9-aminoacridine Research Articles

Competitive Inhibition of a Tumour Cell Surface Protease. A Rapid Technique forin VitroTesting of Selective Targeting Systems

The active centre of a protease on the surface of tumour cells can be located by its affinity for an active site-directed inhibitor, 9-amino acridine. Cells which have uninhibited proteases, bind 9-amino acridine and fluoresce in resin sections. The leukaemic rat was used as a model system to provide tumour cells in a well defined location. Drugs when coupled to a ligand (directed to the active centre of the protease) compete for this binding site with 9-amino acridine. Thus, competitive inhibition of the tumour cell surface protease provides a rapid technique for demonstrating the delivery of liganded molecules to the surface of tumour cells in vitro.

Direct Evidence for the Cell Surface Location of a Protease-Inhibitor Complex on Intact Leukaemia Cells

The interaction of a protease with two fluorescent inhibitors has been studied using intact fixed leukaemia cells as the source of the membrane bound enzyme. Fresh rat leukaemia cells were disrupted and the cytosol collected; this extract was known to contain a protein inhibitor of guanidinobenzoatase (GB) associated with leukaemia cells. All the cytosolic proteins were derivatised with Texas red acid chloride. Leukaemia cells with latent GB failed to bind the Texas red inhibitor protein but did so after activation of GB. Competition experiments with 9-amino acridine (a fluorescent marker for the active site of GB) demonstrated that the Texas red-inhibitor protein could only bind to intact leukaemia cells when the active centre of GB was not already occupied by 9-amino acridine. This competition between these two fluorescent inhibitors demonstrated their specificity for GB. The use of intact leukaemia cells and the high molecular weight of the inhibitor protein precludes the possibility of any interaction between GB and inhibitor within the cells. It is concluded that GB and the GB-inhibitor complex of latent GB are located on the external surface of intact leukaemia cells.

Differential inhibition of plant calcium-dependent protein kinases by long chain fatty acids and other amphiphiles

Ca 2+-dependent protein kinase (CDPK) was partially purified from wheat ( Triticum aestivum) embryo and from wheat and silver beet ( Beta vulgaris) leaves by extensive protocols involving Ca 2+-dependent hydrophobic chromatography on phenyl-Sepharose CL-4B, ion exchange chromatography on DEAE-Sephacel and gel filtration as common procedures. Wheat leaf CDPK is similar to wheat embryo CDPK (purified by application of the same protocol) in Ca 2+-dependence, substrate specificity, molecular size (mol. wt. 81 000) and in inhibition by the calmodulin antagonist trifluoperazine. Both wheat leaf and wheat embryo CDPKs phosphorylate histones (III-S and II-AS preparations), bovine serum albumin, casein and myosin light chains but not the synthetic cyclic AMP-dependent protein kinase substrate, Kemptide. Wheat and silver beet CDPKs are inhibited by amphipathic ligands including even carbon number long chain fatty acids (C 14C 22), odd carbon number long fatty acids (C 15C 21), long chain aliphatic alcohols (C 8C 11), amino acridines and sphingosine. Behenic acid and arachidic acid are potent inhibitors of wheat embryo CDKP (IC 50 values 50 and 60 μM, respectively) but are poor inhibitors of the wheat leaf and silver beet leaf CDPKs.

Acid secretion and membrane reorganization in single gastric parietal cell in primary culture

