Acridine Orange Staining and Fluorescence of Nucleic Acids in Plasmodia and Associated Host Erythrocytes

Acridine orange in daily doses of 1, 2 and 4 mg for 4 days was given to chicks averaging 50 gm in weight. Dosage was started 1, 2 and 3 days after infection with Plasmodium gallinaceum. Such doses were sufficient to stain the parasite in vivo, as shown by its bright fluorescence in UV light, but did not exhibit any antimalarial action. Staining of fresh blood samples from infected chicks with 0.01% acridine orange in Krebs-Ringer containing 0.1 M phosphate buffer (pH 6.0-6.2) resulted in differential fluorescence of the nucleic acids of the plasmodia, to show nuclear DNA bright green and cytoplasmic RNA orange-red. After optimum acid hydrolysis, as used for the Feulgen reaction, staining with 0.1% acridine orange produced intense red fluorescence of the nuclear DNA in the plasmodia. Nuclear DNA of the chick erythrocytes showed bright fluorescence both in vivo and in vitro.     

A Color Image Analysis Method for Assessment of Germination Based on Differential Fluorescence Staining of Bacterial Spores and Vegetative Cells Using Acridine Orange

Color fluorescence image analysis of acridine orange (AO) stained germinating Bacillus subtilis var. niger bacteria revealed a cell population initially dominated by small green spores followed by the emergence of at least three additional discernible subpopulations in response to stimulation with D-glucose. These subpopulations were small, round or oblong red cells; intermediate to large metachromatic cells; and large red rods. Large green rods were rarely observed. An increase in red emissions (i.e., putative RNA synthesis) was sometimes seen as early as 90 min after exposure to D-glucose and uptake of AO at room temperature. This may represent either metabolic recovery from quiescence or RNA synthesis associated with germination. In the absence of D-glucose, or using autoclaved bacteria in the presence of glucose, no relative increase in the red signal was observed despite hours of observation. Digital image analysis was used for relative measurement of red, green and blue signals and to correlate the size of various subpopulations with their fluorescence color emissions over time. Image analysis demonstrated a trend toward increasing size and red emission in the presence of glucose. The average red emission was found to be a good discriminator of the various subpopulations, while the average green emission was approximately equal among the subpopulations making it a poor discriminator. These data suggest that AO staining might be used for rapid computer-assisted discrimination of spores vs. vegetative cells.     

Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and, under severe conformational constraints, is dictated by its one-dimensional geometry. How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.     

Absorption and Fluorescence Spectra of Euchrysine Ggnx and Acridine Orange; Effects of Heparin, NaCl and a Cation Exchange Resin

The absorption and fluorescence spectra of two samples of dye labeled euchrysine were found to differ. One sample, labeled GGNX, had absorption and fluorescence maxima of 435 and 515 nanometers (nm) respectively. The other sample was not further labeled, but had absorption and fluorescence maxima of 492 and 535 nm. The latter values, as well as the shape of both the fluorescence and absorption curves of the second sample were superimposable on a recrystallized sample of acridine orange labeled correctly C. I. 46905. Euchrysine has two free amino groups which are fully methylated in acridine orange, therefore a nitrous acid test can differentiate the two dyes. The sample of euchrysine labeled GGNX gave a reaction, as did acridine yellow, C. I. 46025, but acridine orange, C. I. 46005, did not. Fluorescence metachromasy of euchrysine is less efficient than that of acridine orange in two ways: the shift in the spectrum is smaller by about 40 nm, making the separation of the colors more difficult both visually and by instruments and the metachromatic fluorescence has less than half of the intensity of acridine orange as measured at the peak for each dye. Confusion between these two dyes has occurred because suppliers have used the names interchangeably. For critical studies, the dye used should be identified by its Colour Index number.     

Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments.     

Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions: Implications for the Use of Acridine Orange as a pH Probe

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I⁻, ClO₃⁻, NO₃⁻, Br⁻, or SCN⁻, acridine orange reported lower pH gradients than either quinacrine or [¹⁴C]methylamine. Acridine orange, but not quinacrine, reduced [¹⁴C]methylamine accumulation when NO₃⁻ was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO₃⁻ increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24°C than at 12°C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H⁺, possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO₃⁻/H⁺ symport involved in the export of NO₃⁻ from the vacuole is probably an artefact caused by acridine orange.     

Acridine Orange/exosomes increase the delivery and the effectiveness of Acridine Orange in human melanoma cells: A new prototype for theranostics of tumors

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466?nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

Induction of Oligosporogeneous and Asporogeneous Mutants of Blue-green Alga, Anabaena doliolum by Acriflavine and Acridine Orange

Acriflavine and acridine orange were highly effective in producingmutants defective in spore differentiation in the blue-greenalga, Anabaena doliolum. Acridine orange produced both oligosporogeneous(Osp) and asporogeneous (Sp^–) mutants in high yield withthe frequency ranging from 2.4 to 21 per 10⁴ survivors, whereaswith acriflavine no oligosporogeneous mutants were detected,although it produced non-sporulating (Sp^–) mutants withthe frequency of 1.4 per 10⁴ survivors. Mutants isolated throughthese dyes were stable and showed no tendency towards spontaneousor induced reversion. Behaviour of various mutants indicatedtheir being blocked at different stages of sporulation withdifferent mutagenic sites. Induction of high numbers of Ospand Sp^– mutants after acridine dye treatment indicatesthe involvement of an extrachromosomal determinant in sporulationin Anabaena doliolum. Ohgosporogeneous mutants, asporogeneous mutants, Anabaena doliolum, blue-green algae, mutagenesis, acriflavine, acridine orange     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

Organization of FtsZ Filaments in the Bacterial Division Ring Measured from Polarized Fluorescence Microscopy

Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division.     

Organization of FtsZ Filaments in the Bacterial Division Ring Measured from Polarized Fluorescence Microscopy

Cytokinesis in bacteria is accomplished by a ring-shaped cell-division complex (the Z-ring). The primary component of the Z-ring is FtsZ, a filamentous tubulin homolog that serves as a scaffold for the recruitment of other cell-division-related proteins. FtsZ forms filaments and bundles. In the cell, it has been suggested that FtsZ filaments form the arcs of the ring and are aligned in the cell-circumferential direction. Using polarized fluorescence microscopy in live Escherichia coli cells, we measure the structural organization of FtsZ filaments in the Z-ring. The data suggest a disordered organization: a substantial portion of FtsZ filaments are aligned in the cell-axis direction. FtsZ organization in the Z-ring also appears to depend on the bacterial species. Taken together, the unique arrangement of FtsZ suggests novel unexplored mechanisms in bacterial cell division.     

