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Proceedings of the National Academy of... Mar 1995Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo...
Acridine-induced frameshift mutagenesis in bacteriophage T4 has been shown to be dependent on T4 topoisomerase. In the absence of a functional T4 topoisomerase, in vivo acridine-induced mutagenesis is reduced to background levels. Further, the in vivo sites of acridine-induced deletions and duplications correlate precisely with in vitro sites of acridine-induced T4 topoisomerase cleavage. These correlations suggest that acridine-induced discontinuities introduced by topoisomerase could be processed into frameshift mutations. The induced mutations at these sites have a specific arrangement about the cleavage site. Deletions occur adjacent to the 3' end and duplications occur adjacent to the 5' end of the cleaved bond. It was proposed that at the nick, deletions could be produced by the 3'-->5' removal of bases by DNA polymerase-associated exonuclease and duplications could be produced by the 5'-->3' templated addition of bases. We have tested in vivo for T4 DNA polymerase involvement in nick processing, using T4 phage having DNA polymerases with altered ratios of exonuclease to polymerase activities. We predicted that the ratios of the deletion to duplication mutations induced by acridines in these polymerase mutant strains would reflect the altered exonuclease/polymerase ratios of the mutant T4 DNA polymerases. The results support this prediction, confirming that the two activities of the T4 DNA polymerase contribute to mutagenesis. The experiments show that the influence of T4 DNA polymerase in acridine-induced mutation specificities is due to its processing of acridine-induced 3'-hydroxyl ends to generate deletions and duplications by a mechanism that does not involve DNA slippage.
Topics: Aminacrine; Bacteriophage T4; Base Sequence; DNA Helicases; DNA, Viral; DNA-Directed DNA Polymerase; Escherichia coli; Exodeoxyribonuclease V; Exodeoxyribonucleases; Frameshift Mutation; Genes, Viral; Molecular Sequence Data; Mutagenesis; Viral Proteins
PubMed: 7892253
DOI: 10.1073/pnas.92.6.2234 -
The Journal of Biological Chemistry Oct 1988Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in...
Upon stimulation, the gastric parietal cell secretes a large quantity of isotonic HCl across its apical membrane which must be accompanied by the generation of base in the cytosol. The ability of this cell type to regulate cytosolic pH (pHi) was examined as a function of stimulation of acid secretion by histamine or forskolin. The pHi was estimated from the change of fluorescence of the trapped dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-bis-carboxyethylcarbo xy fluorescein in a purified cell suspension of rabbit parietal cells. Stimulation of the cell suspension raised pHi by an average of 0.13 +/- 0.038 pH units. The H+,K+-ATPase inhibitor, SCH28080 (2-methyl-8-[phenyl-methoxy]-imidazo-(1,2)-pyridine-3-acetonitrile) had only a small effect on the increase of pHi, therefore, was largely independent of H+,K+-ATPase activity. In Na+-free medium, where Na+/H+ exchange would be absent, the rise of pHi was only 0.03 pH units. This increase was blocked by SCH28080, showing that this small increment was the result of acid secretion. In Na+-containing medium, 90% of the increase was inhibited by an inhibitor of Na+/H+ exchange, dimethyl amiloride (DMA). This compound also blocked changes in pHi due to changes in extracellular Na+. Accordingly, most of the change in pHi upon stimulation of acid secretion by histamine and forskolin is due to activation of Na+/H+ exchange in the parietal cell basal-lateral membrane. The addition of DMA to stimulated, but not resting cells, gave a rapid acidification that was blocked by inhibition of anion exchange by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), showing that anion exchange was also activated by stimulation. In single cell recording, canalicular and cytosolic pH were monitored simultaneously using 9-amino acridine and dimethyl carboxyfluorescein, respectively. Cytosolic alkalinization correlated with acid accumulation in the secretory canaliculus until a set point was reached. Thereafter, acidification continued without further change in pHi. To determine the role of Na+/H+ and Cl-/HCO3- exchange in acid secretion, Cl(-)-depleted cells were suspended in medium containing 40 mM Cl-. DMA and DIDS each blocked acid secretion by about 40%, but in combination, acid secretion was blocked by more than 90%. Thus, basal-lateral Na+/H+ and Cl-/HCO3- exchange activities are necessary for acid secretion across the apical membrane of the parietal cell.
Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Aminacrine; Animals; Anti-Ulcer Agents; Bicarbonates; Cell Membrane; Chlorides; Cimetidine; Cytosol; Gastric Acid; Histamine; Hydrogen-Ion Concentration; Imidazoles; In Vitro Techniques; Kinetics; Parietal Cells, Gastric; Rabbits; Sodium
PubMed: 3170561
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1986The ability of 9-aminoacridine to induce mutagenic lesions during DNA replication in vitro was investigated. The ampicillinase gene of pBR322 was replicated in vitro in...
The ability of 9-aminoacridine to induce mutagenic lesions during DNA replication in vitro was investigated. The ampicillinase gene of pBR322 was replicated in vitro in the presence of 9-aminoacridine. Transfection of the replicated DNA into Escherichia coli gave Amps mutants. Determination of the base changes in 76 of these mutants indicated that the spectrum of mutations induced by 9-aminoacridine was consistent with its action in vivo. Both large (407-base) and small (1- and 2-base) deletions were induced at repetitive sequences. The frequency of deletion mutations depended on the identity of the base deleted and sequences surrounding the deletions. The characteristics of the frameshift mutations induced were consistent with the interactions of 9-aminoacridine with DNA. These results establish that 9-aminoacridine can induce frameshift mutations during the replication process and provide an in vitro model of frameshift induction for mechanistic studies.
Topics: Aminacrine; Aminoacridines; Base Sequence; Chromosome Deletion; DNA; DNA Replication; In Vitro Techniques; Mutation
PubMed: 3782116
DOI: No ID Found -
BMC Cancer Jun 2010Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of...
BACKGROUND
Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells.
METHODS
We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT).
RESULTS
We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406) targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512) targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping) and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone.
CONCLUSION
We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.
Topics: Aminacrine; Blotting, Western; Cell Proliferation; Exons; Humans; Introns; Mutagens; Peptide Nucleic Acids; Proto-Oncogene Proteins c-mdm2; RNA Precursors; RNA Splicing; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 20591158
DOI: 10.1186/1471-2407-10-342 -
Biophysical Journal Jun 1992A new general method for the determination of electrostatic potentials at biological surfaces is presented. The approach is based on measurement of the collision...
A new general method for the determination of electrostatic potentials at biological surfaces is presented. The approach is based on measurement of the collision frequency of a charged nitroxide in solution with a nitroxide fixed to the surface at the point of interest. The collision frequency is determined with 14N:15N double label electron-electron double resonance (ELDOR). As a test, the method is shown to give values for phospholipid bilayer surface potentials consistent with the Gouy-Chapman theory, a simple model shown by many independent tests to accurately describe charged, planar surfaces. In addition, the method is applied to determine the electrostatic potential near the surface of DNA. The results indicate that the potential is significantly smaller than that predicted from Poisson-Boltzmann analysis, but is in qualitative agreement with that predicted by Manning's theory of counter ion condensation. The method is readily extended to measurement of surface potentials of proteins.
Topics: Aminacrine; DNA; Electrochemistry; Electron Spin Resonance Spectroscopy; Mathematics; Membrane Potentials; Models, Molecular; Models, Theoretical; Molecular Conformation; Nucleic Acid Conformation; Phosphatidylcholines; Phosphatidylglycerols; Proteins; Spin Labels
PubMed: 1319760
DOI: 10.1016/S0006-3495(92)81950-1 -
Analytical Chemistry Oct 2008A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based approach was developed for the rapid analyses of cellular...
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions.
