Identification of putrescine-responsive mRNAs in Chinese hamster ovary cells using representational difference analysis.
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Animals↗
Biomedical subjects
L Hawel
Publications and source records attributed to L Hawel.
Optimization of cDNA representational difference analysis for the identification of differentially expressed mRNAs.
Representational difference analysis (RDA) is a powerful and sensitive tool for identification of differentially expressed genes (M. Hubank and D. G. Schatz, 1999, Methods Enzymol. 303, 325-349; 1994, Nucleic Acids Res. 22, 5640-5648) that will identify both up- and downregulated genes differentially expressed between two cDNA populations. This manuscript provides a thorough description of an optimized RDA method. This procedure while still based on the traditional RDA originally developed by Lisitsyn and co-workers(N. A. Lisitsyn, 1995, Trends Genet. 11, 303-307; N. A. Lisitsyn, F. S. Leach, B. Vogelstein, and M. H. Wigler, 1994, Cold Spring Harbor Symp. Quant. BioL 59, 585-587; N. Lisitsyn, N. Lisitsyn, and M. Wigler, 1993, 259, 946-951) and modified by Hubank and Schatz for RNA (1994, Nucleic Acids Res. 22, 5640-5648) is improved and requires less starting material than many existing methods. Several key modifications are included (1). Size-exclusion gel-filtration microspin columns are used throughout the procedure to remove the primers and low molecular weight cDNAs. This results in reducing the number of ethanol precipitations required and in improving the yield of desirable amplification products (2). Elimination of the mung bean nuclease treatment in favor of a simple dilution of PCR serves as a means of markedly reducing the single-stranded cDNAs that can interfere with the amplification of differentially expressed products (3). The use of up to six unique noninteracting primers ensures that no anomalous amplification occurs due to carryover of primers or incomplete digestion from the ends of the cDNAs (4). A set of cDNA standards was developed and various concentrations were used to better characterize the ability of representational difference analysis to identify rare messages in a complex cDNA population (5). Integral to this manuscript, a detailed laboratory protocol is available from the authors (craig.byus@ucr.edu) and provides a step-by-step description of the modified procedure.
Base Sequence↗
Characterization of putrescine and cadaverine export in mammalian cells. A pharmacological approach.
We characterized the mechanism(s) involved in the efflux of putrescine/cadaverine from cultured mammalian cells using various pharmacological agents. Verapamil and quinine inhibited putrescine and cadaverine export in monocytic-leukemic RAW 264 and H35 hepatoma cells in a concentration-dependent manner with an IC50 in the micromolar range. Verapamil, which inhibits L-type calcium channels, inhibited putrescine export, regardless of whether calcium was present in the extracellular medium or not. Furthermore, the export of putrescine in the absence of verapamil did not appear to depend upon extracellular calcium. Neither intracellular calcium, external sodium, changes in intracellular pH nor phosphorylation affected the levels of putrescine export independently from changes in intracellular putrescine levels. The data suggest that verapamil and quinine inhibit putrescine/cadaverine efflux from the cell by binding directly to an integral membrane protein.
Animals↗
Selective putrescine export is regulated by insulin and ornithine in Reuber H35 hepatoma cells.
Cultured Reuber H35 rat hepatoma cells under highly viable serum-free conditions were found to selectively export putrescine from inside the cell into the culture medium, but not spermidine, spermine, or their acetylated derivatives. Even untreated cells, with very low intracellular putrescine levels, constitutively exported significant amounts of only putrescine for a 12 h period. Administration of the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) which markedly elevates ornithine decarboxylase (ODC), did not potentiate putrescine export over what was measured in the unstimulated cultures. However, addition of 1 mM ornithine to the cultures resulted in increased intracellular putrescine (maximum at 4 h) with a marked concomitant increase in putrescine export between 0 and 8 h, after which putrescine export again stopped. Treatment with 10(-7) M insulin yielded intracellular putrescine levels that remained elevated for 36 along with a continuous and more rapid export of putrescine over the same 36 h time period. When insulin and ornithine were administered together, even higher levels of intracellular putrescine and putrescine export were observed, with putrescine efflux proceeding over the 36 h time-course at the highest observed rates of 1.5 (0-12 h) and 1.0 (12-36 h) nmol/mg total protein per h. Exposure to DFMO, an inhibitor of ODC, depleted intracellular putrescine stores and effectively suppressed putrescine export. There was not a positive correlation between the time-dependent decreases in the intracellular putrescine concentrations and the respective alterations in the rate of putrescine export under a variety of conditions. Furthermore, the drug verapamil was capable of completely inhibiting putrescine export (IC50 approx. 1 microM) without any change in the level of intracellular putrescine. This data was not consistent with the involvement of simple diffusion of putrescine through the membrane as the major mechanism for putrescine export. The potential mechanisms involved in putrescine export and the role of this process in regulating intracellular polyamine levels, as well as, possible functions of extracellular putrescine are discussed.
