The use of intercalating dye molecules in the study of chromatin structure

This paper reviews the field of chromatin structure studied by means of dye molecules believed to intercalate within DNA. The emphasis is on dyes whose binding properties are the best characterized (ethidium bromide (EB). actinomycin D(AMD), proflavine (PF)) but studies in which less common dye molecules are used are also considered. A comparison is made between the binding of these dyes to purified DNA and to whole or partially deproteinized chromatin in order to investigate both the availability of DNA in chromatin and the localization of chromosomal proteins or the DNA backbone.     

Mutagenesis of acridine orange in mitotic cleavage nuclei of the silkworm, Bombyx mori

The present study was done to determine whether acridine orange (AO) is mutagenic for the mitotic cleavage nuclei in the silkworm. The mutation frequency was estimated by the specific locus method using egg-color genes. AO was injected into the body cavity of marked female pupae (homozygous for pe and re genes) in active vitellogenesis (prophase I oocytes). The moths emerging from the treated pupae were mated to wild type male moths. AO increased the frequency of mosaic type mutations, indicating that AO has a positive mutagenic action on the paternal chromosomes in the mitotic cleavage nuclei in the silkworm.     

Some differences between 470 nm and 510 nm in the acridine-orange-sensitized photodynamic actions on yeast cells

The two wavelengths that correspond to the absorption of two complexes formed in the interactions of acridine orange (AO) with nucleic acids were different in a few aspects of photodynamic action on yeast, Saccharomyces cerevisiae. (1) The dose-survival curve at 470 nm is not the same in shape as that at 510 nm. (2) The efficiency for the induction of gene conversion at 510 nm is higher than that obtained at 470 nm on the basis of both dose and survival.     

The effect of short-term dietary modification on human fecal mutagenic activity

To assess the effect of short-term modification of diet on human fecal mutagenic activity, 6 subjects consumed 2 dietary regimes hypothesized to affect risk of colorectal cancer. After a 7-day baseline period, a 'low-risk' non-meat diet was consumed for 2 weeks followed by 2 weeks on a 'higher risk' diet which emphasized beef and refined grains. Fecal samples were collected at the end of each diet period and assayed for direct-acting mutagens with the fluctuation test for weak mutagens using Salmonella typhimurium TA100 and TA98 as tester strains. Fecal mutagenic activity on TA100 was increased for all subjects during the 'higher risk' period compared to the 'low risk' period. The average mutagenicity on TA98 was also increased, but the trend was not consistent for all subjects. The baseline diet and non-meat diet resulted in approximately equal mean fecal mutagenicity levels. These findings indicate that a diet high in meat and refined grain, as characterized here, increases fecal mutagenic activity within a 2-week period.     

Inhibition of the osteoclast V-ATPase by small interfering RNAs

The multisubunit enzyme V-ATPase harbours isoforms of individual subunits. a3 is one of four 116 kDa subunit a isoforms, and it is crucial for bone resorption. We used small interfering RNA (siRNA) molecules to knock down a3 in rat osteoclast cultures. Labeled siRNA-molecules entered osteoclasts via endocytosis and knocked down the a3 mRNA. Bone resorption was decreased in siRNA-treated samples due to decreased acidification and osteoclast inactivation. Expression of a1 did not respond to decreased a3 levels, suggesting that a1 does not compensate for a3 in osteoclast cultures. Subunit a3 is thus an interesting target for novel nucleic acid therapy.     

Mitotic recombination induced by chemical and physical agents in the yeast Saccharomyces cerevisiae

The treatment of diploid cultures of yeast with ultraviolet light (UV), γ-rays, nitrous acid (NA) and ethyl methane sulphonate (EMS) results in increases in cell death, mitotic gene conversion and crossing-over. Acridine orange (AO) treatment, in contrast, was effective only in increasing the frequency of gene conversion. The individual mutagens were effective in the order UV > NA > γ-rays > AO > EMS. Prior treatment of yeast cultures in starvation medium produced a significant reduction in the yield of induced gene conversion.The results have been interpreted on the basis of a general model of mitotic gene conversion which involves the post-replication repair of induced lesions involving de novo DNA synthesis without genetic exchange. In contrast mitotic crossing-over appears to involve the action of a repair system independent from excision or post-replication repair which involves genetic exchange between homologous chromosomes.     

