Interaction of the fluorescent probe, 1-anilino-8-napthalene sulfonate, with the sulfate transport system of Ehrlich ascites tumor cells.

The addition of the fluorescent dye, ANS, to intact ascites tumor cells results in an enhancement of fluorescence intensity. The increase in fluorescence intensity as a function of time is biphasic which suggests that at least two processes occur. The first associated with the rapid initial rise in fluorescence represents binding to the cell surface while the second or slower phase reflects entrance of ANS into the intracellular phase. The relationship between bound and free ANS in 0.50 mM sulfate medium was used to calculate the apparent dissociation constant of ANS-membrane complex (Kd = 6.53 times 10(-5) M) and the total number of ANS binding sites (4.49 nmoles/mg dry weight). Kinetic analysis of steady state sulfate transport in the presence and absence of ANS suggests that (1) sulfate exchange can be described by Michaelis Menten type kinetics (Km = 2.05 times 10(-3) M), (2) a small fraction of surface associated ANS competitively inhibits sulfate exchange (Ki = 4.28 times 10(-6) M) and (3) the transport system has a higher affinity for ANS than for sulfate. These data are consistent with the hypothesis that inhibition of sulfate exchange is related to the direct, reversible interaction of the negatively charged sulfonate group of ANS with superficial positively charged membrane sites.     

Fluorescence study of lymphocyte plasma membranes]

ANS binding parameters--dissociation constant, number of binding sites, rotation freedom--are measured by fluorescence studies of a complex between ANS and lymph node cell plasma membranes. Divalent ions, Mg++ and Ca++, enhance the complex fluorescence intensity without shifting its maximum wavelength : this enhancement is induced by affinity and quantum yield increases, while the number of binding sites remains constant. The complex fluorescence quenching by ethacrynic acid shows the presence of free SH groups in the ANS binding site. An energy transfer takes place between membrane protein tryptophan residues and bound ANS ; the energy transfer yield is unaffected by Ca++ ions. A correlation of these results is postulated with the biological activity of the membrane.     

Bactericidal action of positive and negative ions in air

Background  

In recent years there has been renewed interest in the use of air ionisers to control of the spread of airborne infection. One characteristic of air ions which has been widely reported is their apparent biocidal action. However, whilst the body of evidence suggests a biocidal effect in the presence of air ions the physical and biological mechanisms involved remain unclear. In particular, it is not clear which of several possible mechanisms of electrical origin (i.e. the action of the ions, the production of ozone, or the action of the electric field) are responsible for cell death. A study was therefore undertaken to clarify this issue and to determine the physical mechanisms associated with microbial cell death.     

The effect of negative air ions on the respiratory organs and blood

The effect of ionized air containing negatively charged ions at a concentration of 320000–350000 ions/cm³ inhaled by rats was studied. It was demonstrated that the inhalation of negative air ions for 60 min activated the secretion of goblet cells without impairing the tracheal mucosa and changing the protein profile of bronchoalveolar lavage. It was also found that the level of spontaneous production of reactive oxygen species by unfractionated blood cells increased after the action of negative air ions in both males and females. However, the intensity of their generation induced by opsonized zymosan increased only in females. Different sensitivities of the female and male blood antioxidant enzymes—superoxide dismutase and glutathione reductase—to negative air ions were observed. These results allow the effect of negative air ions on the respiratory organs and blood to be interpreted as priming and weak activation via a direct action on the mucosa of primary target respiratory organs and then on the blood.     

Effects of air ions on the membrane Na, K-ATPase activity of L 1210 cells

The effect of exposure of L 1210 mouse leukemia cells to artificially generated air ions on the activity of membrane Na, K-ATPase in the cells was investigated. The exposure of cells to air ions of both signs gave identical results, i.e. diminution of transport activity of the enzyme, measured by radioactive 86Rb transport into the cell (ouabain-dependent). Passive ouabain-independent transport of Rb+ into the cells remained unchanged in the air ion-treated cells, as did the passive efflux of the radioisotope from preloaded cells. The possible explanation of the phenomena observed is discussed.     

Influence of calcium ions in the flow cytometric analysis of human CD8-positive cells.

