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P Viallet

Publications and source records attributed to P Viallet.

At least 19 recordsLinked to original sources
PubMed2001

Mag-indo1 affinity for Ca(2+), compartmentalization and binding to proteins: the challenge of measuring Mg(2+) concentrations in living cells.

A physicochemical study of the Mag-indo1 binding to Ca(2+) in solution showed that: (i) the characteristic fluorescence spectra of Ca(2+)-bound and Mg(2+)-bound Mag-indo1 are identical; (ii) two successive equilibria occur for increasing Ca(2+) concentrations; and (iii) the value of the dissociation constant of the first one, as determined by using a probe dilution protocol, amounts to 780 nM. In order to investigate the fluorescence level of Mag-indo1 trapped in cell organelles, fluorescence spectra of Mag-indo1-loaded fibroblasts were recorded before and after a digitonin permeabilization. Their resolution into cation-bound, protein-bound, and free Mag-indo1 characteristic spectra allowed measurement of the fluorescence intensities of these species. The intensities emitted from whole cells were compared to those emitted from organelles (assumed to be endoplasmic reticulum according to a DiOC(6) loading). The cation-bound Mag-indo1 fluorescence resulted partially (20 to 50%) from the cytosol for 30% of the cells, and totally from compartments for 70% of the cells. We found a concentration value of 500 nM for compartmentalized Ca(2+) and concluded that the Mag-indo1 binding to Ca(2+) is likely to affect drastically the Mg(2+) concentration measurements in cells. Moreover, we showed that the amount variation of protein-bound Mag-indo1 also affects Mg(2+) measurements when using the two-wavelength ratio method.

3T3 Cells↗
PubMed1999

Interaction of a protein, BSA, and a fluorescent probe, Mag-Indo-1, influence of EDTA and calcium on the equilibrium.

Recent findings indicate that ion-chelator probes with tetracarboxylate structure bind proteins. It was suggested that these fluorescent probes are valuable tools to gain information on protein structure through the energy transfer from tryptophans to the bound probe. Here, the binding of the fluorescent probe Mag-Indo-1 to bovine serum albumin (BSA) was investigated. Mag-Indo-1 was reported previously to serve as a probe for magnesium cations (Kd = 2.8 x 10(-4) M for zero ionic strength) which can also interact with calcium cations (Kd = 7.5 x 10(-7) M). Probe complexation with protein results in a shift of the emission fluorescence spectrum of the probe from 480 to 457 nm. We used emission fluorescence techniques to monitor this interaction. Computational resolution of the complex fluorescence spectra and a new software to test the theoretical model were developed in our laboratory. This enabled us to calculate the number of interacting sites and the dissociation constants. The fluorescent probe Mag-Indo-1 binds at a singular site with high affinity (Kd = 1.8 x 10(-7) M) to bovine serum albumin (BSA). Since proteins are known to bind several compounds unspecifically, we have studied the influence of EDTA as a competitor of the probe. Our findings suggest that the BSA binding site is identical for both Mag-Indo-1 and EDTA. We found that EDTA binds the protein with Kd = 0.4 x 10(-3) M. We studied the influence of calcium and found that Mag-Indo-1 does not bind the calcium free Apo-protein anymore.

Algorithms↗
PubMed1997

Intracellular accumulation of rhodamine 110 in single living cells.

To gain a better understanding of the internalization of rhodamines, vital staining of living cells in situ by two different rhodamines, R110 and R123, was studied by microfluorometry. These dyes differ strongly in their lipophilic properties because of differences in charge distribution. Microspectrofluorometry was used to study the fluorescence emission spectra of R110-loaded cells to determine reliable loading conditions. Cell uptake and cell efflux studies of R110 were performed by numerical microfluorescence imaging. A slower uptake was observed for R110 (14 hr) vs R123 (2 hr), but the R110 efflux was much more rapid (30 min) than that of R123 (> 24 hr). Although it appeared in the R110 and R123 co-localization study that R110 was able to accumulate in mitochondria, labeling with R110 was lower than with R123. Our results indicate that, rhodamine 110 in its acid cationic form is able to cross the plasma and mitochondrial membrane and to accumulate in cell compartments as does the cationic rhodamine 123. However, because of its acido-basic properties, R110 should be able to decrease the pH of cell compartments, depending on their ability to regulate pH. In such a model, mitochondrial pH should be more greatly decreased than cytosolic pH, leading to a lower mitochondrial accumulation of R110 than of R123. Surprisingly, these effects, which should affect the energetic state of mitochondria, do not influence cell growth, because no cytotoxic effect was observed.