Animals↗
Regulation of putrescine export in lipopolysaccharide or IFN-gamma-activated murine monocytic-leukemic RAW 264 cells.
The regulation of putrescine/polyamine export out of the cell was investigated during activation of monocytic-leukemic RAW 264 cells with LPS and IFN-gamma. The RAW 264 cells exported putrescine constitutively at a significant rate into the culture medium. This export process appeared to be selective for putrescine in that only a small amount of other polyamines (spermidine and N1-acetylspermidine) was found in the culture medium. LPS and IFN-gamma alone and in combination markedly stimulated putrescine export and nitrite production throughout a 24-h period. The efflux of putrescine but not nitrite was further increased by the addition of ornithine (the amino acid precursor of putrescine) to the culture medium. LPS and ornithine also stimulated the intracellular accumulation of putrescine in primary inflammatory macrophages and the export of putrescine into the peritoneal exudate of the mouse. A detailed comparison of the steady state rates of accumulation of intracellular putrescine/polyamines and the rate of putrescine efflux from the cells constitutively and after LPS, IFN-gamma, and ornithine indicated that a surprisingly large fraction of total polyamine biosynthesis is comprised of exported putrescine. The observed dose-dependent inhibition of putrescine export with the drug verapamil implicated the involvement of a specific membrane transport system sensitive to calcium influx in this process. The data are discussed in regard to the potential involvement of putrescine export in the regulation of intracellular polyamine levels, cell differentiation, and macrophage-mediated cytotoxicity.
8-Bromo Cyclic Adenosine Monophosphate↗
Biosynthesis and selective export of 1,5-diaminopentane (cadaverine) in mycoplasma-free cultured mammalian cells.
Macrophage-like RAW 264 and H35 hepatoma cells grown under serum-free conditions exported putrescine and an unidentified diamine into the culture medium. Unlike putrescine, the unknown compound could be detected only extracellularly. Analyses of dansylated polyamine standards and mass spectroscopy confirmed that the unknown compound was cadaverine (1,5-diaminopentane). The cells were free of mycoplasma as evidenced by a negative result using a probe specific for prokaryotic rRNA. After prophylactic treatments with two different mycoplasmacidal agents, the cells continued to export cadaverine. Attempts to "infect" a noncadaverine-exporting cell line with culture medium and cell-free lysates proved unsuccessful, establishing that cadaverine was in fact a bona fide product of these mammalian cells. Cadaverine export by RAW 264 and H35 cells was stimulated by lipopolysaccharide and insulin, respectively. However, administration of exogenous ornithine caused cadaverine export to decrease significantly with concomitant increases in putrescine export. alpha-Difluoromethylornithine, a selective inhibitor of ornithine decarboxylase, inhibited both cadaverine and putrescine export. When cells were labeled with [3H]lysine, the great majority of the radioactivity recovered in exported polyamines was found in cadaverine. The cumulative data suggested that cadaverine formation may be caused by the action of intracellular ornithine decarboxylase upon lysine to produce cadaverine, which is then effluxed from the cell with a high degree of efficiency.
Amino Acids↗
An ion-exchange chromatography procedure for the isolation and concentration of basic amino acids and polyamines from complex biological samples prior to high-performance liquid chromatography.
The original objective of this study was to develop a selective and sensitive method for the analysis and quantification of basic amino acids from biological samples via reversed-phase high-performance liquid chromatography. Using various previously described techniques for the separation of amino acids, we were unsuccessful in measuring levels of histidine, arginine, ornithine, and lysine in biological samples due to the presence of interfering compounds. A "cleanup" procedure for the isolation of the basic amino acids using a weakly acidic cation exchange resin, Biorex-70 (Bio-Rad), is described in detail. Upon separation from the bulk of the neutral and acidic amino acids, the basic amino acids were subjected to precolumn fluorescence derivatization using 9-fluorenylmethyl chloroformate (FMOC) and the fluorescent derivatives were separated by RP-HPLC. The advantages of this method over previously described amino acid analysis techniques are (i) isolation and stable recovery (greater than 95%) of the desired basic amino acids, (ii) sensitivity of detection (low pmol range), (iii) complete resolution of derivatized amino acids via HPLC, (iv) limited amount of sample required for analysis, and (v) samples readily concentrated by lyophilization or rotoevaporating. This ion-exchange cleanup procedure was also adapted for the analysis of polyamines in concentrated culture media samples and proved additionally advantageous by eliminating the use of costly C-18 extraction columns required by previously described techniques.
Amino Acids, Diamino↗