BACKGROUND: The CD8 co-receptor is an important marker used to identify various lymphocyte subsets. A significant decrease in CD8alpha staining intensity was observed in the presence of divalent cation chelators. METHODS: Peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers were treated with calcium chelators, stained with different anti-human CD8 mAbs, and analyzed by flow cytometry. RESULTS: Calcium chelators caused a dose-dependent decrease in fluorescence intensity, using specific anti-human CD8alpha mAbs. This phenomenon was not due to CD8 internalization and could be reversed by the addition of calcium ions. In contrast, calcium depletion increased staining intensity with one anti-CD8beta mAb. CONCLUSIONS: Divalent cation chelators are used as cell anti-clumping agents in MACS or FACS applications. Researchers should be aware that such treatment could lead to the almost complete loss of fluorescence with selected anti-human CD8alpha mAbs. Since CD8 staining is used in conjunction with tetramer staining to identify antigen-specific cytotoxic human T cells, the effect of calcium depletion should be taken into account in experimental design.     

The interaction of 1-anilino-8-naphthalenesulfonate with the membranes of the sarcoplasmic reticulum and various lipid compounds

Summary (1) The enzymatic removal of lipids from the vesicular membranes of the sarcoplasmic reticulum does not interfere with the fluorescence of the 1-anilino-8-naphthalenesulfonate (ANS) vesicular complex. (2) The fluorescence intensity of the ANS vesicular complex is considerably (50%) reduced by oleic acid (0.5mm) because it displaces ANS from its binding sites. (3) Stearic acid, which also combines with the membranes, interferes neither with ANS binding nor with ANS fluorescence. (4) Of all lipid compounds tested, oleylamine produces the most pronounced fluorescence enhancement of ANS. (5) The complexes formed between oleic acid and cetyltrimethyl ammonium salts or between oleic acid and polylysine produce a much higher fluorescence enhancement than the isolated components. (6) Low concentrations of ether added to ANS-containing vesicular suspensions reduce their fluorescence intensity. It returns to the initial intensity when the ether is removed. (7) A small cyclic change of the fluorescence of the vesicular ANS complex takes place during active calcium uptake.     

A fluorescent probe response to the interaction of pyocin R1 with sensitive cells.

Additon of pyocin R1, a bacteriocin of Pseudomonas aeruginosa, to sensitive cells caused a fluorescence increase of 8-anilino-1-naphthalenesulfonate (ANS) in the cell suspension. The reaction was rapid, starting with a short time lag after adsorption of pyocin onto the cells and finishing within several minutes. The fluorescence response was attributed to the interaction of the cell body and ANS, not to that of the medium outside the cells and ANS. The maximal amplitude of fluorescence after pyocin addition was dependent on temperature, and the relation appeared to be biphasic. Similarly, Arrhenius plots of the initial rate of fluorescence change were biphasic. The transition of slopes in both cases occurred in the temperature range between 18 and 19 degrees. These results suggest that ANS interacts with lipids in the cell envelope and that pyocin causes a structural change of the cell envelope leading to increased fluorescence of ANS.     

Effect of air ions on submicron t1 bacteriophage aerosols

The effect of a high concentration of ionized air molecules on sampling T1 phage aerosols of submicron particle size was evaluated by comparing the phage recoveries of all-glass impingers (AGI-4) and type 6 filter papers. Sampler recoveries of all ionized aerosols were less than the recoveries of nonionized control aerosols. These reductions in recovery were greater with positive ions than with negative ions or ions of mixed polarity. The AGI-4 allowed considerable slippage, which was not affected by the air ions. Type 6 filter paper recoveries were less than AGI-4 recoveries. The air ions did not appear to affect the aerosol particle size as determined by an electron microscope.     

The influence of diffusion potentials across liposomal membranes on the fluorescence intensity of 1-anilinonaphthalene-8-sulphonate

The influence of diffusion potentials across different phospholipid membranes on the fluorescence intensity of 1-anilinonaphthalene-8-sulphonate (ANS) was studied. With liposomes or chloroform spheres covered with a monolayer of egg lecithin, no specific effects were found. With liposomes of soy-bean phospholipids, generation of a diffusion potential leads to an enhancement or decrease, depending on the direction of the potential, of the intensity of ANS fluorescence. This effect is mainly due to a change in quantum yield of the bound ANS. These data support a mechanism according to which ANS molecules are pushed into or pulled out of the membrane by a potential, but not an electrophoretic one in which the potential causes movement of ANS across the membrane.     

