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Scientific Reports Jul 2018Biomolecule abundance levels change with the environment and enable a living system to adapt to the new conditions. Although, the living system maintains at least some...
Biomolecule abundance levels change with the environment and enable a living system to adapt to the new conditions. Although, the living system maintains at least some characteristics, e.g. homeostasis. One of the characteristics maintained by a living system is a power law distribution of biomolecule abundance levels. Previous studies have pointed to a universal characteristic of biochemical reaction networks, with data obtained from lysates of multiple cells. As a result, the spatial scale of the data related to the power law distribution of biomolecule abundance levels is not clear. In this study, we researched the scaling law of metabolites in mouse tissue with a spatial scale of quantification that was changed stepwise between a whole-tissue section and a single-point analysis (25 μm). As a result, metabolites in mouse tissues were found to follow the power law distribution independently of the spatial scale of analysis. Additionally, we tested the temporal changes by comparing data from younger and older mice. Both followed similar power law distributions, indicating that metabolite composition is not diversified by aging to disrupt the power law distribution. The power law distribution of metabolite abundance is thus a robust characteristic of a living system regardless of time and space.
Topics: Aminacrine; Animals; Liver; Male; Mice; Mice, Inbred C57BL; Models, Biological; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 29985415
DOI: 10.1038/s41598-018-28667-5 -
Molecular Cancer Jun 2008Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of...
BACKGROUND
Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis.
RESULTS
To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice.
CONCLUSION
Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.
Topics: Aminacrine; Animals; Breast Neoplasms; Cell Line, Transformed; Cell Line, Tumor; Chromosomal Instability; Female; Frameshift Mutation; Genes, Tumor Suppressor; Humans; Mammary Neoplasms, Experimental; Mice; Models, Biological; Mutagenesis; Neoplasm Transplantation; Nitrogen Mustard Compounds; Nucleic Acid Hybridization; RNA Stability; Spectral Karyotyping
PubMed: 18534021
DOI: 10.1186/1476-4598-7-51 -
The Journal of Biological Chemistry May 1987A demonstration is made of pyrophosphate's use as a precipitating anion in studies of Ca2+ release from isolated sarcoplasmic reticulum (SR). Not only does pyrophosphate...
A demonstration is made of pyrophosphate's use as a precipitating anion in studies of Ca2+ release from isolated sarcoplasmic reticulum (SR). Not only does pyrophosphate speed up the rate at which Ca2+ can be preloaded into SR, but it also allows the accumulated Ca2+ to be released in response to agents such as caffeine. Because so much Ca2+ can be preloaded into SR with pyrophosphate present, more experiments can be performed with a given amount of SR material, and even rapid Ca2+ release rates (greater than 1 mumol/mg X min) are maintained for many seconds. These rates can easily be quantified using conventional spectrophotometric and isotopic methods, without the need for expensive rapid mixing equipment. Caffeine-induced Ca2+ release is exhibited by triadic and terminal cisterna SR subfractions but not by light SR. Caffeine specifically increases the rate of unidirectional 45Ca2+ efflux. This increased efflux is blocked by ruthenium red at submicromolar concentrations and by tetracaine, 9-aminoacridine, or Ba2+ at submillimolar concentrations.
Topics: Aminacrine; Animals; Barium; Caffeine; Calcium; Diphosphates; Fluorides; Oxalates; Oxalic Acid; Rabbits; Ruthenium Red; Sarcoplasmic Reticulum; Tetracaine
PubMed: 2437114
DOI: No ID Found -
The Journal of General Physiology Sep 1984The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the...
The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.
Topics: Acetamides; Aminacrine; Aminoacridines; Culture Techniques; Electrophysiology; Ion Channels; Kinetics; Neuroblastoma; Sodium; Time Factors
PubMed: 6090578
DOI: 10.1085/jgp.84.3.361 -
Nucleic Acids Research 2004A series of peptide nucleic acid (PNA) oligomers targeting the mdm2 oncogene mRNA has been tested for the ability to inhibit the growth of JAR cells. The effect of these...
A series of peptide nucleic acid (PNA) oligomers targeting the mdm2 oncogene mRNA has been tested for the ability to inhibit the growth of JAR cells. The effect of these PNAs on the cells was also reflected in reduced levels of the MDM2 protein and increased levels of the p53 tumor suppressor protein, which is negatively regulated by MDM2. Initially, PNA oligomers were delivered as DNA complexes with lipofectamine, but it was discovered that PNA conjugated to the DNA intercalator 9-aminoacridine (Acr) (Acr-PNA) could be effectively delivered to JAR cells (as well as to HeLa pLuc705 cells) even in the absence of a DNA carrier. Using such lipofectamine-delivered Acr-PNA conjugates, one PNA targeting a cryptic AUG initiation site was identified that at a concentration of 2 microM caused a reduction of MDM2 levels to approximately 20% (but no reduction in mdm2 mRNA levels) and a 3-fold increase in p53 levels, whereas a 2-base mismatch control had no such effects. Furthermore, transcriptional activation by p53 was also increased (6-fold), and cell viability was reduced to 80%. Finally, this PNA acted cooperatively with camptothecin treatment both with regard to p53 activity induction as well as cell viability. Using this novel cell delivery system, we have identified a target on the mdm2 mRNA that appears sensitive to antisense inhibition by PNA and therefore could be used as a lead for further development of mdm2-targeted antisense (PNA and other) gene therapeutic anticancer drugs.
