermia etiology, Ovarian Follicle physiopathology, Ovary chemistry, Ovary pathology, Seminal Vesicles chemistry, Testis chemistry, Testis pathology, gamma-Glutamyltransferase genetics, gamma-Glutamyltransferase physiology, Infertility, Female etiology, Infertility, Male etiology, Reproduction, gamma-Glutamyltransferase deficiency
- Abstract
Mice deficient in gamma-glutamyl transpeptidase (GGT) are growth retarded as a result of cysteine deficiency secondary to excessive glutathione excretion in urine and display coat color defects and cataracts. Although GGT is widely expressed throughout the mouse reproductive axis, little is known about its role in reproduction. Here, we present an analysis of the reproductive phenotypes of GGT-deficient mice. Mutant male mice have reduced testis and seminal vesicle size and suppressed serum insulin-like growth factor I and FSH levels and are infertile. Although these mice are severely oligospermic, histological analysis of testes reveals grossly normal stages of spermatogenesis, including late stage spermatids, but the tubule diameter is reduced. GGT-deficient female mice are also hypogonadal and infertile. At 6 weeks of age, the ovaries of mutant mice are histologically indistinguishable from those of its wild-type counterpart. However, the absence of antral follicles and corpora lutea and follicular degeneration are apparent by 11-13 weeks. In addition, immature female mutant mice (at 21-23 days) are insensitive to exogenous gonadotropin administration and fail to superovulate, suggesting an intraovarian defect. Consistent with these mutant phenotypes, HPLC analysis of adult mutant testes and ovaries showed a reduction in intracellular cysteine levels. Administration of N-acetylcysteine in the drinking water beginning on day 21 to mutant mice for 2 weeks restored testis, seminal vesicle, and ovary sizes to values comparable to those in wild-type mice. Furthermore, N-acetylcysteine-fed (continuously) mutant male and female mice were fertile and produced normal numbers of offspring when mated to wild-type control mice. These results demonstrate that GGT itself is not necessary for reproductive function. However, GGT plays an important role in cysteine homeostasis within the mouse reproductive axis.
- Published
- 2000
- Full Text
- View/download PDF
45. The MRP2/cMOAT transporter and arsenic-glutathione complex formation are required for biliary excretion of arsenic.
- Author
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Kala SV, Neely MW, Kala G, Prater CI, Atwood DW, Rice JS, and Lieberman MW
- Subjects
- Animals, Anion Transport Proteins, Biological Transport, Magnetic Resonance Spectroscopy, Rats, Rats, Wistar, Arsenic metabolism, Arsenicals metabolism, Bile metabolism, Carrier Proteins physiology, Glutathione analogs & derivatives, Glutathione metabolism
- Abstract
Worldwide, millions of people are exposed to arsenic in drinking water that exceeds the World Health Organization standard of 10 microg/liter by as much as 50-300-fold, yet little is known about the molecular basis for arsenic excretion. Here we show that transport of arsenic into bile depends on the MRP2/cMOAT transporter and that glutathione is obligatory for such transport. Using reversed phase liquid chromatography/mass spectrometry, we demonstrate that two arsenic-glutathione complexes not previously identified in vivo, arsenic triglutathione and methylarsenic diglutathione, account for most of the arsenic in the bile. The structure of the compounds was also confirmed by nuclear magnetic resonance spectroscopy. Our findings may help explain the increased susceptibility of malnourished human populations to arsenic.
- Published
- 2000
- Full Text
- View/download PDF
46. gamma-glutamyltranspeptidase-deficient knockout mice as a model to study the relationship between glutathione status, mitochondrial function, and cellular function.