A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based approach was developed for the rapid analyses of cellular glycerophospholipids. Through multiplexed solvent-enabled optimization of analyte-matrix interactions during the crystallization process, over a 30-fold increase in S/N was achieved using 9-aminoacridine as the matrix. The linearity of response (r(2) = 0.99) and dynamic range of this method (over 2 orders of magnitude) were excellent. Moreover, through multiplexing ionization conditions by generating suites of different analyte-matrix interactions in the absence or presence of different alkali metal cations in the matrix, discrete lipid classes were highly and selectively ionized under different conditions resulting in the de facto resolution of lipid classes without chromatography. The resultant decreases in spectral complexity facilitated tandem mass spectrometric analysis through high energy fragmentation of lithiated molecular ions that typically resulted in informative fragment ions. Anionic phospholipids were also detected as singly negatively charged species that could be fragmented using MALDI tandem mass spectrometry leading to structural assignments. Collectively, these results identify a rapid, sensitive, and highly informative MALDI-TOF MS approach for analysis of cellular glycerophospholipids directly from extracts of mammalian tissues without the need for prior chromatographic separation.
Topics: Aminacrine; Animals; Cardiolipins; Crystallization; Glycerophospholipids; Male; Mice; Mice, Inbred C57BL; Myocardium; Phosphatidylcholines; Solvents; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tissue Extracts; Triglycerides
PubMed: 18767869
DOI: 10.1021/ac801200w -
Proceedings of the National Academy of... Apr 1984We investigated the effects of the well-known mutagenic agents ethyl methanesulfonate (EtMes), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and ICR-191 on colonies of the...
We investigated the effects of the well-known mutagenic agents ethyl methanesulfonate (EtMes), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), and ICR-191 on colonies of the Chinese hamster ovary line CHO cultured on a semisolid substrate. These agents induced heterogeneity in diameter and integrated optical density of colonies as determined by computer-assisted photography and subsequent analysis of the images of the colonies. When CHO colonies were exposed to agents such as urethane that are not known to be mutagenic in mammalian systems or to activation-requiring mutagens such as cyclophosphamide, there was no noticeable effect on the distribution of colony diameter and volume. Similarly, nonmutagenic agents such as dimethyl sulfoxide (Me2SO) also did not induce heterogeneity in colony diameter and integrated optical density. Our observations recommend the use of agar-grown mammalian cell colonies for predictive testing of chemical mutagens and carcinogens in a simple, in vitro mammalian cell assay. This assay system, unlike other mammalian cell culture assays, allows detection and measurement of the simultaneous effects of chemical mutagens on several genetic and non-genetic targets and, thus, may emulate more closely the potential hazards of these agents in vivo.
Topics: Aminacrine; Animals; Cell Division; Cell Line; Cell Survival; Clone Cells; Cricetinae; Cricetulus; Cyclophosphamide; Dimethyl Sulfoxide; Ethyl Methanesulfonate; Female; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutagens; Mutation; Nitrogen Mustard Compounds; Ovary; Urethane
PubMed: 6585791
DOI: 10.1073/pnas.81.7.2112 -
The Biochemical Journal Feb 19811. The addition of chelators to a suspension of mitochondria in a low-cation medium containing 9-aminoacridine caused a decrease in 9-aminoacridine fluorescence. The...
1. The addition of chelators to a suspension of mitochondria in a low-cation medium containing 9-aminoacridine caused a decrease in 9-aminoacridine fluorescence. The chelators removed bivalent cations from the membranes and allowed more 9-aminoacridine to move into the diffuse layer. The relative effect of EGTA and EDTA on the fluorescence suggested that the mitochondria are isolated with about equal amounts of Ca2+ and Mg2+ on the membranes. 2. The removal of the bivalent ions by chelators resulted in the inhibition of NADH oxidation. The inhibition could not be removed by adding sufficient decamethylenebistrimethylammonium ion (DM2+) to screen the fixed charges on the membranes and restore the fluorescence of 9-aminoacridine. This observation suggests that bivalent metal ions have a specific role in the oxidation of NADH. 3. Ca2+ and not Mg2+ reversed the inhibition of NADH oxidation caused by EGTA, whereas both reversed the inhibition caused by EDTA. This suggests that Ca2+ plays a specific role and that Mg2+ reverses the inhibition caused by EDTA by displacing the bound calcium from the chelator. 4. The results are interpreted as showing that Ca2+ plays a specific role in the oxidation of external NADH in addition to its ability to screen electrostatically or bind to the fixed charges associated with the surface of the membrane.