Topics: Aminacrine; Antineoplastic Agents; Base Sequence; Camptothecin; Cell Line; Cell Line, Tumor; Cell Survival; DNA Damage; DNA, Antisense; Down-Regulation; HeLa Cells; Humans; Molecular Sequence Data; Nuclear Proteins; Peptide Nucleic Acids; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; RNA, Messenger; Transfection; Tumor Suppressor Protein p53
PubMed: 15371552
DOI: 10.1093/nar/gkh820 -
Bioorganic & Medicinal Chemistry Jan 2006A parallel synthetic strategy to the 9-aminoacridine scaffold of the classical anti-malarial drug quinacrine (2) is presented. The method features a new route to...
A parallel synthetic strategy to the 9-aminoacridine scaffold of the classical anti-malarial drug quinacrine (2) is presented. The method features a new route to 9-chloroacridines that utilizes triflates of salicylic acid derivatives, which are commercially available in a variety of substitution patterns. The route allows ready variation of the two diversity elements present in this class of molecules: the tricyclic aromatic heterocyclic core, and the disubstituted diamine sidechain. In this study, a library of 175 compounds was designed, although only 93 of the final products had purities acceptable for screening. Impurity was generally due to incomplete removal of 9-acridones (18), a degradation product of the 9-chloroacridine synthetic intermediates. The library was screened against two strains of Plasmodium falciparum, including a model of the drug-resistant parasite, and six novel compounds were found to have IC(50) values in the low nanomolar range.
Topics: Aminacrine; Animals; Antimalarials; Cells, Cultured; Chloroquine; Drug Resistance; Erythrocytes; Humans; Plasmodium falciparum
PubMed: 16216519
DOI: 10.1016/j.bmc.2005.08.017 -
Plant Physiology Sep 2000Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron...
Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron acceptor, ferricyanide and the DeltapH indicator, 9-aminoacridine, was used to measure simultaneously electron transport rates and DeltapH solely by changes in the fluorescence of 9-aminoacridine. This method yields values for the rate of electron transport that are comparable with those obtained by established methods. Using this method a relationship between the rate of electron transport and DeltapH at various uncoupler concentrations or light intensities was obtained. In addition, the method was used to study the effect of reducing the disulfide bridge in the gamma-subunit of the chloroplast ATP synthase on the relation of electron transport to DeltapH. When the ATP synthase is reduced and alkylated, the threshold DeltapH at which the ATP synthase becomes leaky to protons is lower compared with the oxidized enzyme. Proton flow through the enzyme at a lower DeltapH may be a key step in initiation of ATP synthesis in the reduced enzyme and may be the way by which reduction of the disulfide bridge in the gamma-subunit enables high rates of ATP synthesis at low DeltapH values.
Topics: Adenosine Triphosphate; Aminacrine; Electron Transport; Fluorescence; Fluorescent Dyes; Hydrogen-Ion Concentration; Indicators and Reagents; Proton-Translocating ATPases; Spinacia oleracea; Thylakoids
PubMed: 10982453
DOI: 10.1104/pp.124.1.407 -
Proceedings of the National Academy of... Dec 19912fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by alpha interferon (IFN-alpha). Two IFN-alpha-unresponsive...
2fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by alpha interferon (IFN-alpha). Two IFN-alpha-unresponsive mutants were isolated previously at a low frequency (ca. 10(-8)) by selecting mutagenized 2fTGH cells in selective medium containing 6-thioguanine and IFN-alpha. By using five rounds of mutagenesis, mutants can be isolated at an appreciably higher frequency, greater than 3 x 10(-7). Five new mutants have been isolated, and all are recessive, as are the two mutants we described previously. The seven mutants are in four complementation groups (U1-U4). Since several different types of mutants unresponsive to IFN-alpha have been isolated with high frequency, related approaches may succeed with other cytokines or growth factors. Mutants in the two new complementation groups U3 and U4 are unresponsive to IFN-alpha and, surprisingly, also unresponsive to IFN-gamma. They are also partially defective in response to double-stranded RNA. These results indicate that the signaling pathways for the two types of IFN and double-stranded RNA share common components or that their function depends on common enzymes or transcription factors. IFN receptors are unaffected in mutants U3A and U4A. A major defect appears to be in the synthesis or activation of E, the transcription factor mediating the primary response to type I (alpha/beta) IFNs. Band-shift complementation assays show that U3A contains the E gamma subunit but does not contain an active E alpha subunit after treatment with IFN-alpha.
Topics: Aminacrine; Cell Fusion; Cell Line; Flow Cytometry; Gene Expression Regulation, Enzymologic; Genetic Complementation Test; Humans; Hypoxanthine Phosphoribosyltransferase; Interferon-alpha; Interferon-beta; Interferon-gamma; Kinetics; Mutagenesis; Mutagens; Nitrogen Mustard Compounds; RNA, Messenger; Receptors, Immunologic; Receptors, Interferon; Transcription, Genetic
PubMed: 1837150
DOI: 10.1073/pnas.88.24.11455 -
The Biochemical Journal Mar 1989Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from...
Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.
Topics: Adenosine Triphosphatases; Aminacrine; Animals; Ca(2+) Mg(2+)-ATPase; Cattle; Cerebral Cortex; Fluorescent Dyes; Glutamates; Glutamic Acid; Hydrogen-Ion Concentration; Isoxazoles; Membrane Potentials; Protons
PubMed: 2565109
DOI: 10.1042/bj2580499 -
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