Protein damage by photoproducts of merocyanine 540.

Exposure of certain photoactive dyes to light prior to their use in biological systems (preactivation) has been shown to result in formation of long-lived cytotoxic photoproducts. The cytotoxic species responsible for the biological activity of preactivated merocyanine 540 (pMC540) appears to be a hydroperoxide generated by oxidation of ground-state dye by singlet molecular oxygen, formed via energy transfer from triplet excited-state dye to oxygen. A positive correlation (r = .93) exists between the levels of hydroperoxides and percent of tumor cells killed upon exposure to pMC540. Exposure of bovine serum albumin (BSA) (0.5 mg/mL) to pMC540 (0.2 mg/mL-1 mg/mL) results in loss of tryptophan fluorescence and 345 nm emission, suggesting a probable role of either hydroxyl (.OH) or .OH + superoxide (O2-). Polyacrylamide gel electrophoresis indicates fragmentation of treated BSA. Aggregation of pMC540-treated BSA is not detected. Bityrosine production is not observed. A dose-dependent decrease in BSA solubility is observed in treated samples, suggesting an increase in hydrophobicity. Amino acid analysis of BSA treated with pMC540 shows loss of some amino acids residues. The data presented here suggest that photoproducts of MC540 derived via the process of preactivation may mediate their effect (at least in part) by reactive oxygen species.     

Response of the electrochromic dye,merocyanine 540, to membrane potential in rat liver mitochondria

Summary We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca^2–] _a -dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca^2–-channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca²⁺-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K^–] _a , nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba²⁺] _a . All these data are consistent with the concept that PTX may act on Ca^2– channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca²⁺ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca²⁺ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX-treated cells. Our measurements of whole cell inward Ca²⁺ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca²⁺ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca²⁺-channel conductance (6 nS/cell at 2.6mm [Ca²⁺] _a ). PTX evoked the activation of a new class of Ca²⁺-selective channels (5 pS in 25mm [Ca²⁺]_pipet), which are rather insensitive to membrane potential. We have termed theseG-type calcium channels. These data suggest that treatment with PTX not only increases the probability of L-type Ca²⁺-channel activation at more negative potentials, but also increases the probability of opening of an entirely new, voltage-independent, Ca²⁺ channel. These actions of PTX should promote Ca²⁺ entry and might explain the stimulation by the toxin of CA secretion from medullary chromaffin cells in culture.     

Staining of Plasmodium yoelii-infected mouse erythrocytes with the fluorescent dye rhodamine 123

The cationic permeant fluorescent dye rhodamine 123 (R123) was used to stain Plasmodium yoelii-infected mouse erythrocytes. Fluorescence microscopic observations demonstrated that the parasite, but not the matrix of the infected erythrocyte, accumulated the dye. Differences in fluorescence intensity could not be found at the various developmental stages of the parasite; however, quantitation of the cell-associated dye revealed an increase in R123 uptake with parasite development. The retention of the parasite-associated dye, as measured by fluorescence microscopy and spectrophotometry after extraction of R123 with butanol, was markedly reduced by treatment of the infected erythrocytes with a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an inhibitor of proton ATPase, dicyclohexylcarbodiimide (DCCD). These results indicate that the accumulation and retention of R123 in P. yoelii reflect the parasite membrane potential and suggest that the parasite plasma membrane has a membrane potential-generating proton pump.     

A chemometric approach to the estimation of the absorption spectra of dye probe merocyanine 540 in aqueous and phospholipid environments

Merocyanine 540 (MC540) is a widely used dye probe for membranous environments. However, fundamental knowledge of the spectral features of this dye in aqueous and hydrophobic environments is still lacking. Such knowledge is important because biomembranes involve a hydrophobic environment surrounded by a hydrophilic environment. Because many investigations so far have been performed based on indistinct spectral estimations, the interpretation of the data obtained using this dye as a fluorescent transmembrane probe remains controversial. In order to determine the exact spectra in both aqueous and hydrophobic environments, we adopted principal factor analysis (PFA), a method of multivariate analysis. The PFA method can also determine the number of molecular species present in the reaction mixture, which is three in pure water and two in phospholipid suspension. Two of the species in both water and phospholipid suspension were the monomer and dimer. The third species in water was the trimer, but its amount was so small at 10 microM MC540 solution that the spectral data in water can be approximated neglecting this molecular species. The monomer spectrum changed its form markedly with a bathochromic shift when transferred from the water to phospholipid environment, whereas the dimer remained similar in its shape except for a remarkable red shift. In water, the dissociation constants, K(1) and K(2), for the assumed stacking-model reactions, M+M <--> M(2) and M+M(2) <--> M(3), were 3.1 x 10(-4) M and 5.7 x 10(-4) M, respectively. In the phospholipid environment, the dissociation constant K* for the assumed stacking-model reaction, M(*)+M(*) <--> *M(2), was 1.9x10(-5)M. The fluorescent intensities of MC540 were also measured in both water and phospholipid environments. A comparison based on the absorption and fluorescence spectra suggested that the temporal increase in the amount of the monomer on the excitable membrane contributes to the fluorescent intensity change observed in the transmembrane potential change.     