Topics: Aminacrine; Calcium; Edetic Acid; Egtazic Acid; Helianthus; Magnesium; Mitochondria; NAD; Oxidation-Reduction
PubMed: 6796061
DOI: 10.1042/bj1940487 -
Molecular and Cellular Biology Aug 1991Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlled by the upstream region of interferon...
Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlled by the upstream region of interferon (IFN)-stimulated human gene 6-16. After mutagenesis of 2fTGH and selection with 6-thioguanine and IFN-alpha, we isolated 11.1, a recessive mutant that does not respond to IFN-alpha. We now describe U2, a second recessive mutant, selected similarly, that complements 11.1. U2 had no response to IFN-alpha or IFN-beta, and its response to IFN-gamma was partially defective. Although many genes did respond to IFN-gamma in U2, the 9-27 gene did not and the antiviral response of U2 cells to IFN-gamma was greatly reduced. Band shift assays showed that none of the transcription factors normally induced in 2fTGH cells by IFN-alpha (E and M) or IFN-gamma (G) were induced in U2. However, extracts of untreated U2 cells gave rise to a novel band that was increased by treatment with IFN-gamma but not IFN-alpha. Band shift complementation assays revealed that untreated and IFN-gamma-treated U2 cells lack the functional E gamma subunit of transcription factor E and that IFN-alpha-treated U2 cells do contain the functional E alpha subunit.
Topics: Aminacrine; Cell Fusion; Cell Line; Genes, Recessive; Genetic Complementation Test; Humans; Interferon Type I; Interferon-gamma; Mutagenesis; Mutagens; Nitrogen Mustard Compounds; RNA, Messenger; Thioguanine; Transcription, Genetic
PubMed: 1906577
DOI: 10.1128/mcb.11.8.4189-4195.1991 -
Nucleic Acids Research Feb 1990Theoretical computations are performed of the comparative binding affinities of five polymethylene carboxamide derivatives of 9-aminoacridine to a series of...
Theoretical computations are performed of the comparative binding affinities of five polymethylene carboxamide derivatives of 9-aminoacridine to a series of double-stranded hexanucleotides. The purpose of this investigation is to ascertain whether minor groove recognition of a guanine base adjacent to the intercalation site can occur, and be preferentially stabilized, for a given length of the polymethylene side chain, encompassing from n = 2 up to n = 6 methylene groups. For that purpose, several representative sequences were investigated, in which intercalation of the 9-aminoacridine chromophore occurred at a central d(CpG) or d(TpA) step. Investigated were the self-complementary sequences d(CGCGCG)2, d(GCCGGC)2, d(TATATA)2 and d(ATTAAT)2, as well as the 'mixed' sequences d(ACTAAT) .d(ATTAGT) and d(TGTATA). d(TATACA). For n = 3 up to n = 6, such a recognition was enabled only when the guanine base was located downstream of the intercalation site, i.e. with steps d(CGG) and d(TAG). It occurred by means of a bidentate interaction involving, on the one hand, H(N2) and N3 of the base, and, on the other hand, the carbonyl oxygen and the cis amino hydrogen of the terminal formamide moiety of the ligand. Because of the flexibility of the side chain, however, alternative binding modes were also found to occur competitively, involving backbone-only interactions of the side chain. On the basis of the present computations, upon binding to the sequence d(GCCGGC)2, an optimal value of n = 5 could be derived, with the corresponding acridine derivative eliciting both a significant prevalence of the bidentate over backbone only binding mode, and the most favourable energy balance within the investigated series. This privileged value of n = 5 is fully consistent with the experimental results of Markovits et al. and Gaugain et al. The very flexibility of the side chain, however, hampered any preferential recognition of a triplet sequence with a downstream guanine, such as d(CGG) or d(TAG), to be elicited over sequences such as d(TAA), d(TAT) or d(TAC).
Topics: Aminacrine; Aminoacridines; Base Sequence; Chemical Phenomena; Chemistry; Intercalating Agents; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides
PubMed: 2315037
DOI: 10.1093/nar/18.4.711