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Biomedical subjects

J M Salmon

Publications and source records attributed to J M Salmon.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1994

Use of quantitative image microfluorometry to follow fluorescent ricin internalization in single living cells.

We used microspectrofluorometry and videomicrofluorometry to follow the binding and internalization of fluorescein-labeled toxic lectin ricin in living Zajdela hepatoma cells. Microspectrofluorometry showed that when ricin was specifically labeled on its B-chain with one molecule of fluorescein (ABF), its fluorescence spectrum did not alter during its binding to the cell surface and subsequent internalization. This enabled us to use image analysis to follow cell internalization of labeled ricin. Accordingly, we measured the appropriate fluorescent cell parameters, comprising total fluorescence intensity, cell surface area, mean fluorescence intensity and its standard error, and used the measurements for mono- and biparametric studies of cell fluorescence distribution. The results showed that (a) ricin binds two different subpopulations of Zajdela hepatoma cells, (b) Zajdela hepatoma cells internalize ricin rapidly and after a relatively stable period of 1-2 hr, internalization starts again at 4 hr, and (c) the distribution of intracellular fluorescence is heterogeneous and ABF accumulates in certain cellular localizations. Our results demonstrate that quantitative microfluorometry is an effective and interesting approach for real-time studies of macromolecule internalization in living cells.

Animals↗
PubMed1993

Staining with Hoechst 33342 and rhodamine 123: an attempt to detect multidrug resistant phenotype cells in leukemia.

Development of resistance is the major cause of failure in chemotherapeutic treatments. We have previously shown that the level of labeling with Hoechst 33342 and rhodamine 123 in established cell lines was decreased in cells with 'classic' MDR phenotype. This functional test was carried out using fluorescence image cytometry on living cells. We applied this protocol to patients with chronic lymphocytic leukemia. Although a large variability of the labeling is observed in cells from healthy donors, this approach seems to be useful for early detection of P-gp-dependent resistance in leukemia cells and for identification of new reversing agents on patient lymphocytes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
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

Cloning and expression in yeast of a plant potassium ion transport system.

A membrane polypeptide involved in K+ transport in a higher plant was cloned by complementation of a yeast mutant defective in K+ uptake with a complementary DNA library from Arabidopsis thaliana. A 2.65-kilobase complementary DNA conferred ability to grow on media with K+ concentration in the micromolar range and to absorb K+ (or 86Rb+) at rates similar to those in wild-type yeast. The predicted amino acid sequence (838 amino acids) has three domains: a channel-forming region homologous to animal K+ channels, a cyclic nucleotide-binding site, and an ankyrin-like region.

Amino Acid Sequence↗
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↗
PubMed1992

The effect of irrigating solutions on the strength of the cement-bone interface.

Irrigating solutions are commonly used in the preparation of bony surfaces before total joint replacement components are cemented. Using an experimental cemented arthroplasty model, metal pseudo-prostheses were cemented to a cadaveric bone surface prepared with a specific irrigating solution. A tension load was then applied to the prosthesis until failure occurred at the cement-bone interface. A quantitative analysis of the effect of three commonly used irrigating solutions on the strength of the cement-bone interface is presented. Sixty trials were performed with normal saline, povidone iodine and hydrogen peroxide. Irrigation with hydrogen peroxide afforded statistically superior cement fixation in this arthroplasty model when compared with either normal saline or povidone iodine (P less than 0.01 Chi-squared test).

Animals↗
PubMed1991

DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains.

A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the Km values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.

Amino Acid Sequence↗
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

Effect of Sugar Transport Inactivation in Saccharomyces cerevisiae on Sluggish and Stuck Enological Fermentations.

Sluggish and stuck (i.e., very delayed or incomplete) fermentations have been often observed in wine making. Some of them appeared to be associated with insufficient levels of yeast nutrients such as assimilable nitrogen. In these conditions, sugar transport catabolite inactivation, which is triggered by the protein synthesis arrest, may account in part for the inhibition of fermentation. Moreover, this mechanism of inhibition may explain the failure of added ammoniacal nitrogen to nitrogen-limited musts to restore or elevate rate of fermentation after the early yeast growth phase.

Journal Article↗
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↗
PubMed1988

[Use of 1,4 diacetoxy-2,3 dicyanobenzol in microspectrofluorometry for determining the intracellular pH of isolated living cells].

Microspectrofluorometry allows to obtain the fluorescence spectrum of an isolated living cell. When cells are preincubated with 1,4 diacetoxy-2,3 dicyanobenzol the cellular fluorescence spectrum can be resolved in its components i.e. the characteristic fluorescence spectrum of each ionized forms of the probe and the intrinsic cell fluorescence spectrum due to NAD(P)H. This allows the determination of the intracellular pH with good accuracy. Furthermore, comparison between the intensity of the intrinsic cell fluorescence and the probe fluorescence intensity offers us an opportunity to monitor the intracellular amount of the drug.

Animals↗