A digitally-enhanced videomicroscopy study of rabbit gastric parietal cells in primary culture was performed using alternate observations with differential interference contrast and fluorescence optics of cells mounted and perfused on a temperature-controlled microscope stage. The effect of histamine, a physiological effector of acid secretion, was followed. Isolated parietal cells possess an internal apical vacuole, which kept the cell in a pseudopolarized state. This apical vacuole is a site of acid secretion. This was demonstrated by the direct visualization of the uptake of the fluorescent weak base 9-amino acridine and of the concomitant enormous swelling of the acid vacuole which reached an estimated size of 3–7 times the normal cell volume. This morphological change of shape and acidification of apical vacuoles was fully reversible and cells could respond to successive stimulations. A quantitative study of these events provided a value of the acid accumulation index for each single cell in response to histamine. Individual cell response varied within a factor of 7. The cellular localization of the proton pump complex responsible for acid secretion and of the major components of the secretory microvilli, actin and ezrin, a histamine-dependent phosphorylation target of protein kinase A, were detected by indirect immunofluorescence microscopy in resting and stimulated cells. Both actin and ezrin colocalized at the apical vacuole membrane in resting and stimulated cells, whereas the proton pump shifted from an intracytoplasmic pool to the apical vacuole membrane upon stimulation.

The Protective Role of a Natural Inhibitor in the Fluorescent Location of Cells Possessing a Latent form of Cell Surface Protease

Leukaemia cells possess a latent form of a cell surface protease referred to as guanidinobenzoatase. Latency is due to complex formation between an inhibitor protein and the cell surface enzyme which is stable under acid conditions but is dissociated with formaldehyde treatment. The latent form of the cell surface protease has been used as a protecting mechanism during a preliminary step to stain all the nuclei of cells with haematoxylin. The enzyme-inhibitor complex was then dissociated and a combination of 9-amino acridine and propidium iodide employed to enable the fluorescent location of cells possessing active guanidinobenzoatase. We were thus able to visualise the nuclei by conventional light microscopy and simultaneously visualise the cell surface of leukaemia cells by fluorescent microscopy. This simple model system has provided technology applicable to the more complex analysis of neoplastic cells in cervical smears.

Endpoint of first stage of zona pellucida-induced acrosome reaction in mouse spermatozoa characterized by acrosomal H+ and Ca2+ permeability: population and single cell kinetics.

The acrosome reaction induced by the mouse egg's zona pellucida in mouse sperm has been shown to proceed in two stages as characterized empirically by sequential changes in patterns of chlortetracycline fluorescence on the sperm plasma membrane surfaces. The chlortetracycline fluorescence pattern characteristic of fully intact sperm is designated B; in sperm bound to structurally intact zonae that induce the acrosome reaction, the B pattern changes first to an intermediate pattern S and then to a terminal pattern AR characteristic of the completed acrosome reaction. In the same study, it was shown, using a 9-amino acridine fluorescent pH probe, that completion of the first stage was characterized by increase in H+ permeability such that the H+ gradient between sperm head and medium was dissipated. In this study, we show that the fluorescent pH probe 9-N-dodecylamino acridine and the intracellular Ca2+ fluorescent probe fura-2 are both localized to the anterior part of the sperm head encompassing the acrosomal compartment in intact sperm, and the fluorescence associated with each probe is lost as the first stage of the acrosome reaction is completed. Loss of the pH probe fluorescence, pattern N, corresponds to onset of H+ permeability, and loss of fura-2 fluorescence, pattern F, corresponds to onset of Ca2+ permeability. Localization of intracellular fura-2 fluorescence to the acrosomal compartment required extracellular Mn2+ to quench surface-bound fura-2 AM, the tetra-acetoxymethyl ester of fura-2 used to load the cells. Loss of acrosomal fura-2 fluorescence is due to quenching by tracer Mn2+ accompanying Ca2+. Onset of membrane permeability to both H+ and Ca2+, as seen by loss of patterns N and F, occurred in synchrony in populations of sperm bound to isolated, structurally intact zonae, with an overall time course of 210 min postbinding. The loss of pattern N in individual sperm cells bound to zonae was rapid, with a half time of 2.1 min. Concomitant with this rapid loss of pattern N was a shift in the amplitude of flagellar motion from large to small. The lag times to pattern N loss in 50 individual cells ranged from 30 to 140 min. The variable lag times determine the population kinetics; the rate of the endpoint reaction seen in the individual cells is rapid and constant. Dissipation of the H+ gradient with immediate loss of pattern N was readily achieved by addition of nigericin with no change in the time course of the onset of Ca2+ permeability of the membranes enclosing the acrosome.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino acridines action on Friend's retrovirus in relation to their molecular ionization

Amino acridines action on Friend's retrovirus in relation to their molecular ionization

Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell.