Birefringence and DNA Condensation of Liquid Crystalline Chromosomes

DNA can self-assemble in vitro into several liquid crystalline phases at high concentrations. The largest known genomes are encoded by the cholesteric liquid crystalline chromosomes (LCCs) of the dinoflagellates, a diverse group of protists related to the malarial parasites. Very little is known about how the liquid crystalline packaging strategy is employed to organize these genomes, the largest among living eukaryotes—up to 80 times the size of the human genome. Comparative measurements using a semiautomatic polarizing microscope demonstrated that there is a large variation in the birefringence, an optical property of anisotropic materials, of the chromosomes from different dinoflagellate species, despite their apparently similar ultrastructural patterns of bands and arches. There is a large variation in the chromosomal arrangements in the nuclei and individual karyotypes. Our data suggest that both macroscopic and ultrastructural arrangements affect the apparent birefringence of the liquid crystalline chromosomes. Positive correlations are demonstrated for the first time between the level of absolute retardance and both the DNA content and the observed helical pitch measured from transmission electron microscopy (TEM) photomicrographs. Experiments that induced disassembly of the chromosomes revealed multiple orders of organization in the dinoflagellate chromosomes. With the low protein-to-DNA ratio, we propose that a highly regulated use of entropy-driven force must be involved in the assembly of these LCCs. Knowledge of the mechanism of packaging and arranging these largest known DNAs into different shapes and different formats in the nuclei would be of great value in the use of DNA as nanostructural material.DNA molecules are the indispensable genetic material of every organism. Apart from having the encoding power of a 4-base double-stranded polymer, DNA also has an enormous capacity to be condensed. It is probably this ability that allowed the evolution of increasing genome sizes in the eukaryotes. Histone-mediated nucleosome-based chromatin is the prevailing method for DNA packaging, but it is not the only way that DNA is condensed in the eukaryotes. Highly compact liquid crystalline DNA has been reported in several animal sperm nuclei (11, 28) and was also found in the nucleosomeless liquid crystalline chromosomes (LCCs) of dinoflagellates. Neither animal sperm nuclei nor dinoflagellate nuclei employ histones. In fact, the histone core octamer may hinder the attainment of ultrahigh levels of condensation due to its restrictive volume. In the sperm model, protamine is a sperm-specific DNA-binding protein (∼7 kDa) that is an order of magnitude smaller than the histone core octamer, and it adopts a structural role in the organization of the male gametic genome (1, 3). It is this very reduction in the protein-to-DNA ratio that may well enable the high DNA compaction into a liquid crystalline state. The dinoflagellate chromosomes are known to have a protein-to-DNA ratio even smaller than those of the prokaryotes (24). The typical eukaryotic genome has a protein/DNA ratio of 1:1, whereas the dinoflagellate genome has a ratio of 1:10 (23, 24).The dinoflagellates, counterintuitively, have the largest known genomes among all living organisms, with a DNA content per genome ranging from 1.5 pg to 200 pg per haploid cell (19, 25, 43). An extraordinarily high level of DNA condensation must be attained in order to sequester these genomes within the bounds of the nucleus, and this is achieved through the form of LCCs. The concentration of the DNA in the dinoflagellate nucleus was estimated to be ∼200 mg ml⁻¹ (up to 80 times more than a human cell) (24), falling well within the range observed for in vitro cholesteric liquid crystalline DNA formation (40). By observing ultrathin sections of dinoflagellate chromosomes using high-resolution transmission electron microscopy (TEM), plectonemic structures with nested series of arches and bands were seen (8, 12, 39). This architecture resembles the molecular architecture of thin-section TEM in vitro cholesteric liquid crystals (12, 39, 43).Knowledge of the condensation process would not only be of interest in terms of eukaryotic genome packaging, but would also provide new insights for the use of DNA as building blocks for nanotechnology applications. Self-assembly is increasingly understood to be of great significance in the regulation of various macromolecules in the crowded environment of living cells (7, 15, 16, 35, 36). Interestingly, it was also recently reported that nucleosomes and polynucleosomes themselves can be self-assembled (17), and in liquid crystalline form (33). The formation of LCCs probably also relies upon the associated chromosomal proteins, while self-assembly of DNA molecules via neutralization of the negatively charged polyelectrolytes by counterions and the crowding effects within the permanently closed nucleus must also contribute significantly (36, 49, 50). Most of the previous studies of dinoflagellate LCCs were focused on the chromosomes of Prorocentrum micans (27, 29). Though it has been reported that not all dinoflagellate chromosomes show the same degree of birefringence (5), the relationships of different species and their DNA contents, densities, or compaction ratios to birefringence have not been reported.The presence of two indices of refraction and optical anisotropy confer on the liquid crystalline DNA the property of birefringence, or double retardance. This optical phenomenon describes the phase differences between the two resultant light rays of a given light path that pass through the liquid crystal according to its refractive indices. For any given anisotropic material, the birefringence is a direct result of its nanoarchitecture. The property of birefringence and polarizing microscopy have long been employed to study the submicroscopic molecular organizations of different biological samples, such as mitotic spindles (5), filamentous actins (22), and microtubules (38), in living cells. Polarizing microscopy also allows the documentation of dynamic cellular behavior and measurements of cellular structures to be performed in a repeated noninvasive manner, even over extended periods of time (22). Taking advantage of this, we used a recently developed automatic rotating polarizing light microscope (the Metripol system) to study the relative amounts of birefringence and the ultrastructures of the chromosomes in several dinoflagellate species. Highly diverse chromosome structures were observed among the species, and surprisingly, not all dinoflagellates exhibited birefringent chromosomes, even though they are apparently constructed with similar liquid crystalline architectures, as seen in TEM ultrathin sections. The use of a semiautomatic Metripol polarizing microscope allowed the quantification of the retardance (20, 21, 47) of individual chromosomes of different dinoflagellate species in a fixative-free environment, and the relationships between the absolute retardance, DNA content, DNA condensation, and chromosome architecture in the dinoflagellate chromosomes are established in this study.     