Sequential disassembly of myofibrils induced by myristate acetate in cultured myotubes

The phorbol ester TPA induces the sequential disassembly of myofibrils. First the alpha-actin thin filaments are disrupted and then, hours later, the myosin heavy chain (MHC) thick filaments. TPA does not induce the disassembly of the beta- and gamma-actin thin filaments of stress fibers in presumptive myoblasts or fibroblasts, nor does it block the reemergence of stress fibers in 72-h myosacs that have been depleted of all myofibrillar molecules. There are differences in where, when, and how myofibrillar alpha-actin and MHC are degraded and eliminated from TPA-myosacs. Though the anisodiametric myotubes have begun to retract into isodiametric myosacs after 5 h in TPA, staining with anti-MHC reveals normal tandem A bands. In contrast, staining with mAb to muscle actin fails to reveal tandem I bands. Instead, both mAb to muscle actin and rhophalloidin brilliantly stain numerous disk-like bodies approximately 3.0 micron in diameter. These muscle actin bodies do not fuse with one another, nor do they costain with anti-MHC. All muscle actin bodies and/or molecules disappear in 36-h myosacs. The collapse of A bands is first initiated in 10-h myosacs. Their loss correlates with the appearance of immense, amorphous MHC patches. MHC patches range from a few micrometers to over 60 micron in size. They do not costain with antimuscle actin or rho-phalloidin. While diminishing in number and fluorescence intensity, MHC aggregates are present in 30% of the 72-h myosacs. Myosacs removed from TPA rapidly elongate, and after 48 h display normal newly assembled myofibrils. TPA reversibly blocks incorporation of [35S]methionine into myofibrillar alpha-actin, MHC, myosin light chains 1 and 2, the tropomyosins, and troponin C. It does not block the synthesis of beta- or gamma-actins, the nonmyofibrillar MHC or light chains, tubulin, vimentin, desmin, or most household molecules.     

Electrostatic interaction between merocyanine 540 and liposomal and mitochondrial membranes

Summary The fluorescence of merocyanine 540 (MC) in liposomal and mitochondrial suspensions was measured under various conditions. Under a given condition, both the amount of dye bound to the membrane and the zeta potential were determined simultaneously. It was found that the fluorescence intensity was proportional to the amount of bound dye and correlated with the zeta potential of particles. The fluorescence intensity was represented quantitatively in terms of the Langmuir adsorption isotherm, when the electrostatic interaction acting between MC and membrane surface was properly taken into account. It was concluded that the changes in MC fluorescence in the liposomal and mitochondrial suspensions are mainly attributed to the changes in the surface potential of the membranes.     

Identification of normal and leukemic granulocytic cells with merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

Mechanism and kinetics of merocyanine 540 binding to phospholipid membranes

The physicochemical mechanism for merocyanine 540 (M540) binding to unilamellar phosphatidylcholine (PC) vesicles was examined by steady-state and dynamic fluorescence and fluorescence stopped-flow methods. At 530-nm excitation, aqueous M540 has an emission peak at 565 nm, which red shifts to 580 nm with formation of membrane-bound monomers (M); bound dimers (D) are nonfluorescent. Equilibrium fluorescence titrations show that 50% of total M540 partitions into the membrane to form D at [M540]/[PC] (Rm/p)_approximately 0.6. M and D concentrations are equal at Rm/p approximately 0.05. For Rm/p less than 0.1, M540 has a single fluorescence lifetime (tau), which decreases with Rm/p [tau-1 (ns-1) = 0.48 + 3.3Rm/p], indicating a rapid collisional rate between M to form D. Dynamic depolarization studies show that hindered rotation of M (r infinity = 0.13 at Rm/p = 0.006) becomes more rapid (rotational rate 0.2-1.9 ns-1) with increasing Rm/p (0.006-0.075). The efficiencies of energy transfer between n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12, 16) and bound M540 suggest that M is oriented parallel to the phospholipids near the membrane surface; studies of efficiencies of n-AF quenching by D are consistent with an orientation of D perpendicular to the phospholipids. In stopped-flow fluorescence measurements in which M540 is mixed with PC vesicles, there is a rapid (1 ms) followed by a slower (10-50 ms) concentration-dependent fluorescence increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-induced permeabilization and merocyanine 540 staining of mouse spleen cells