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.

Conformations of duplex structures formed by oligodeoxynucleotides covalently linked to the intercalator 2-methoxy-6-chloro-9-aminoacridine.

A family of covalent complexes between oligonucleotides and derivatives of the intercalating agent 9-amino acridine has been synthesized (Asseline, U., Thuong, N.T. and Helene, C. (1983) C.R.Acad. Sci. (Paris) 297 (III), 369-372) and studied (Lancelot, G., Asseline, U., Thuong, N.T., and Helene, C. (1985) Biochemistry 24, 2521-2529; Lancelot, G., Asseline, U., Thuong, N.T., and Helene, C. (1985) J. Biomol. Str. Dyn. 3, 913-921) with a view to understand nucleic acid-nucleic acid recognition. In order to understand the nature of interactions between the intercalator and the oligonucleotides in such complexes and the sensitivity of such interactions to the polymorphic form of the DNA, we have carried out molecular mechanics simulations on duplex deoxyoligonucleotides d(A)6.d(T)6 (A and B forms) and d(TATC).d(GATA) (B form) covalently bound to 2-methoxy-6-chloro-9-aminoacridine through a pentamethylene linker chain. Structures in which the acridine derivative is end stacked (at the 3' and 5' ends) and in which the dye is intercalated between the terminal base pairs (at both the ends) and between second and third base pairs from the 3' end are all of reasonably low energy in both A and B forms of DNA. Our studies on 3' end complexes find that in the B form, intercalation of the dye between the second and third base pairs is preferred over the other two modes of binding, while in the A form, intercalation between the terminal base pairs is preferred. In the 5' end A and B form complexes, outside stacking and intercalation between the terminal base pairs are preferred, respectively. Our calculations suggest the possibility that the presence of the dye attached covalently to the DNA can induce conformational transitions in the DNA. For example, intercalation of the dye two base pairs from the end could induce an A----B transition.

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.

The role of inhibitors in the fluorescent staining of benign naevus and malignant melanoma cells with 9-amino acridine and acridine orange.

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.

Oligo-[alpha]-deoxynucleotides covalently linked to an intercalating agent. Double helices with parallel strands are formed with complementary oligo-[beta]-deoxynucleotides.

An oligo-[alpha]-deoxynucleotide of sequence (5')d(TCTAAACTC) (3') was synthesized using the alpha-anomers of deoxynucleosides and its 5'-phosphate was covalently linked to a 9-amino acridine derivative via a pentamethylene linker. Two oligo-[beta]-deoxynucleotides containing the complementary sequence in either the 5'----3' or the 3'----5' orientation were synthesized using natural [beta]-deoxynucleosides. Complex formation was investigated by absorption and fluorescence spectroscopies. No change in spectroscopic properties was detected with the anti-parallel [beta] sequence. Absorption changes were induced in the visible absorption band of the acridine derivative at 2 degrees C when the acridine-substituted oligo-[alpha]-deoxynucleotide was mixed in equimolecular amounts with the complementary [beta]-sequence in the parallel orientation. Hypochromism was observed in the UV range. The fluorescence of the acridine derivative was quenched by the guanine base present in the second position of the complementary sequence. Cooperative dissociation curves were observed and identical values of melting temperatures were obtained by absorption and fluorescence. An increase in salt concentration stabilized the complex with a delta Tm of 8 degrees C when NaCl concentration increased from 0.1 to 1 M. These results demonstrate that an oligo-[alpha]-deoxynucleotide covalently linked to an intercalating agent is able to form a double helix with an oligo-[beta]-deoxynucleotide. The two strands of this [alpha]-[beta] double helix adopt a parallel 5'----3' orientation. The acridine ring is able to intercalate between the first two base pairs on the 5'-side of the duplex structure.