Snake Venom Cardiotoxins-Structure,Dynamics, Function and Folding

Abstract

Snake cardiotoxins are highly basic (pI>10) small molecular weight (～6.5 kDa), all β-sheet proteins. They exhibit a broad spectrum of interesting biological activities. The secondary structural elements in these toxins include antiparallel double and triple stranded β-sheets. The three dimensional structures of these toxins reveal an unique asymmetric distribution of the hydrophobic and hydrophilic amino acids. The 3D structures of closely related snake venom toxins such as neurotoxins and cardiotoxin-like basic proteins (CLBP) fail to show similar pattern(s) in the distribution of polar and nonpolar residues. Recently, many novel biological activities have been reported for cardiotoxins. However, to-date, there is no clear structure-function correlation(s) available for snake venom cardiotoxins. The aim of this comprehensive review is to summarize and critically evaluate the progress in research on the structure, dynamics, function and folding aspects of snake venom cardiotoxins.     

Assessment of the metabolic activity of Acremonium chrysogenum using Acridine Orange

A method is described for the assessment of the metabolic activity of the filamentous fungus Acremonium chrysogenum using the fluorescent dye Acridine Orange. Changes in metabolic activity are indicated by a reversible red-green shift in the colour of the dye, quantifiable by image analysis. A. chrysogenum mycelia exhibited an overwhelmingly green colour in circumstances leading to high substrate uptake, while under carbon starvation they appeared orange-red. It is believed these colour changes reflected changes in internal pH. The method provides a visual tool for the investigation of the metabolic behaviour of filamentous fungi.     

Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy

Abstract The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial biofilm community developed from attached single cells and distinct microcolonies via a more confluent structure characterized by various filamentous bacteria to a mature biofilm rich in polymeric material with fewer cells on a per-area basis after 56 days. During the different stages of biofilm development, characteristic microcolonies and cell morphotypes could be identified as typical features of the investigated lotic biofilms. In situ analysis using a comprehensive suite of rRNA-targeted probes visualized individual cells within the alpha-, beta-, and gamma-Proteobacteria as well as the Cytophaga–Flavobacterium group as major parts of the attached community. The relative abundance of these major groups was determined by using digital image analysis to measure specific cell numbers as well as specific cell area after in situ probing. Within the lotic biofilm community, 87% of the whole bacterial cell area and 79% of the total cell counts hybridized with a Bacteria specific probe. During initial biofilm development, beta-Proteobacteria dominated the bacterial population. This was followed by a rapid increase of alpha-Proteobacteria and bacteria affiliated to the Cytophaga–Flavobacterium group. In mature biofilms, alpha-Proteobacteria and Cytophaga–Flavobacteria continued to be the prevalent bacterial groups. Beta-Proteobacteria constituted the morphologically most diverse group within the biofilm communities, and more narrow phylogenetic staining revealed the importance of distinct phylotypes within the beta1-Proteobacteria for the composition of the microbial community. The presence of sulfate-reducing bacteria affiliated to the Desulfovibrionaceae and Desulfobacteriaceae confirmed the range of metabolic potential within the lotic biofilms. Received: 24 September 1998; Accepted: 17 February 1999