3T3 Cells↗
PubMed1995

Is reduced accumulation of Hoechst 33342 in multidrug resistant cells related to P-glycoprotein activity?

Although bisbenzimidazole-DNA interactions have been studied in solution, little information has been available in living cells. The reduced accumulation of the nuclear dye Hoechst 33342 (H342) in cells with multidrug resistant (MDR) phenotype suggested its possible use in a functional test for detection of these cells. We performed experiments to elucidate the mechanisms involved in the H342-exclusion from resistant cells. As contradictory results have been reported in literature, we compared the entire fluorescence spectra of H342 in solution and in intact living cells under different experimental conditions. The study was performed by fluorescence image cytometry. This technique allow accurate quantification of the amount of H342 bound to DNA in living cells. The dye uptake was followed in sensitive and resistant cells, a lymphoblastoid cell line, CCRF-CEM, and its resistant variant selected with vinblastine CEM/VLB100 under conditions that could modulate H342-cell binding. Competition experiments with sodium azide, verapamil, and vinblastine indicated that resistant cells did not differ in the number of possible binding sites for H342. The obtained results ruled out the possibility of discriminating cells on the basis of a spectral shift. Two modes of binding, differing in their affinity for the dye, seem to co-exist in intact cells. Although it clearly appeared that the P-glycoprotein expressed in MDR cells was mainly responsible for the H342-exclusion, other mechanisms might also be involved.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Modulation of rhodamine 123 uptake by nigericin in sensitive and multidrug resistant leukemic cells.

We have investigated the effect of the ionophore nigericin (NIG) in multidrug resistant (MDR) cells, using intracellular accumulation of the fluorescent dye rhodamine 123 (R123). NIG increased the accumulation of R123 in half of the murine MDR RFLC3 population but not in the human MDR CEM/VLB 100 cells. Co-treatment of RFLC3 with NIG plus verapamil showed additive effect on the accumulation of R123. The increase in R123 accumulation observed in RFLC3 was not the consequence of a direct effect of NIG on P-glycoprotein and was accompanied by a redistribution of the dye throughout the cell and a high cytotoxicity, which prevents the use of NIG as a resistance modulating agent.

Animals↗
PubMed1994

Measurement of intracellular magnesium concentration in 3T3 fibroblasts with the fluorescent indicator Mag-indo-1.

Mag-indo-1, a fluorescent probe for measuring intracellular magnesium concentration, was used in 3T3 fibroblasts with microspectrofluorometry. The complex emission fluorescence spectrum emitted by a single living cell was analyzed with a computerized method, making it possible to evaluate the contribution of each species of Mag-indo-1 to the total fluorescence. The dye self-association observed in solutions at high dye concentration was not encountered in cells. The model of equilibria of Mag-indo-1 (monomer form) with protons, magnesium, and protein was then applied to calculate the intracellular magnesium concentration. The spectral analysis evaluated the contribution of each fluorescent species of Mag-indo-1 (a deprotonated, a magnesium-bound, and a protein-bound form) and of the cell autofluorescence to the total cell fluorescence. This method permitted accurate and reproducible measurements of intracellular magnesium concentration. Finally, this method was applied to the measurement of intracellular magnesium concentration in a 3T3 fibroblast population in exponential growth phase.

3T3 Cells↗
PubMed1994

Are intracellular ionic concentrations accessible using fluorescent probes? The example of Mag-indo-1.