Calibration of the response of 9-amino acridine fluorescence to transmembrane pH differences in bacterial chromatophores

The spectral characteristics of absorption and fluorescence emission of 9-amino acridine are not altered by the interaction with bacterial chromatophores, except for the attenuation of both the absorption and emission following the formation of a protonic gradient. The lifetime of fluorescence of the dye is significantly affected in the presence of membranes, and even more following illumination. The shortening of the lifetime induced by light is reversible and prevented by nigericin and K +. The onset kinetics of the fluorescence quenching following the generation of an artificial transmembrane pH difference is temperature dependent, with an activation energy of 17 ± 3 kcal/mol. The effect of pH on the rate constants is consistent with a model assuming that the diffusion of the unprotonated species is the limiting step in the quenching phenomenon. The response of 9-amino acridine to artificially imposed ΔpH's has been utilized as a calibration method for the measurements of the light-induced protonic gradient. The apparent inner volume of chromatophores, evaluated from the extraplation of the response at ΔpH = 0, was found to be much larger (15- to 40-fold) than the true osmotic volume, indicating that most of the dye is bound to the membrane when accumulated into the inner lumen.

Electric field selective optical data storage using persistent spectral hole burning

The electric field domain is used as a storage dimension in optical data storage by persistent spectral hole burning. The memory locations in the electric field domain are addressed with the voltage applied to the sample consisting of the amorphous polymer polyvinyl-butyral doped with the dye 9-amino acridine. The information is written by burning spectral holes at different electric field strengths with a HeCd laser and read by detecting the presence or absence of holes with weak laser intensity.

Fluorescent location of guanidinobenzoatase on cell surfaces by the competitive inhibitor 9-amino acridine

Fluorescent location of guanidinobenzoatase on cell surfaces by the competitive inhibitor 9-amino acridine

The ascidian sperm reaction: Evidence for Cl − and HCO −3 involvement in acid release

Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na + concentration of the medium. The optimal [Na +] for acid release is 20 m M with excellent correlation between acid release and initiation of morphological changes. Increasing the [K +] to around 20 m M inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K + inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl −-free (NO − 3 or glutamate substituted) seawater (SW). Cl − efflux accompanies H + efflux with twice as many Cl − being released as H +. Both H + and Cl − release in Cl −-free SW are dependent upon CO 2 being present in HCO − 3-free medium, suggesting that H + efflux is in part Cl − and HCO − 3-mediated. However, the chloride channel blocking agent SITS has no effect on H + release and augments Cl − release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na +-dependent acidification system of unreacted sperm and the Cl −- and HCO − 3-mediated H + release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process.

Essais d'interprétation des déplacements des spectres électroniques de diverses amino-9 acridines en solution par leurs interactions avec des solvants aprotiques

The absorption and fluorescence spectra of nine differently substituted 9-amino acridines in 20 aprotic solvents were recorded. Using the empirical parameters π * and β determined by Kamlet and coworkers the spectral shifts are related to polarity and polarizability effects between solvent and solute molecules and also partly to the formation of hydrogen bonds in which the amino group acts as a proton donor. The influence of different substituents of these molecules is studied here. A comparison of the results obtained for the shift of absorption spectra with those obtained for the shift of fluorescence spectra shows a difference which could be related to solvation variations between the ground state and a fully relaxed excited state.