- Author
-
Will Y, Fischer KA, Horton RA, Kaetzel RS, Brown MK, Hedstrom O, Lieberman MW, and Reed DJ
- Subjects
- Adenosine Diphosphate analysis, Adenosine Triphosphate analysis, Adenosine Triphosphate biosynthesis, Animals, Glutathione analysis, Liver cytology, Liver ultrastructure, Mice, Mice, Knockout, Microscopy, Electron, Oxygen Consumption, Glutathione metabolism, Mitochondria, Liver physiology, gamma-Glutamyltransferase deficiency
- Abstract
gamma-Glutamyltranspeptidase (GGT)-deficient mice (GGT(-/-)) display chronic glutathione (GSH) deficiency, growth retardation, and die at a young age (<20 weeks). Using livers from these mice, we investigated the relationship between GSH content, especially mitochondrial, and mitochondrial and cellular function. We found that the GSH content of isolated liver mitochondria was diminished by >/=50% in GGT(-/-) mice when compared with wild-type mice. Respiratory control ratios (RCRs) of GGT(-/-) mice liver mitochondria were =60% those of wild-type mice primarily as a result of impaired state 3 respiration. Mitochondrial adenine nucleotide content was decreased by >/=40% in mitochondria obtained from GGT(-/-) mice. We observed a strong correlation between mitochondrial GSH content and RCRs. Even moderate decreases (<50%) correlated with adverse effects with respect to respiration. Electron microscopy revealed that livers from GGT(-/-) knockout mice were deprived of fat and glycogen, and swollen mitochondria were observed in animals that were severely deprived of GSH. Thus, GGT(-/-) mice exhibit a loss of GSH homeostasis and impaired oxidative phosphorylation, which may be related to the rate of adenosine triphosphate (ATP) formation and subsequently leads to progressive liver injury, which characterizes the diseased state. We also found that supplementation of GGT(-/-) mice with N-acetylcysteine (NAC) partially restored liver GSH, but fully restored mitochondrial GSH and respiratory function. Electron microscopy revealed that the livers of NAC-supplemented GGT(-/-) mice contained fat and glycogen; however, slightly enlarged mitochondria were found in some livers. NAC supplementation did not have any beneficial effect on the parameters examined in wild-type mice.
- Published
- 2000
- Full Text
- View/download PDF
47. Altered gene expression in the liver of gamma-glutamyl transpeptidase-deficient mice.
- Author
-
Habib GM, Shi ZZ, Ou CN, Kala G, Kala SV, and Lieberman MW
- Subjects
- ATP Binding Cassette Transporter, Subfamily B genetics, ATP-Binding Cassette Transporters genetics, Animals, Cystathionine gamma-Lyase genetics, Enzymes metabolism, Glutathione biosynthesis, Glutathione metabolism, Liver enzymology, Mice, Mice, Inbred C57BL, Oxidoreductases metabolism, Phosphorylation, Proto-Oncogene Proteins c-jun metabolism, RNA, Messenger metabolism, Stress, Physiological metabolism, Sulfhydryl Compounds metabolism, Gene Expression, Liver physiology, gamma-Glutamyltransferase deficiency
- Abstract
We used mice deficient in gamma-glutamyl transpeptidase (GGT) to analyze the effects of GGT deficiency and altered thiol levels on gene expression in liver. GGT-deficient mice have markedly reduced levels of glutathione (GSH), cysteine, methionine, and cysteinylglycine in liver. Steady-state RNA levels of the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis, are elevated 4-fold in these mice, while those for glutathione synthetase (GSH syn) are elevated 2-fold. RNA levels of cystathionase (cystathionine gamma-lyase), a key enzyme in the synthesis of cysteine from methionine, are elevated approximately 3.5-fold. In contrast, levels of RNA coding for multidrug resistance protein 2 (MRP2), which transports GSH into bile, are half wild-type values. We found no change in RNA levels of enzymes related to oxidative injury (CuZn and Mn superoxide dismutases [SOD], catalase, and glutathione peroxidase). Similarly, RNA levels of glutathione reductase and ribonucleotide reductase were unchanged. Furthermore, in contrast to previous in vitro results, methyl methanesulfonate did not induce stress-activated signal transduction as measured by c-jun phosphorylation in livers of GGT-deficient mice, despite further depletion of GSH by buthionine sulfoximine. Our findings indicate that GGT deficiency itself and/or altered thiol levels regulate expression of genes involved in GSH metabolism, but have no effect on the expression of other antioxidant genes.
