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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 -
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 Physiology Mar 19951. N-methyl-D-aspartate (NMDA) receptor responses were recorded from rat hippocampal neurons grown in dissociated culture, using whole-cell, outside-out and nucleated...
1. N-methyl-D-aspartate (NMDA) receptor responses were recorded from rat hippocampal neurons grown in dissociated culture, using whole-cell, outside-out and nucleated patch recording techniques. Rapid perfusion was used to study voltage-dependent block of NMDA receptors by 9-aminoacridine (9-AA) and by Mg2+. 2. Large amplitude tail currents were evoked on depolarization to +60 mV after application at -100 mV of NMDA and 9-AA but not NMDA and Mg2+. These tail currents were resistant to block by competitive antagonists to the glutamate and glycine binding sites on NMDA receptors and were not evoked when either NMDA or 9-AA were applied alone. 3. The decay kinetics of the tail current were dependent on agonist affinity; the time required for 80% charge transfer was 10-fold briefer for NMDA than for glutamate and 7-fold briefer for L-alanine than for glycine. These results are in accord with a sequential model for block of NMDA receptors by 9-AA, in which neither glutamate nor glycine can dissociate from the open-blocked state of the receptor. 4. Tail current responses had amplitudes 2- to 4-fold larger than responses to maximally effective concentrations of glutamate and glycine, indicating that NMDA receptor channels accumulate in the open-blocked state during co-application of agonist and 9-AA. The rise time and decay kinetics of tail current responses were faster than the response to brief applications of a maximally effective concentration of glutamate. Together, these results suggest that at +60 mV recovery from block by 9-AA occurs faster than the rate of opening of NMDA receptors in response to glutamate. 5. Our experiments suggest that open channel block of NMDA receptors can provide a novel approach for measurement of both open probability and the first latency distribution for ion channel opening in response to the binding of agonists, and provide additional evidence suggesting that the delayed opening of NMDA receptor channels underlies slow activation and deactivation of responses to glutamate.
Topics: Aminacrine; Animals; Animals, Newborn; Binding, Competitive; Electrophysiology; Embryo, Mammalian; Female; Glutamic Acid; Glycine; Hippocampus; Ion Channel Gating; Kinetics; Magnesium; Neurons; Pregnancy; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Time Factors
PubMed: 7650609
DOI: 10.1113/jphysiol.1995.sp020591 -
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 -
Enhancement of mutagenic activity of 9-aminoacridine by introducing a nitro group into the molecule.Bioscience, Biotechnology, and... Aug 1994Mutagenic activity and DNA intercalation were examined for 9-aminoacridine (9-AA) and its derivatives. Introduction of a nitro group into the 9-AA molecule was found to...
Mutagenic activity and DNA intercalation were examined for 9-aminoacridine (9-AA) and its derivatives. Introduction of a nitro group into the 9-AA molecule was found to enhance the activity enormously as was detected by the Ames test. Acetylation of amino group at 9-position of acridine ring inhibited the intercalation, the frameshift activity disappearing. Rat liver S9 converted 9-AA metabolically to 9-amino-2-hydroxyacridine.
Topics: Acetylation; Aminacrine; Animals; Chromatography, Thin Layer; DNA Damage; Hydroxylation; Magnetic Resonance Spectroscopy; Microsomes, Liver; Mutagenesis; Mutagenicity Tests; Mutagens; Nitro Compounds; Rats; Spectrophotometry, Ultraviolet
PubMed: 7765272
DOI: 10.1271/bbb.58.1420 -
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 -
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 -
Genetics Dec 1991As the most nucleophilic site in DNA, the guanine N7 atom is a major site of adduction by a large number of alkylating mutagens and carcinogens. Aflatoxin B1, a powerful...
As the most nucleophilic site in DNA, the guanine N7 atom is a major site of adduction by a large number of alkylating mutagens and carcinogens. Aflatoxin B1, a powerful mutagen, is believed to act through its reaction with this DNA site. On the basis of the specificity of base substitutions induced by various adduct forms of aflatoxin, we have proposed that bulky guanine N7 adducts elicit base substitutions by two mechanisms. The first mechanism is similar to that observed for a number of bulky noninstructive lesions, whereas the second mechanism invokes mispairing between N7-adducted guanine and thymine. A prediction of the mispairing hypothesis is that diverse bulky guanine N7 adducts (regardless of structural similarities with the aflatoxins) should induce predominantly G-to-A transitions. Accordingly, we have recently observed that base substitutions induced by the acridine half-mustard ICR-191 in the M13 double-stranded DNA transfection system are predominantly G:C-to-A:T transitions. Here, by transfecting ICR-191-treated M13 AB28 single-stranded DNA into Escherichia coli, we show that base substitutions are predominantly targeted to guanines. Since the N7-adducted-guanine:thymine mispairing is proposed to require N1 deprotonation promoted by the primary N7 lesion, guanine imidazole ring-opening should abolish this mispairing property, and thereby alter the specificity of mutagenesis. Here, we show that the incubation of ICR-191-treated RF DNA at pH 10.5 results in a significant reversal of the specificity of G:C-targeted substitutions such that G-to-T transversions predominated over G-to-A transitions.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Aminacrine; Bacteriophages; Base Composition; Base Sequence; DNA Damage; DNA, Single-Stranded; DNA, Viral; Escherichia coli; Frameshift Mutation; Guanine; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Mutagens; Nitrogen Mustard Compounds; Transfection
PubMed: 1783299
DOI: 10.1093/genetics/129.4.981 -
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 -
The Journal of Biological Chemistry May 1991Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of...
Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.
Topics: Aminacrine; Animals; Barium; Biological Transport, Active; Calcium; Calcium Channels; Calcium-Transporting ATPases; Cell Compartmentation; Cell-Free System; Dithiothreitol; Glutathione; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Membranes; Permeability; Rabbits; Ranidae; Ruthenium Red; Sarcoplasmic Reticulum; Tetracaine
PubMed: 1709160
DOI: No ID Found