Some quantitative aspects of Acridine Orange fluorescence in unfixed, sucrose-isolated mammalian nuclei

Synopsis Nuclei were isolated from mouse liver and central nervous system (CNS). These nuclei were fluorochromed without fixation in a 0.25m sucrose medium containing 5.5×10^–5 m Acridine Orange and measured with an incident microfluorometric system. In the case of mouse CNS nuclei, the major and minor axes of the nuclei were measured with a filar micrometer. Three modal values were obtained from the hepatocyte suspension corresponding to 2C, 4C, and 8C nuclei, respectively. While the CNS nuclei displayed substantial variability in size, the Acridine Orange emission values at 530 nm were nearly constant. The data suggest that under these conditions, Acridine Orange fluorescence reflects DNA content. Further, the 530 nm fluorescence emission is not affected by chromatin condensation or proteins complexed with DNA.     

Evidence for plasmid-mediated toxin production inBacillus cereus BIS-59

A strain ofBacillus cereus, isolated from shrimps pasteurized by radiation, harboured a 7.6mda plasmid. When cells were cured with Acridine Orange, they still produced the haemolytic toxin but not the non-haemolytic one. The results therefore suggest that the plasmid encodes the gene(s) responsible for the non-haemolytic toxin.     

The relationship between DNA strand-scission and DNA synthesis inhibition in HeLa cells treated with neocarzinostatin.

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drug's ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.     

Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli.

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.     

p-Aminophenol induced DNA damage and cytotoxicity enhanced by autoxidation

p-Aminophenol inhibits DNA synthesis and alters the structure of DNA. A decrease in sedimentation of nucleoids from cells treated with p-aminophenol was observed and this decrease in sedimentation was considerably less when cells were incubated with p-aminophenol in an atmosphere of nitrogen or at lower pH values. This compound was also shown to be cytotoxic to cells in culture. These results demonstrate that conditions retarding the autoxidation of p-aminophenol lead to reduced effects on DNA structure and a lesser cytotoxic effect.     

An application of Acridine Orange fluorescent staining to the micronucleus test

Acridine Orange fluorescent staining was applied to the micronucleus test in mice and rats. Micronuclei emitted bright green fluorescence and were easily distinguished from micronucleus-like inclusions or contaminants. In rat bone-marrow cells, micronuclei with green fluorescence could be easily distinguished from granules accidentally dispersed from broken mast cells, which showed bright red fluorescence. Therefore, it is recommended that the Acridine Orange staining method be used to provide more reliable data in the micronucleus test.     

Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

Abstract We have proposed that guanosine tetraphosphate produced in Escherichia coli cells subjected to an isoleucine limitation inhibits pBR322 DNA replication [1]. In E. coli relA which cannot synthesize guanosine tetraphosphate (ppGpp) upon amino acid limitation pBR322 DNA is amplified after arginine starvation. The yield of plasmid DNA amplified either by chloramphenicol (Cm) or by arginine limitation is compared. The plasmid yield per cell is equal in amino acid-starved cells and in cells treated with Cm. To increase the plasmid content per ml of cell suspension the growth medium was supplemented with increasing amounts of nutrients. Plasmid DNA can be isolated in large quantities by this procedure. This simple method can be used for the enrichment of pBR325 DNA which cannot be amplified by Cm treatment. Our results indicate that E. coli relA strains might be suitable hosts for the amplification of pBR322 and related plasmids in E. coli .     

Behavior of theophylline-sensitive T lymphocytes in viral A, B, non-A, non-B hepatitis. I. Methodological aspects

To evaluate the behaviour of the Theophylline-sensitive T lymphocytes subpopulation some modifications of the standard procedure are proposed. Lymphoprep purified lymphocytes were counted in a Neubauer hemocytometer after Acridine Orange stain, viability was evaluated by Ethidium Bromide counterstain and monocytes contamination was evaluated by the peroxidase stain. Sheep red blood cells were treated with AET, Theophylline was used at 3 mM (final concentration) and the results compared with untreated lymphocytes; the enumeration of the rosetting lymphocytes was facilitated by adding Acridine Orange prior to the resuspension. The modifications described were able to increase the % of rosetting T lymphocytes, to eliminate differences depending by different lots of sheep red blood cells and to decrease differences depending by subjective evaluation of the rosetting T lymphocytes.     