Merocyanine 540 (M540) is a potential-sensitive, hydrophobic dye that preferentially incorporates into the 'fluid' domains of cellular membranes, distinguishing between hemopoietic cells according to their differentiation state. A bright staining with M540 is usually achieved by UV illumination of the cells during staining. We show by flow cytometric analysis that: (1) staining is greatly enhanced by UV illumination of mouse spleen cells before addition of the dye; (2) UV treatment causes an increased permeability toward propidium iodide and intracellular fluorescein as well; (3) the increment in M540 fluorescence precedes permeabilization to propidium iodide, while the latter precedes leakage of fluorescein. We also describe an overshoot and accelerated recovery of M540 fluorescence after photobleaching by a 514 nm laser beam. It is suggested that penetration of M540 to the more fluid inner membrane structures explains the fluorescence increment in both experiments.     

Fluorescent properties of merocyanine 540 in solutions of sialogangliosides

The spectral modifications in the absorption and emission properties of merocyanine 540 have been evaluated in solvents of varying dielectric constants. The fluorescence behavior of dye in solutions of low dielectric constant has offered a possibility for monitoring the micropolarity of sialoganglioside micelles in aqueous solutions. Our results demonstrate that sialic acid residues markedly influence the aggregation properties of gangliosides in solution as well as the nature of dye binding to the micellar structures.     

Separation of viable and nonviable animal cell using dielectrophoretic filter

Selective separation of cells using dielectrophoresis (DEP) has recently been studied and methods have been proposed. However, these methods are not applicable to large‐scale separation because they cannot be performed efficiently. In DEP separation, the DEP force is effective only when it is applied close to the electrodes. Utilizing a DEP filter is a solution for large‐scale separation. In this article, the separation efficiency for viable and nonviable cells in a DEP filter was examined. The effects of an applied AC electric field frequency and the gradient of the squared electric field intensity on a DEP velocity for the viable and nonviable animal cells (3‐2H3 cell) were discussed. The frequency response of the DEP velocity differed between the viable and the nonviable cells. We deducted an empirical equation that can be used as guiding principle for the DEP separation. The results indicate that the viable and the nonviable cells were separated using the DEP filter, and the best operating conditions such as the applied voltage and the flow rate were discussed. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cholesterol-free phospholipid domains may be the membrane feature selected by N epsilon-dansyl-L-lysine and merocyanine 540

We have used N epsilon-dansyl-L-lysine as a fluorescent membrane probe, to study cells taken from tissues concerned with immune function. There is a striking similarity between the staining selectivity of this compound and that reported by others for merocyanine 540. Both compounds stain leukemic, human, peripheral leukocytes, an erythroleukemia line, and some mouse bone marrow cells, suggesting common selectivity for a membrane feature of hemopoietic cells. Both compounds fail to stain red blood cells, normal human leukocytes, mouse spleen and thymus cells. We have recently reported that dansyl-lysine apparently selects for cholesterol-free phospholipid domains in liposomes and now report similar selectivity for merocyanine 540 staining of liposomes.     

Photodynamic action of merocyanine 540 on carcinoma of cervix cells

Results of the studies carried out on localization and photodynamic action of merocyanine 540 (MC540) on carcinoma of cervix (HeLa) cells are presented. Fluorescence microscopic study showed that when HeLa cells were incubated with MC540 in dark, the dye localized in plasma membrane of cells. Photoirradiation of cells in presence of MC540 led to enhancement of dye uptake, intracellular localization of dye and a dose dependent decrease in cell survival. Clonogenic assay showed 96% cell killing at a light dose of 42 kJ/m2. Photosensitization of cells resulted in loss of membrane integrity, decrease in plasma membrane fluidity and reduction in mitochondrial dehydrogenase activity as measured by tetrazolium reduction (MTT) assay. At a given light dose, the relative change in plasma membrane properties was higher than the reduction in activity of mitochondrial enzyme. These results suggest plasma membrane is a primary target of photosensitization of HeLa cells by MC540.     

Direct observation of singlet oxygen production by merocyanine 540 associated with phosphatidylcholine liposomes

The production of singlet molecular oxygen (1O2) by the photosensitizing dye merocyanine 540 (MC540) bound to phosphatidylcholine liposomes has been demonstrated by direct detection of 1O2 luminescence at 1268 nm. 1O2 phosphorescence emission was enhanced in deuterated buffer and upon saturation of the sample with oxygen and could be quenched by the addition of sodium azide to the external medium. No 1O2 luminescence was detected in nitrogen-saturated samples, in the absence of dye, or with MC540 in aqueous solution. Photobleaching of liposome-bound MC540 was also observed to be dependent on oxygen concentration. These studies are consistent with 1O2 intermediacy in the mechanism of MC540-mediated photosensitization.     

