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The Biochemical Journal Jan 1985Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized...
Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Aminacrine; Animals; Asialoglycoproteins; Ca(2+) Mg(2+)-ATPase; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Centrifugation, Density Gradient; In Vitro Techniques; Liver; Monensin; Protons; Rats; Subcellular Fractions; Transferrin
PubMed: 2983664
DOI: 10.1042/bj2250051 -
Bioorganic & Medicinal Chemistry Letters Aug 2009The cytotoxicity and mechanism of action of a series of substituted 9-aminoacridines is evaluated using topoisomerase I and cancer cell growth inhibition assays. In...
The cytotoxicity and mechanism of action of a series of substituted 9-aminoacridines is evaluated using topoisomerase I and cancer cell growth inhibition assays. In previous work, compounds of this type were shown to catalytically inhibit topoisomerase II, leading to a G1-S phase arrest of the cell cycle and apoptosis in pancreatic cancer cells in vitro and in vivo. The present study expands the potential utility of these compounds in the development of cancer therapeutics by showing that these compounds inhibit proliferation of cell lines derived from the nine most common human cancers. Further results show that at least one of the compounds effectively stabilizes topoisomerase I-DNA adduct formation in intact cells. RNA interference experiments, however, indicate that this interaction does not contribute to the drug-induced killing of cancer cells indicating the compounds may be non-lethal poisons of topoisomerase I.
Topics: Acridines; Aminacrine; Antineoplastic Agents; Cell Line; Chemistry, Pharmaceutical; DNA Topoisomerases, Type I; Dose-Response Relationship, Drug; Doxorubicin; Drug Design; Drug Screening Assays, Antitumor; Etoposide; Humans; Models, Chemical; Neoplasms; RNA, Small Interfering
PubMed: 19501511
DOI: 10.1016/j.bmcl.2009.05.037 -
European Journal of Biochemistry Nov 1981The response of human platelets to stimulation by a specific aggregant such as thrombin has been postulated to proceed sequentially via induction of response at the... (Comparative Study)
Comparative Study
The response of human platelets to stimulation by a specific aggregant such as thrombin has been postulated to proceed sequentially via induction of response at the membrane, followed by execution of shape change, secretion, and aggregation of the platelets. We have shown earlier that the platelet response includes a depolarization of the membrane which starts within less than 5 s and is thrombin-dose-dependent up to 4.5 nM alpha-thrombin. This depolarization may be measured by the distribution of either a fluorescent or a tritium-labeled lipophilic cation. We present here an adaptation of techniques for intracellular pH measurements to the human platelet. These show that stimulation with thrombin also induces a rapid change in the platelet transmembrane pH gradient as measured using either a weak base or a fluorescein derivative as a probe. The pH gradient undergoes a time-dependent and thrombin-dose-dependent change which parallels that exhibited by the membrane potential and by serotonin secretion.
Topics: Aminacrine; Blood Platelets; Cytoplasm; Fluoresceins; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Potentials; Platelet Aggregation; Serotonin; Thrombin
PubMed: 7318826
DOI: 10.1111/j.1432-1033.1981.tb05703.x -
The Journal of Biological Chemistry Jan 1995Previous studies have shown that proteins are transported across the chloroplast thylakoid membrane by two very different mechanisms, one of which requires stromal... (Comparative Study)
Comparative Study
Previous studies have shown that proteins are transported across the chloroplast thylakoid membrane by two very different mechanisms, one of which requires stromal factors and ATP, whereas the other mechanism is ATP independent but completely reliant on the thylakoidal delta pH. We have examined the role of the delta pH in the latter mechanism by simultaneously monitoring the magnitude of delta pH (by 9-aminoacridine fluorescence quenching) and the rate of import of the 23-kDa photosystem II protein into isolated pea thylakoids. We show that protein import can take place, at low but significant rates, at very low values of delta pH (in the region of 1.2-1.4), and that plots of the rate of protein import against proton concentration gradient are probably hyperbolic in nature. There is no evidence for a threshold level of delta pH which is required to drive translocation of the 23-kDa protein. Addition of uncouplers midway during import incubations results in a rapid and complete inhibition of translocation, showing that the continuous presence of the delta pH is required for translocation to take place. During import into intact chloroplasts, the intermediate-size 23-kDa protein substrate for the thylakoidal protein transport machinery is found only in the stromal fraction at all values of delta pH, suggesting that the initial interaction with the machinery is relatively weak, reversible and delta pH-independent. We therefore propose that the delta pH is required for both the initiation and completion of translocation; these roles are in marked contrast to the roles of protonmotive force in mitochondrial and sec-dependent bacterial protein transport.
Topics: Adenosine Triphosphate; Aminacrine; Bacterial Proteins; Chloroplasts; Fabaceae; Fluorescent Dyes; Hydrogen-Ion Concentration; Intracellular Membranes; Kinetics; Plant Proteins; Plants, Medicinal; Thermodynamics
PubMed: 7829499
DOI: 10.1074/jbc.270.4.1657 -
Biochimica Et Biophysica Acta Apr 1995During growth a cyclic exposure of anionic phospholipids to the external surface of the plasma membrane was found. The surface charge density (sigma) increased gradually...