Structural perturbation and enhancement of the chaperone-like activity of alpha-crystallin by arginine hydrochloride

Structural perturbation of alpha-crystallin is shown to enhance its molecular chaperone-like activity in preventing aggregation of target proteins. We demonstrate that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side-chains, increases the chaperone-like activity of calf eye lens alpha-crystallin as well as recombinant human alphaA- and alphaB-crystallins. Arginine-induced increase in the chaperone activity is more pronounced for alphaB-crystallin than for alphaA-crystallin. Other guanidinium compounds such as aminoguanidine hydrochloride and guanidine hydrochloride also show a similar effect, but to different extents. A point mutation, R120G, in alphaB-crystallin that is associated with desmin-related myopathy, results in a significant loss of chaperone-like activity. Arginine restores the activity of mutant protein to a considerable extent. We have investigated the effect of arginine on the structural changes of alpha-crystallin by circular dichroism, fluorescence, and glycerol gradient sedimentation. Far-UV CD spectra show no significant changes in secondary structure, whereas near-UV CD spectra show subtle changes in the presence of arginine. Glycerol gradient sedimentation shows a significant decrease in the size of alpha-crystallin oligomer in the presence of arginine. Increased exposure of hydrophobic surfaces of alpha-crystallin, as monitored by pyrene-solubilization and ANS-fluorescence, is observed in the presence of arginine. These results show that arginine brings about subtle changes in the tertiary structure and significant changes in the quaternary structure of alpha-crystallin and enhances its chaperone-like activity significantly. This study should prove useful in designing strategies to improve chaperone function for therapeutic applications.     

Effect of quinacrine treatment on the activity of acid hydrolases (phosphatase, N-acetyl-beta-glucosaminidase, glucosidase, galactosidase and esterase) in Tetrahymena.

The effect of phospholipase A2 (PLA2) inhibitor, quinacrine, on the activity of hydrolytic enzymes in Tetrahymena pyriformis homogenate, was investigated. The activity of all of the enzymes studied (acid phosphatase, N-acetyl-beta-glusosaminidase, glucosidase, galactosidase and esterase) was significantly reduced in the presence of quinacrine. Since there are no data on the inhibitory effect of PLA2 and PLA2 influenced metabolic pathways to the hydrolytic enzymes, the direct effect of quinacrine on the hydrolytic enzymes (of Tetrahymena) can be supposed. This is supported by the fact that the other PLA2 inhibitor, 4-bromophenacyl bromide, did not influence phosphatase activity.     

The absolute configuration of the enantiomers of 6-chloro-9-(4′-diethylamino-1′-methylbutyl)-amino-2-methoxyacridine (quinacrine)

Absolute configurations have been assigned to the enantiomers of the antimalarial drug quinacrine dihydrochloride. Condensation of (?)-(R)-4-amino-1-diethylaminopentane (from L -glutamic acid) with 6,9-dichloro-2-methoxyacridine gave (?)-(R)-6-chloro-9-(4′-diethylamino-1′-methylbutyl) amino-2-methoxyacridine [(?)-(R)-quinacrine] while (+)?(S)-quinacrine was obtained from the enantiomeric diamine.     

Dynamics of ribosomal activity and protein production in peripheral blood lymphocytes during an immune response

Microscopic spectrofluorimetry with acridine orange and 1-anilino-8-naphthalene sulfonate was used to assess the levels of translationally active rRNA (parameter α’, red/green emission ratio) and protein synthesis (parameter β, blue/green) in peripheral blood lymphocytes during rabbit immunization with ovalbumin. The averaged α’ and β changed synchronously and increased maximally after the third booster injection. At the height of the immune response, the distributions of lymphocytes in either parameter were broad and polymodal, suggesting differential activation of particular subpopulations. This two-dye, three-color assay holds much promise for monitoring the functional state of various cells.     