Effect of negative air ions on respiratory organs and blood

The effect of negatively charged ions on respiratory organs and blood of rats has been studied. It was shown that the inhaling of negative air ions (NAI) for 60 min with a concentration of NAI at the place of location of animals 320-350 000 ions/cm2 activated the secretion of goblet cells without damaging the mucosa of the trachea and changed the spectrum of proteins of bronchopulmonary lavage. It was also found that the spontaneous production of reactive oxygen species (ROS) by cells of nonfractionated blood after the exposure to NAI increased in both males and females; the intensity of ROS generation induced by opsonized zymosan increased only in females. Different sensitivity of the antioxidant enzymes superoxide dismutase and glutathione reductase of blood to NAI in females and males was revealed. These results enable one to consider the effect of NAI as priming and a weak activation of the respiratory organs through the direct action on the mucosa of the primary target organs of the respiratory tract and then on the blood.     

Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells

The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino-1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.     

Ionic changes in the mitotic apparatus at the metaphase/anaphase transition

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane- associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.     

Detection of conformational changes in chloroplast coupling factor 1 by 8-anilino-1-naphthalene-sulphonate fluorescence changes

Chloroplast coupling factor 1 (CF1) contains a high-affinity binding site for 8-anilino-1-napthalene sulphonate (ANS,Kd = 5-6 microM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue-shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1. Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 microM to 22 microM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n = 1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals. The affinity of latent CF1 for ADP (Kd = 12 microM) is considerably higher than for ATP (Kd = 95 microM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 microM. Mg increases the apparent affinity for ATP (Kd = 28 microM) but not for ADP or Pi. Binding of ATP to the tight-sites does not inhibit the ADP or Pi-induced fluorescence quenching but decreases the affinity for ADP (Kd = 34 microM) and for inorganic phosphate (Kd = 320 microM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites. Activation of a Mg-specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis. ATPase activation also influences the ANS responses to Ca and Mg. Ca-ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg-ATPase activation specifically increases the Mg-induced fluorescence enhancement. The fluorescence of CF1-bound ANS is enhanced by Dio-9 and quenched by phloridzin, quercetin, Nbf-Cl and FITC. Nbf-Cl and FITC completely inhibit the ADP-induced fluorescence quenching whereas Dio-9 inhibits the Mg-induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site. ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.     

Mast cell-lysosomal cationic protein interaction: effects of metabolic inhibitors and decay of activated cells on enhanced fluorescence of anilinonaphthalene sulfonate

It has been shown earlier that the interactions of the isolated rat peritoneal mast cells with cationic protein from rabbit neutrophil lysosomes (band 2 protein) can be studied using anilinonaphthalene sulfonate (ANS) as a fluorescent probe. In the present communication, binding of ANS dye to the mast cells interacting of histamine release by metabolic inhibitors was found to have no effect on enhancement of ANS fluorescence. On the other hand, inhibition of histamine release at high concentration of Ca2+ (14.4 mM) was accompanied by the decrease in enhance fluorescence. In the presence of 7.2 mM of Sr2+, the release of histamine was enhanced with small but significant increase in ANS fluorescence. The cells heated to 42 degrees C partially lost their capacity to release histamine without the loss of enhanced fluorescence. The mast cells interacting with B2 at 10 degrees C for various time intervals showed time-dependent loss in histamine releasing capacity with concomitant loss in enhanced fluorescence. These studies suggest that the enhancement of ANS fluorescence is associated with the early events of the cell membrane caused by interaction of B2 with the cells. The extracellular cations significantly influence this early event.     