Echinocytosis and microvesiculation of human erythrocytes induced by insertion of merocyanine 540 into the outer membrane leaflet

Echinocytosis and release of microvesicles from human erythrocytes treated with the impermeant fluorescent dye merocyanine 540 (MC540) has been correlated with the extent of dye binding to intact cells and ghosts. At 20 degrees C binding appeared to saturate at about 9.3.10(6) molecules per cell (3.6 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 2.7%. Stage 3 echinocytes were formed upon binding of (3-4).10(6) molecules of MC540/cell (about 1.3 mol/100 mol phospholipid), equivalent to an expansion of the outer leaflet lipid area of about 1.0%. Negligible release of microvesicles was observed with MC540 at 20 degrees C. Binding of MC540 to permeable ghosts was approximately twice that to cells suggesting that there was no selective binding to the unsaturated (more fluid) phospholipids which are concentrated in the inner lipid leaflet of the membrane. At 37 degrees C apparent maximal binding of MC540 was about 3.2 mol/100 mol phospholipid and correlated with the maximal release of microvesicles from the cells as measured by release of phospholipid and acetylcholinesterase. These results are discussed in relation to the bilayer couple hypothesis of Sheetz and Singer (Proc. Natl. Acad. Sci. USA 71 (1974) 4457-4461).     

Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling

AIMS: The aim of this study was to use confocal laser scanning microscopy (CLSM) to examine the spatial distribution of both viable and nonviable bacteria within microcosm dental plaques grown in vitro. Previous in vivo studies have reported upon the distribution of viable bacteria only. METHODS AND RESULTS: Oral biofilms were grown on hydroxyapatite (HA) discs in a constant-depth film fermenter (CDFF) from a saliva inoculum. The biofilms were stained with the BacLight LIVE/DEAD system and examined by CLSM. Fluorescence intensity profiles through the depth of the biofilm showed an offset between the maximum viable intensity and the maximum nonviable intensity. Topographical differences between the surface properties of the viable and nonviable biofilm virtual surfaces were also measured. CONCLUSIONS: The profile of fluorescence intensity from viable and nonviable staining suggested that the upper layers of the biofilm contain proportionally more viable bacteria than the lower regions of the biofilm. SIGNIFICANCE AND IMPACT OF STUDY: Viability profiling records the transition from predominantly viable to nonviable bacteria through biofilms suggesting that this technique may be of use for quantifying the effects of antimicrobial compounds upon biofilms. The distribution of viable bacteria was similar to that found in dental plaque in vivo suggesting that the CDFF produces in vitro biofilms which are comparable to their in vivo counterparts in terms of the spatial distribution of viable bacteria.     

Staining of Viable and Nonviable Myotubes and of Myofibrils by the Fluorescent Dye Merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5–10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Temperature-jump studies of merocyanine 540 relaxation kinetics in lipid bilayer membranes

The temperature-jump technique was used to study the rapid kinetics of merocyanine 540 (M-540) interactions with single-walled phosphatidylcholine (PC) vesicles. The absorption spectrum of M-540 in PC vesicles has an isosbestic point at 560 nm at low [PC]/[M-540], where solution M-540 and membrane-bound M-540 dimers are present, and an isosbestic point at 548 nm at high [PC]/[M-540], where membrane-bound M-540 monomers and dimers are present. In response to a 15-kV discharge across a solution containing M-540 and PC vesicles (2.5 degrees C temperature increment), there was a rapid increase in absorbance at 575 nm (less than 5 microseconds) followed by a slower (approximately 1 ms), monoexponential relaxation process of opposite sign and approximately equal amplitude to the initial rise. The amplitude of the slower process was wavelength-dependent and reversed sign at approximately 540 nm. The slower relaxation time constant decreased as [PC] was increased at constant [M-540]. A proposed model for the potential sensitivity of M-540 involves intramembrane reorientation of dye molecules and dimerization. The results obtained here suggest that reorientation of dye molecules is the rate-limiting step, with a rate constant for reorientation from parallel to perpendicular to the plane of the membrane of 1340 +/- 200 s-1 at 23 degrees C.     

Separation of viable from nonviable cells using a discontinuous density gradient

Summary When a suspension containing a mixture of viable and nonviable cells is layered over a dense ficoll-metrizoate solution and centrifuged, most of the viable cells are retained at the interface above the dense solution; whereas most of the nonviable cells are distributed in other fractions. The cells revovered for the interface are capable of subsequent growth in culture.