Broadening by acridine orange of the thermal transition of DNA

Acridine Orange, at appropriate intermediate concentrations, causes a substantial broadening of the thermal transitions of Bacillus subtilis DNA and of dAT. Experiments in which the two polymers are healed together show that the broadening is the result of the transfer of acridine orange molecules from denatured to native DNA molecules.     

Further evidence for the DNA base specificity of light-induced banding

Summary Experiments have been carried out to investigate the DNA base specificity of light-induced banding (LIB) produced by photo-oxidation of chromosomes followed by Acridine Orange staining to detect denatured DNA. Nuclei of different base composition, human and onion, and fluorochromes of different base specificities and modes of binding to DNA were used. Our results indicate that specific destruction of guanine residues is the main effect of photo-oxidation under the conditions used, and that LIB is a base-specific phenomenon. In addition, photo-oxidation may also cause DNA-protein cross-linking which affects the binding of some dyes, while prolonged photo-oxidation appears to cause more general damage to DNA.     

Transformation of Xenorhabdus nematophilus.

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.     

Transformation of Xenorhabdus nematophilus

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.     

栉孔扇贝血细胞吞噬和包囊化作用实验方法的改进

为了建立一种快速、准确观察血细胞吞噬和包囊化作用的实验方法,通过抽取栉孔扇贝(Chlamysfarreri)血淋巴,与杆菌或茶花花粉作用30 min,制片,吖啶橙染色,用荧光显微镜观察吞噬或包囊化现象。结果表明,在荧光显微镜下可以明显看到扇贝血细胞呈现绿色,杆菌和茶花花粉呈现红色,两者颜色反差大,易于观察和计数。是研究血细胞吞噬和包囊化作用一种效果更好的实验方法。     

Optimization of an Acridine Orange–Bisbenzimide Procedure for the Detection of Apoptosis-Associated Fluorescence Colour Changes in Etoposide-Treated Cell Cultures

This study was initiated in order to investigate the possibility of improving fluorescence microscopy as a method for evaluating apoptosis in cells by combining two fluorescent dyes with different staining characteristics. Cells were vitally stained with bisbenzimide (1.3 microM) and Acridine Orange (6.6 microM) and observed using the following filter configuration: excitation 380 nm, beamsplitter 395 nm and longpass filter 397 nm. Control cells exhibited clear blue fluorescent nuclei and red fluorescing lysosomes. In cells treated with etoposide to induce apoptosis, two distinct occurrences were observed: a change in the spectrum of emitted light from bisbenzimide bound to the nuclear region and an increase in lysosomal Acridine Orange fluorescence. The two occurrences together permit a more unbiased detection of apoptosis than most assays. Only one filter set is required for evaluation and the resulting images can be easily evaluated visually or processed further by image analysis.     

Fluorescence microscopic study of cells and polykaryons in the early periods following polyethylene glycol treatment

A fluorescence-microscopical study is made of cultured murine fibroblasts (L-cells) in early periods after the treatment with polyethylene glycol (PEG). Optimal conditions of fusion procedure were found under which the effectiveness of fusion was the highest and the toxical effect of PEG the lowest. The number of dead cells after the treatment with PEG did not exceed 10%. No significant changes in chromatin cytochemical properties (Acridine Orange and Olivomycin binding) were observed in the early periods of PEG treatment, that allows to use PEG for studying chromatin properties in hybrid cells obtained by PEG fusion. By means of PEG fusion, the hybrid cells with prematurely condensed chromosomes and also hybrids between animal and yeast cells have been obtained.     