The interaction of the anionic fluorescence probe, 1-anilinonaphthalene-8-sulfonate, with hepatocytes and hepatoma tissue culture cells

The fluorescence probe 1-anilinonaphthalene-8-sulfonate (ANS) has been used to characterize the anion transport properties of normal hepatocytes and hepatoma tissue culture cells. Incubation of hepatocytes in the presence of ANS (20 micron) resulted in a 35-fold enhancement of fluorescence and a 50 nm blue shift. The time course of this process is biphasic. A rapid initial fluorescence enhancement suggests ANS binding to the plasma membrane, and a slower component reflects the uptake of ANS into intracellular compartments. Analysis of ANS uptake showed this latter process to be saturable, with a Km of 10 micron, to be temperature dependent and to occur only in viable cells. The above observations suggest a carrier-mediated anion transport mechanism. Incubation of hepatoma tissue culture cells with ANS (20 micron) gave a fluorescence emission spectrum similar to that obtained from purified plasma membranes. The kinetics of this interaction only exhibited a rapid initial binding of ANS. The second slow component was now absent, suggesting that ANS transport by the malignant cell system was greatly reduced. Transport of ANS could, however, be stimulated in the presence of the local anesthetic tetracaine. The observed transport was now saturable, temperature dependent, and as in normal hepatocytes, required viable cells, again indicating a carrier-mediated transport system. These studies suggest a significant alteration in membrane function in hepatoma tissue culture cells resulting in a major defect in anion transport.     

Microirradiation of cells with energetic heavy ions

The ion microprobe SNAKE at the Munich 14 MV tandem accelerator achieves beam focussing by a superconducting quadrupole doublet and can make use of a broad range of ions and ion energies, from 20 MeV protons to 200 MeV gold ions. Because of these properties, SNAKE is particularly attractive for biological microbeam experiments. Here we describe the adaptation of SNAKE for microirradiation of cell samples. This includes enlarging of the focal distance in order to adjust the focal plane to the specimen stage of a microscope, construction of a beam exit window in a flexible nozzle and of a suitable cell containment, as well as development of procedures for on-line focussing of the beam, preparation of single ions and scanning by electrostatic deflection of the beam. When irradiating with single 100 MeV ¹⁶O ions, the adapted set-up permits an irradiation accuracy of 0.91 µm (full width at half maximum) in the x-direction and 1.60 µm in the y-direction, as demonstrated by retrospective track etching of polycarbonate foils. Accumulation of the repair protein Rad51, as detected by immunofluorescence, was used as a biological track detector after irradiation of HeLa cells with geometric patterns of counted ions. Observed patterns of fluorescence foci agreed reasonably well with irradiation patterns, indicating successful adaptation of SNAKE. In spite of single ion irradiation, we frequently observed split fluorescence foci which might be explained by small-scale chromatin movements.     

Generation of negative air ions by wheat seedlings in a high voltage electrization of soil

It was shown that plantlets of wheat (Triticum vulgare) are capable of generating negative aeroions during the electrization of soil by high-voltage impulses. Soil electrization was carried out either from the moment of planting of seeds or from the appearance of the first seedlings. The concentration of negative ions was measured in the air at a distance of 50 cm from plants. In both variants, similar growth-related changes in the concentration of negative ions were observed. The generation of negative ions began on day 6 after the planting of seeds and reached a concentration of 380 x 10(3) ion/cm3. During the next three days, this level remained unchanged. On day 10, the generation of negative aeroions increased abruptly; on days 10-14, it was twofold as high as on days 7-9. The level of generation of negative aeroions by plants stimulated from the moment of appearance of plantlets was 5-8% higher than by plants stimulated from the moment of planting. The intensity of generation of negative aeroions upon additional illumination and in full darkness remained unchanged.     

Ans fluorescence and electrokinetic potential of activated lymphocytes by con A or LPS in vivo

The $\text{in vivo}$ effect of Concanavalin A (Con A), or bacterial lipopolysaccharide (LPS) on mouse spleen cell populations was investigated. The membrane fluorescence changes of activated splenic lymphocytes were studied during two weeks after the injection of polyclonal immunogens. Experiments were performed with the hydrophobic fluorescent probe: 1-anilino-8-naphthalene sulphonate (ANS). The kinetic studies further indicated that the course of fluorescence changes may considerably vary depending on both immunogens. These fluorescence intensity changes would be in direct relation to the electrokinetic surface potential changes of activated lymphocytes, as assessed by the electrophoretic mobility analysis. By comparison with the inverse relationship observed in our previous study, it would be concluded that the relation (direct or inverse) between ANS fluorescence and electrokinetic potential depends on the net electrical charge of the antigens used.     

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

Fluorescence photomicrographs show that the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 cells. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.