The study of the physicochemical properties of Mag-indo-1, a fluorescent probe used for intracellular magnesium measurements, has shown that in a biological environment the deprotonated form of this probe is in simultaneous equilibrium with a protonated form, a protein and a magnesium-bound form. The complex emission fluorescence spectrum emitted by a single living cell was analyzed using a computerized method, allowing the evaluation of the contribution of each species of the Mag-indo-1 to the cellular fluorescence. This approach used to evaluate intracellular Mg2+ concentration has also shown the variability of the important participation of protein-bound Mag-indo-1 to the cellular fluorescence. Thus the widely used ratioing method, unable to take into account this variability, cannot afford a reliable evaluation of [Mg2+]. Whatever the technique used for investigation (microfluorimetry, flow cytometry, etc.) the evaluation of [Mg2+]i using the fluorescent probe Mag-indo-1 requires a method able to quantify, in complex fluorescence, the fluorescence intensity of the forms involved in the equilibrium with Mg2+.

3T3 Cells↗
PubMed1993

Nile red labeling of single living cells for contour delineation to quantify and evaluate the distribution of rhodamine 123 with fluorescence image cytometry.

Simultaneous study of intracellular quantification and distribution of fluorescent probes is difficult when cell staining is not homogeneous. This occurs after mitochondrial staining with rhodamine 123 (R123). Classical techniques for evaluation of intracellular R123 fluorescence, such as flow cytometry, are based on measurement of the global fluorescence intensity but do not take into account parameters that reflecting cellular distribution of the probe. For simultaneously studying intracellular quantification and distribution of R123 with fluorescence image analysis, we delineated a mask of the cell, generated from a fluorescent image of the plasma membrane stained by nile red (NR). After a preliminary study of the fluorescence characteristics of R123 and NR to avoid artifacts and optimize conditions of staining, quantification and distribution of intracellular R123 studies were performed by superimposition of the mask on the R123 fluorescence image. This protocol was applied to leukemic cells and allowed estimation of individual cell parameters such as mean fluorescence intensity and standard deviation, the latter providing information of the cellular distribution of R123. Moreover, it permitted demonstration of the redistribution of R123 in the whole cell when coincubated in the presence of nigericin.

Animals↗
PubMed1992

Investigation of noncalcium interactions of fura-2 by classical and synchronous fluorescence spectroscopy.

Several authors have reported unexpected intracellular spectra of both indo-1 and fura-2. One of the major methodological problems in the evaluation of calcium concentration using fluorescent probes is that it is assumed that only two forms of the dyes are detectable within the cells. We show in this study of fura-2 properties that this calcium probe is pH-sensitive and able to bind to cellular proteins. The excitation spectra of protonated and protein-bound forms of fura-2 exhibit a maximum in the same region as that associated with the calcium-free form (i.e., near 365 nm). The very small shift in the excitation spectra upon proton or protein binding precludes the use of classical methods to determine the spectral composition of mixtures of several forms of fura-2. We therefore used the synchronous fluorescence technique to detect the protein-bound form of fura-2 selectively, in order to assess the pH dependence of the fura-2/protein interaction. The nonspecific binding of fura-2 to proteins is reinforced at acidic pH and inhibited by calcium. The fact that the same type of interaction was found between fura-2 and poly-L-lysine suggests that it could be mediated by basic amino acids. Because of the strong overlap of the excitation spectrum of the unprotonated free fura-2 with those associated with the protonated and protein-bound forms, a cytoplasmic acidification may lead to an artifactual measurement of low calcium levels.

Calcium↗
PubMed1992

Identification of multi-drug resistant cells in sensitive Friend leukemia cells by quantitative videomicrofluorimetry.

The cellular resistance to cytotoxic drugs, particularly to anthracyclines, remains a major problem in cancer chemotherapy. A number of biochemical mechanisms have been described, one of them being a lower accumulation of drugs in resistant cells. The accumulation of Ho33342 in sensitive and resistant Friend leukemia cells was studied by quantitative fluorescence image analysis, a method which allows investigations to be made on living tissues and cells. The intensity of fluorescence is related to the amount of Ho33342 accumulated into the cells and has been found to be more intense in sensitive cells than in resistant ones. Moreover, the retention of this vital dye was inversely related to the degree of resistance in the three resistant cell lines. The addition of verapamil, which is known to reverse resistance to anthracyclines, resulted in an increase of the amount of Ho33342 accumulated in the resistant cells. Ho33342 presents a higher quantum yield than any other anthracyclines, such as adriamycin and can be used as a microfluorimetric probe to identify the resistant cells in a heterogeneous cell population.