Generation, modulation and maintenance of the plasma membrane asymmetric phospholipid composition in yeast cells during growth: their relation to surface potential and membrane protein activity.
During growth a cyclic exposure of anionic phospholipids to the external surface of the plasma membrane was found. The surface charge density (sigma) increased gradually reaching a maximum in the first 5 h of growth and returned gradually to their initial value at the end of the logarithmic phase of growth (10-12 h). Phosphatidylinositol, that determines to a large extent the magnitude of the sigma, increased 83% in the yeast cells during the first 4 h of growth and returned gradually to their initial level at 10-12 h. During the stationary phase (12-24 h), both sigma and the anionic/zwitterionic phospholipid ratio, remained without any significant variation. The high-affinity H-linked glutamate transport system that behaves as a sensor of the changes in the membrane surface potential (phi) increased its activity in the first 5 h and then decreased it, following with great accuracy the sigma variations and remained without changes during the stationary phase of growth. The phosphatidylserine (PS) relative concentration in the cells (9.0%) did not significantly change during the whole growth curve, but their asymmetric distribution varied, contributing to the changes in sigma. PS facing the outer membrane surface increased 2.45-times during the first 5 h of growth and then returned to their original value at the end of the log phase (12 h). Phosphatidylcholine (PC) remained constant during the whole growth curve (50%), while phosphatidylethanolamine (PE) decreased 3-fold in the first 4 h and then increased to its original value at 10 h. Interestingly, PE at the outer membrane surface remained constant (3% of the total phospholipids) during the whole growth curve. During growth yeast cells change their phospholipid composition originating altered patterns of the plasma membrane phospholipid composition and IN-OUT distribution. This dynamic asymmetry is involved in the regulation of the surface potential and membrane protein activity.
Topics: Aminacrine; Cell Membrane; Glutamates; Membrane Lipids; Membrane Potentials; Membrane Proteins; Phospholipids; Saccharomyces cerevisiae; Time Factors
PubMed: 7718598
DOI: 10.1016/0005-2736(94)00311-c -
Journal of Radiation Research Sep 2007The endogenous tonB gene of Escherichia coli was used as a target for 9-aminoacridine-induced mutations that were identified in recA(-) and uvrA(-) cells. The...
The endogenous tonB gene of Escherichia coli was used as a target for 9-aminoacridine-induced mutations that were identified in recA(-) and uvrA(-) cells. The cytotoxicity of 9-aminoacridine was enhanced in the uvrA and recA strains compared to the wild-type strain, and the mutagenicity of 9-aminoacridine in the uvrA and recA strains was similar to that in the wild type. For all three strains, the most common mutations were minus frameshifts in repetitive G:C base-pairs followed by minus frameshifts in nonrepetitive G:C base-pairs. 9-aminoacridine-induced minus frameshifts in the wild-type strain were distributed with several hot and warm spots. These sites were also hot and warm spots for minus frameshifts in the recA and uvrA stains. Furthermore, they were hot and warm sites in a 9-aminoacridine-treated strain carrying the target tonB gene oriented in the opposite direction. 9-Aminoacridine is known to interact with DNA to form intercalations which are involved in minus frameshift mutagenesis. In this study, we therefore argue that 1) 9-aminoacridine can induce bulky DNA lesions which are excised by nucleotide excision repair and not involved in mutagenesis, 2) the presence or absence of a recA-dependent repair pathway does not influence the mutagenic effect of 9-aminoacridine, and 3) both leading strand and lagging strand replication equally produce minus frameshifts, therefore gene orientation is not an important determinant of the formation of hot and warm spots by 9-aminoacridine.
Topics: Aminacrine; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Escherichia coli; Frameshift Mutation; Radiation Tolerance; Rec A Recombinases; Ultraviolet Rays
PubMed: 17611351
DOI: 10.1269/jrr.07036 -
Nucleic Acids Research Apr 1980We have quantitatively examined the unwinding angles for the complexes of a related series of acridine and quinoline derivatives with DNA. Ethidium bromide was used as a...
We have quantitatively examined the unwinding angles for the complexes of a related series of acridine and quinoline derivatives with DNA. Ethidium bromide was used as a control for determining superhelix densities at different ionic strengths. Relative to ethidium, 9-aminoacridine and quinacrine had an essentially constant unwinding angle of approximately 17 degrees at all ionic strengths tested. The apparent unwinding angle for chloroquine and 9-amino-1,2,3,4-tetrahydroacridine was found to be ionic strength dependent, increasing with increasing ionic strength. This suggests that competitive nonintercalative binding at low ionic strengths causes an apparent lowering of the quinoline unwinding angle. This can also explain why 4-aminoquinaldine, examined at low ionic strength, gives a quite low apparent unwinding angle. Quinacrine along with chloroquinine and 9-aminoacridine approaches a limiting value for their unwinding angle of approximately 17 degrees. 4-aminoquinaldine and 9-amino-1,2,3,4-tetrahydroacridine could not be examined at an ionic strength above 0.03 because of their very low equilibrium binding constants.