In vitro packaging of bacteriophage T4 DNA

Bacteriophage T4 can be formed in an in vitro DNA packaging system which requires addition of exogenous T4 DNA. Extracts prepared from bacteria infected with mutants 20 cs, 17 cs, 16 am, 17 am, or 16 am–17 am contain processed proheads. These appear to be efficient DNA acceptors in the in vitro system. Packaging of mature T4 DNA is more efficient than of concatemeric T4 DNA. The phage yield is proportional to the quantity of DNA and prohead-containing extract added. Activity requires addition of nucleoside triphosphate, Mg 2+, and is inhibited by 9-amino acridine. gpl6 and gp17, the primary T4 DNA packaging gene products, are essential, but 16 am and 17 am mutants appear not to complement each other in vitro. Many T4 recombinants, including multiple recombinants, appear to be generated by in vitro recombination in this system. In vitro T4 DNA packaging conforms to expectations based upon analysis of T4 mutants and head assembly in vivo and displays fundamental similarity to that of other complex double-stranded DNA phages.

Effects of some antimalarials and related substances on intralysosomal proteolysis

The antimalarials primaquine, chloroquine, quinine, and quinacrine and the related substances 6-chloro-9-(3′-dimethylamino-2′-hydroxypropylamino)-2-methoxyacridine and 9-amino acridine inhibited intralysosomal proteolysis at pH 8 in mouse kidney subcellular suspensions containing [ 125I]-labeled albumin-filled phagolysosomes at concentrations from 5 × 10 −5 (quinacrine) to about 2.5 × 10 −4M (quinine). All these substances inhibited rat liver cathepsin B 1 but had no effects on cathepsins A, C or D. These inhibitions were greater on the alkaline side of the pH optimum of cathepsin B 1. The drugs also partially inhibited the stimulatory effect of ATP on intralysosomal proteolysis at pH 8 which we have interpreted previously to reflect an energy-dependent proton pump for maintenance of intralysosomal acidity. Preincubation of [ 125I]-labeled albumin-filled phagolysosomes for 10 min at pH 8 in media containing 0.1 mM quinacrine or chloroquine or 0.5 mM quinine sulfate, followed by washing to remove these drugs, partially inhibited the stimulatory effect of ATP on further incubation in antimalarial-free media but had no effects on intralysosmal proteolysis. Preincubation in 1 mM chloroquine completely abolished the stimulatory effect of ATP but also partially inhibited proteolysis. Antimalarials also inhibited rat liver lysosomal (tritosomal) membrane ATPase suggesting that this activity may be related to the stimulatory effects of ATP on intralysosomal proteolysis. Quinine was the most effective of the inhibitors tested. These studies suggest that [ 125I]-labeled albumin-filled mouse kidney phagolysosomes may provide a convenient model system for rapid screening of drugs potentially useful for the treatment of diseases caused by organisms possessing a lysosome-like digestive system.

Comparison of the electrochemical proton gradient and phosphate potential maintained by Rhodospirillum rubrum chromatophores in the steady state

A technique for the estimation of light-induced membrane potential in chromatophores is described. It is based on measurement of light-induced enhancement in fluorescence of 8-anilinonaphthalene sulfonic acid, which is calibrated by known K + diffusion potentials. The electrochemical proton gradient (Δ μ H+ − ) formed during lightinduced electron transport in Rhodospirillum rubrum chromatophores amounts to 250 mV, which is almost equally distributed between the membrane potential and the pH gradient as measured by changes in the fluorescence of anilinonaphthalene sulfonate and 9-amino acridine. Addition of the permeant anion, NaSCN, or of NH 4Cl reduces the overall Δ μ H+ − by less than 20% but changes its distribution between the pH gradient and the membrane potential so that with NaSCN it is composed mainly of the first and with NH 4Cl mainly of the second. Initiation of phosphorylation causes a drop of about 50 mV in the measured Δ μ H+ − . In the absence of salts, the drop is observed in both components, although two-thirds of it are reflected in the membrane potential. In the presence of NaSCN or NH 4Cl the 50-mV drop is exclusively recorded in the pH gradient or in the membrane potential, respectively. The steady-state phosphate potential maintained during electron transport was found to change in parallel to the Δ μ H+ − , but exceeded it by 60 to 80 mV when based on a stoichiometry of two protons translocated per ATP synthesized.