Benzimidazoles↗
PubMed1992

Microspectrofluorometry as a tool for investigation of non-calcium interactions of Indo-1.

Indo-1 is a fluorescent calcium probe used to measure intracellular free calcium concentrations. These measurements are often performed by comparing the fluorescence intensities of Indo-1-treated cells at two selected wavelengths corresponding to the maxima of the fluorescence spectra of the calcium-bound and calcium-free forms. In this study, we used an optical multichannel analyser to numerise the fluorescence emitted by a single cell. A computerised resolution of numerised spectra was used on intracellular Indo-1 fluorescence. Calculation of numerical and graphic estimators allows us to evaluate the fit of the resolution. Different sets of characteristic spectra were compared using this method. It appeared that no linear combination of the two known forms of Indo-1 and of the cell autofluorescence can fit with spectra of Indo-1-treated cells. In addition, a study of the physico-chemical properties of Indo-1 shows the existence of two other forms of the molecule: a protonated form (maximum emission at 455 nm) and a form in interaction with proteins (maximum emission at 438 nm). Taking into account the contribution of these two new forms leads to an improved spectral resolution of the fluorescence of Indo-1-treated living cells and, therefore, improves calcium measurements. Moreover, quantification of the amount of the protonated form of Indo-1 allows a measurement of intracellular pH at the same time as calcium determination.

3T3 Cells↗
PubMed1991

Fluorescent image cytometry: from qualitative to quantitative measurements.

Image analysis is being increasingly used in biology and medicine; however, in order to obtain truly quantitative data and thus avoid errors in interpretation, a certain number of precautions must be taken when the image is digitized, well before any attempt is made to analyse or interpret the data. This is particularly true for image microfluorometry. In this article we will examine an image analysis system for fluorescent images composed of a mercury lamp, a microscope, a high sensitivity video camera and an image analyser and evaluate the principal sources of random and non-random errors, various constraints, and their relative importance. A signal correction protocol is proposed to minimize non-random errors during digitalization. A few examples are given to illustrate its efficiency.

Analog-Digital Conversion↗
PubMed1990

Influence of D-galactosamine on the kinetics of metabolic processes for two intermediate metabolites, 9-hydroxybenzo(a)pyrene and 3-hydroxybenzo(a)pyrene, in 3T3 and RTG2 cells.

PAH metabolism is known to proceed in two successive steps, the first step resulting in the production of activated metabolites which are subsequently transformed by the different pathways involved in the second step. Microspectrofluorometry enables the study of the kinetics of these steps in living intact cells into which no imbalance has been artificially introduced. We used this technique to check the influence of pre-incubation with D-galactosamine on the kinetics of the detoxification step. 9- and 3-hydroxybenzo(a)pyrene (OH-B(a)P) were selected as fluorescent substrates because they are potential substrates for the different pathways of the second step. The physiological cell status was controlled at the level of the intrinsic cellular fluorescence. Pre-incubation with D-galactosamine results in a strong decrease of the experimental rate constants characteristic of the metabolism of 9- and 3-OH-B(a)P in both RTG2 and 3T3 cells. Moreover, such pre-incubation leads to a strong decrease of the transitory intracellular accumulation of 3-O-glucuronide when 3-OH-B(a)P is used as substrate for 3T3 cells. Nevertheless, it cannot be said that both phenols cannot be used as substrates by MFOs and STase, at least in rigorous experimental conditions.

Animals↗
PubMed1989

Relevance of the chemical charge of rhodamine dyes to multiple drug resistance.