- Published
- 2000
- Full Text
- View/download PDF
48. Glutathione synthesis is essential for mouse development but not for cell growth in culture.
- Author
-
Shi ZZ, Osei-Frimpong J, Kala G, Kala SV, Barrios RJ, Habib GM, Lukin DJ, Danney CM, Matzuk MM, and Lieberman MW
- Subjects
- Animals, Apoptosis, Blastocyst cytology, Blastocyst drug effects, Cell Division drug effects, Cell Line, Fetal Death, Gastrula physiology, Glutamate-Cysteine Ligase deficiency, Glutamate-Cysteine Ligase genetics, Glutathione deficiency, Glutathione pharmacology, Heterozygote, Homozygote, Mesoderm physiology, Mice, Mice, Knockout, Acetylcysteine pharmacology, Blastocyst physiology, Embryonic and Fetal Development, Glutamate-Cysteine Ligase metabolism, Glutathione biosynthesis
- Abstract
Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of gamma-glutamylcysteine synthetase (gammaGCS-HS(tm1)), an essential enzyme in GSH synthesis. Embryos homozygous for gammaGCS-HS(tm1) fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.
- Published
- 2000
- Full Text
- View/download PDF
49. Mouse leukotriene A4 hydrolase is expressed at high levels in intestinal crypt cells and splenic lymphocytes.
- Author
-
Habib GM, Cuevas AA, Barrios R, and Lieberman MW
- Subjects
- Amino Acid Sequence, Animals, DNA chemistry, DNA genetics, DNA, Complementary chemistry, DNA, Complementary genetics, Exons, Gene Expression Regulation, In Situ Hybridization, Intestine, Small cytology, Introns, Male, Mice, Mice, Inbred Strains, Molecular Sequence Data, RNA genetics, RNA metabolism, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Spleen cytology, Tissue Distribution, Epoxide Hydrolases genetics, Intestine, Small enzymology, Lymphocytes enzymology, Spleen enzymology
- Abstract
LTA4 hydrolase (EC 3.3.2.6) is a dual-function enzyme that is essential for the conversion of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) and also possesses an aminopeptidase activity. To characterize the expression of this unusual enzyme, we have cloned the mouse LTA4 hydrolase cDNA. The deduced amino acid sequence revealed 92% identity with the human sequence. Cloning and analysis of genomic sequences of mouse LTA4 hydrolase indicated that it is a single-copy gene spanning over 40kb and containing 20 exons. LTA4 hydrolase is widely expressed, with the highest levels of expression occurring in the small intestine, followed by the spleen. In situ hybridization revealed that LTA4 hydrolase is localized in the crypt cells of the small intestine, white pulp of the spleen, bronchiolar epithelium of the lung, myocardium, adrenal cortex, epithelium of the seminal vesicles, proximal tubules and the collecting ducts of the kidney, and occasional hepatocytes. Thus the widespread distribution of LTA4 hydrolase in various cell types in the tissues suggests that LTB4 may possess biological activities other than those known at present. It is also plausible that the widespread occurrence of LTA4 hydrolase in various tissues may correspond more with its function as an aminopeptidase than its function as an LTA4 hydrolase.
- Published
- 1999
- Full Text
- View/download PDF
50. Effect of growth temperature and vector ends phosphorylation in the cloning efficiency of specific DNA fragments.
- Author
-
Sepulveda AR and Lieberman MW
- Subjects
- Alkaline Phosphatase, Animals, DNA Probes, Deoxyribonucleases, Type II Site-Specific, Mice, Molecular Weight, Phosphorylation, Promoter Regions, Genetic genetics, Temperature, Cloning, Molecular methods, Plasmids genetics, gamma-Glutamyltransferase genetics
- Published
- 1999
- Full Text
- View/download PDF
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