Paraquat and roundup effects on nucleases activities of alfalfa seedlings and alfalfa nucleases activities on paraquat-treated and roundup-treated nucleic acids

The specific activities of acid (pH 5.5) and neutral (pH 7) DNases and RNases were determined in alfalfa (Medicago sativa L.) seedlings grown in the dark in the presence of 3.7 mM paraquat (PQ) or 1 mM roundup (RD). Seedlings were taken at 0, 1, 3, and 5 days. Plant growth parameters (plant height and fresh weight) were dramatically reduced under these conditions of growth comparing to the control (grown in water). The DNase and RNase specific activities of herbicide-treated seedlings were reduced. The reduction of activities ranged by about 50–90 and 15–70% in PQ- and RD-treated seedlings, respectively. In vitro, PQ- and RD-treated nucleic acids [single-stranded DNA (ssDNA), RNA, and plasmid DNA (pl-DNA)] were incubated with acid and neutral nucleases. Both enzymes were isolated and purified from alfalfa seedlings. Electrophoretic analysis on agarose gel of the above incubated mixtures revealed the following: (a) neutral nuclease (pH 7) was capable of hydrolyzing PQ-treated ssDNA while acid nuclease (pH 5.5) was incapable. This could be due to the fact that acid and neutral nucleases displayed different base linkage specificity toward ssDNA; (b) RD formed strong complexes with ssDNA that were unable to be hydrolyzed by both nucleases; (c) in contrast, both enzymes were capable of hydrolyzing PQ- or RD-treated RNA; (d) neutral nuclease was capable of nicking and linearizing both PQ- and RD-treated pl-DNA while acid nuclease had the same activity only toward the PQ-treated pl-DNA; (e) the enzyme activities were not inhibited in the presence of both herbicides. The data suggest that the complexes of PQ or RD with DNA should not be functional substrates of nucleases, and consequently cell processes (e.g., metabolism of nucleic acids, gene expression, replication), in which DNA and nucleases are involved, could be disturbed.     

Microbiological Comparison of Surface Soil and Unsaturated Subsurface Soil from a Semiarid High Desert

Thirty-two chemoheterotrophic bacteria were isolated from unsaturated subsurface soil samples obtained from ca. 70 m below land surface in a high desert in southeastern Idaho. Most isolates were gram positive (84%) and strict aerobes (79%). Acridine orange direct counts of microbes in one subsurface sample showed lower numbers than similar counts performed on surface soils from the same location (ca. 5 × 10⁵ versus 2 × 10⁶ cells per g [dry weight] of soil), but higher numbers than those from plate counts performed on the subsurface material. Another sample taken from the same depth at another location showed no evidence of colonies under identical conditions. Soil analyses indicated that subsurface sediments versus surface soils were slightly alkaline (pH 7.9 versus 7.4), had a higher water content (25.7 versus 6.3%), and had lower organic carbon concentrations (0.05 to 0.17 versus 0.25% of soil dry weight). Analyses of biologically relevant gases from the unsaturated subsurface indicated an aerobic environment. As in other unsaturated soil environments, either a high proportion of bacteria in these subsurface sediments are not viable or they are incapable of growth on conventional media under aerobic conditions. The presence and numbers of bacteria in these deep sediments may be influenced by colonization opportunities afforded by periodic percolation of surface water through fractures in overlying strata.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Nalidixic Acid

During the conjugal transfer of the R64-11 plasmid at 42 C from donor cells thermosensitive for vegetative deoxyribonucleic acid (DNA) synthesis to recipient minicells, the plasmids are conjugally replicated in the donor cells. This conjugal replication is inhibited by nalidixic acid, and the degree of inhibition is comparable to the reduction in the amount of plasmid DNA transferred to the recipient minicells in the presence of the drug. In addition, the size of DNA transferred to the minicells and the fraction of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA under alkaline conditions are both reduced by nalidixic acid. When the drug is added to a mating that is underway, the rate of conjugal replication is immediately reduced. This change is accompanied by a reduction in the amount of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA. Furthermore, conjugally replicated plasmid DNA that is not associated with the donor cell membrane becomes membrane bound after the addition of nalidixic acid.