Previously, we have shown that multiple drug resistant (MDR) Friend leukemia cells (FLC) are cross-resistant to the positively-charged dye, Rhodamine 123 (Rho 123), and that this resistance can be reversed by verapamil (VER). In the present study we used two zwitterionic rhodamine analogs, Rhodamine 116 and Rhodamine 110, and another positively-charged analog, Rhodamine 6G, to determine whether drug accumulation, resistance and modulation were affected by changes in the charge of these compounds. While there was no differential sensitivity between sensitive and resistant FLC to zwitterionic rhodamines, there was marked differential toxicity between these cell types for the positively-charged analogs. The IC50 values were 1000- and 100-fold greater in resistant than in sensitive cells for Rho 123 and Rho 6G respectively. Intracellular drug accumulation was significantly higher in sensitive as compared to resistant cells for both Rho 123 and Rho 6G, but little difference in drug uptake between these two cell types was observed for Rho 110 and Rho 116. It was also found that the intracellular to extracellular ratio of the positively-charged compounds was greater than unity in both sensitive and resistant cells whereas for the zwitterionic analogs this ratio was less than 1. Furthermore, this ratio of drug uptake was found to be significantly higher for Rho 6G than for Rho 123, which correlated with the high oil:water partition coefficient of Rho 6G (115.6). In MDR cells, verapamil increased Rho 123 and Rho 6G accumulation by 9.4- and 8.6-fold respectively. In addition, IC50 values in resistant cells were reduced greater than 100-fold for Rho 6G and greater than 1000-fold for Rho 123 in the presence of 10 micrograms/ml of verapamil. In contrast, less than 2-fold reduction of IC50 values for both of the zwitterionic analogs could be obtained under the same conditions. These results indicate that the chemical charge of rhodamines plays an important role in their differential accumulation, cytotoxicity and sensitivity to modulators such as verapamil, in sensitive and multi-drug resistant cells. The data also suggest that increased lipophilicity of the positively-charged rhodamines may increase their ability to accumulate in, and subsequently kill, MDR cells.

Animals↗
PubMed1988

Influence of long term treatment with 3-methylcholanthrene or carbaryl on mixed-function oxidase activity in 3T3 fibroblasts: studies by microspectrofluorimetry on single cells.

Using benzo(a)pyrene (BaP) as a probe for aryl hydrocarbon hydroxylase (AHH) activity, differences in mixed-function oxidase (MFO) activity were observed using microspectrofluorimetry in single living cells during long term treatment with 3-methylcholanthrene (3-MC) or carbaryl. Although these two compounds differ in chemical structure, similar effects were observed in 3T3 cell populations. The results suggest that the two compounds activate the same enzymatic system and that individual cells of a supposed homogeneous cell population are not equally sensitive to xenobiotics, i.e. subpopulations were observed which have differences in AHH activity.

Animals↗
PubMed1988

Resolution of complex fluorescence spectra recorded on single unpigmented living cells using a computerised method.

The identification and quantification of fluorescent compounds in a complex fluorescence spectra are always difficult, especially in the case of low signal:noise ratio. We propose a computerised method that allows the resolution of low light level complex fluorescence spectra into its components. Based on a linear combination of N possible characteristic fluorescence spectra, and using N weighting functions, this method allows the integration of fluorescence intensities over the entire fluorescence spectra and the generation of n equations with N unknowns. The compounds that participate in complex fluorescence spectra are identified and quantified. Because fluorescence intensities can be integrated we can resolve complex fluorescence spectra presenting a low signal:noise ratio. The reliability and sensitivity of our method are shown through examples of resolution of complex intracellular fluorescence of single living cells pretreated with benzo(a)pyrene. Depending on the cell type and treatment, two, four, or five components can be identified in the complex fluorescence spectra.

Animals↗
PubMed1988

6-Amino-chrysene, a potent inhibitor of transferase activity in single living RTG2 cells.

Previous reports on the inhibitory effect of 6-amino-chrysene (6AC) on benzo(a)pyrene (BP) metabolism using single living cells have suggested that aryl hydrocarbon hydroxylase (AHH) is not the only pathway for 6AC metabolism. We present here results demonstrating that direct glucuronidation may constitute an alternative pathway for 6AC elimination. First, we describe the conjugate of 6AC to UDP-glucuronic acid (UDPGA) in solution. We performed competition experiments between 6AC and monohydroxy BP, which are known to be good substrates for glucuronic transferase (GT), in RTG2 cells, using microspectrofluorimetry. Because of intracellular accumulation of fluorescent metabolites during BP metabolism, RTG2 cells can be used as a tool for simultaneous study of AHH and GT activities. When RTG2 cells have been simultaneously treated with BP and 6AC, GT appeared to be a more specific target for 6AC than AHH in these cells. Therefore, 6AC can be expected to act as a more specific inhibitor for GT than for AHH activity.

Animals↗