Topics: Aminacrine; Aminoacridines; Animals; Cattle; Chemical Phenomena; Chemistry; Chloroquine; DNA; Ethidium; Kinetics; Molecular Conformation; Nucleic Acid Conformation; Osmolar Concentration; Quinacrine; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Thymus Gland; Viscosity
PubMed: 7191995
DOI: 10.1093/nar/8.7.1613 -
Proceedings of the National Academy of... Aug 1987The neighbor-exclusion principle is one of the most general and interesting rules describing intercalative DNA binding by small molecules. It suggests that such binding...
Molecular mechanical simulations on double intercalation of 9-amino acridine into d(CGCGCGC) X d(GCGCGCG): analysis of the physical basis for the neighbor-exclusion principle.
The neighbor-exclusion principle is one of the most general and interesting rules describing intercalative DNA binding by small molecules. It suggests that such binding can only occur at every other base-pair site, reflecting a very large negative cooperativity in the binding process. We have carried out molecular mechanics and molecular dynamics simulations to study intercalation complexes between 9-amino acridine and the base-paired heptanucleotide d(CGCGCGC) X d(GCGCGCG), in which the neighbor-exclusion principle was both obeyed and violated. Our studies find no stereochemical preference that favors the neighbor-exclusion-obeying structures over the neighbor-exclusion-violating structures. Alternative explanations for the existence of the neighbor-exclusion principle are vibrational entropy effects that we calculate to favor the more flexible neighbor-exclusion models over the more rigid neighbor-exclusion-violating models and polyelectrolyte (counterion release) effects.
Topics: Aminacrine; Aminoacridines; Models, Molecular; Nucleic Acid Conformation; Polydeoxyribonucleotides
PubMed: 3475700
DOI: 10.1073/pnas.84.16.5735 -
Cellular & Molecular Biology Letters Mar 2014The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify...
The recently described method of cell electroporation by flow of cell suspension through localized direct current electric fields (dcEFs) was applied to identify non-toxic substances that could sensitize cells to external electric fields. We found that local cationic anesthetics such as procaine, lidocaine and tetracaine greatly facilitated the electroporation of AT2 rat prostate carcinoma cells and human skin fibroblasts (HSF). This manifested as a 50% reduction in the strength of the electric field required to induce cell death by irreversible electroporation or to introduce fluorescent dyes such as calcein, carboxyfluorescein or Lucifer yellow into the cells. A similar decrease in the electric field thresholds for irreversible and reversible cell electroporation was observed when the cells were exposed to the electric field in the presence of the non-toxic cationic dyes 9-aminoacridine (9-AAA) or toluidine blue. Identifying non-toxic, reversibly acting cell sensitizers may facilitate cancer tissue ablation and help introduce therapeutic or diagnostic substances into the cells and tissues.
Topics: Aminacrine; Anesthetics; Animals; Cations; Cell Line, Tumor; Electricity; Electroporation; Fibroblasts; Fluoresceins; Humans; Rats; Surface Properties
PubMed: 24415057
DOI: 10.2478/s11658-013-0114-z -
Nucleic Acids Research Aug 1997Four nitrogen mustards have been used in this study to examine protein-DNA interactions in intact human cells, specifically at the locus control region hypersensitive...
Four nitrogen mustards have been used in this study to examine protein-DNA interactions in intact human cells, specifically at the locus control region hypersensitive site-2 (LCR HS-2) of the human beta-globin locus. Three of these nitrogen mustards are DNA-targeted by attachment of an acridine or amsacrine intercalating chromophore, while the fourth (chlorambucil) is a non-targeted mustard. The ligation-mediated PCR technique was used to determine the sites of damage at base pair resolution on DNA sequencing gels. A densitometric comparison was made between DNA damaged in intact erythroid K562 cells and in purified DNA. The intensity of DNA damage sites in the LCR HS-2 were found to differ significantly between intact K562 cells and purified DNA. At the NF-E2/AP-1 motif, pronounced damage protection was observed in DNA derived from drug treated cells. The nuclear factor- erythroid 2 (NF-E2) protein factor is thought to bind at this NF-E2/AP-1 motif in K562 cells. Other sites of protection and enhancement that corresponded to known transcription factor binding sites were also detected. These nitrogen mustards are therefore very effective compounds for detection of transcription factor binding to DNA in intact cells and are superior to other commonly used agents. The sequence selectivity of the compounds was determined using plasmid DNA and compared to that found in intact cells. The acridine-based nitrogen mustard had a preference for forming adducts at guanine bases, while the two amsacrine-based nitrogen mustards and chlorambucil formed adducts at both guanine and adenine bases.
Topics: Aminacrine; Amsacrine; Cell Line; Chlorambucil; Chromatin; DNA Damage; DNA-Binding Proteins; Gene Expression Regulation; Globins; Humans; Mechlorethamine; Promoter Regions, Genetic
PubMed: 9241238
DOI: 10.1093/nar/25.16.3255