The longevity and activity of the primary root of maize

The longevity of the main root cylinder and the laterals of the primary root of maize plants was determined under controlled greenhouse conditions by means of nuclear staining with acridine orange. The cortex of the main root was found to be alive for the whole life-span of the plant, whereas the life-span of the root hairs was only 2 to 3 days as evidenced by electronmicroscopical examination of cell integrity. The onset of senescence of laterals was observed at the older part of the main root at the 6-leaf stage of the plant. Senescence of 1st and 2nd order laterals commenced near the root tip a few days after their protrusion and advanced towards the basal region of the root. In any root segment death of the cortex cells preceeded that of the stele. At the late grain filling stage all laterals along the main root exhibited advanced senescence, but stainable nuclei were seen in the root tissues of the basal part of 1^st order laterals (both cortex and stele) as well as of the 2^nd order laterals which emerged from that root segment. The pattern of the dying of the root tissue is discussed with regard to the P-nutrition of the shoot system by the primary root.     

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine

Summary 1. Site directed mutagenesis was used to alter the structure ofTorpedo californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed inXenopus laevis oocytes injected within vitro synthesized mRNA and the whole cell currents induced by acetylcholine (ACh) were recorded by two electrode voltage clamp.2. A series of mutations of a highly conserved Arg at position 209 of the subunit ofTorpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (R209K) or His (R209H) at position 209 shifted the EC₅₀ for ACh slightly from 5µM to 12µM and increased the normalized maximal channel activity 8.5-and 3.2-fold, respectively.3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in both wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition.4. Further mutations at different positions of the subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibition whereas mutationsI210A andL212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine.5. These findings support a hypothesis that the quinacrine binding site is located in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the beginning of M1 region might be sheet structure.     

The expression of p75 neurotrophin receptor protects against the neurotoxicity of soluble oligomers of beta-amyloid

In this paper, evidence is provided that p75 neurotrophin receptor (p75NTR) exerts an opposite role on the cytotoxic function of β-amyloid (Aβ) depending on the different state of the peptide, fibrillar or oligomeric soluble form. Previous work in our laboratory has shown that the expression of p75NTR is required for cell death in vitro by Aβ peptides in fibrillar form (G. Perini, V. Della-Bianca, V. Politi, G. Della Valle, I. Dal-Pra, F. Rossi, U. Armato. Role of p75 neurotrophin receptor in the neurotoxicity by beta-amyloid peptides and synergistic effect of inflammatory cytokines. J. Exp. Med. 195 (2002) 907-918). In the present study, performed by using the same cell clones and procedures as in previous paper, we show that: (a) soluble oligomers of Aβ(1-42) exert a cytotoxic activity independent of p75NTR, (b) the expression of p75NTR exerts a protective role against the toxic activity of soluble oligomers, (c) this role is due to an active function of the juxtamembrane sequence of the cytoplasmic region of p75NTR and (d) the protective function is mediated by phosphatidylinositide 3-kinase (PI3K) activity.     

Alteration of the enzymatic properties of smooth muscle myosin by a monoclonal antibody against subfragment 2.

The fluorescent dye 10-N-nonyl acridine orange (NAO), known as specifically associated with mitochondria, has been reported to have a cytotoxic effect when high doses were applied to cells. Presently, the biochemical basis of its toxicity was investigated on isolated rat liver mitochondria. At low concentrations, NAO strongly inhibited state 3 respiration and ATP synthesis. At high concentrations, electron transport, ATP hydrolysis, P_i-transport and adenine nucleotide activities were also decreased. All these inhibitions can be explained by probe-cardiolipin interactions which could induce the collapse of energy conversion and/or the modification of membrane fluidity.     

Specific interaction of the new fluorescent dye 10-N-nonyl acridine orange with inner mitochondrial membrane: A lipid-mediated inhibition of oxidative phosphorylation

The fluorescent dye 10-N-nonyl acridine orange (NAO), known as specifically associated with mitochondria, has been reported to have a cytotoxic effect when high doses were applied to cells. Presently, the biochemical basis of its toxicity was investigated on isolated rat liver mitochondria. At low concentrations, NAO strongly inhibited state 3 respiration and ATP synthesis. At high concentrations, electron transport, ATP hydrolysis, P_i-transport and adenine nucleotide activities were also decreased. All these inhibitions can be explained by probe-cardiolipin interactions which could induce the collapse of energy conversion and/or the modification of membrane fluidity.