Biomedical subjects

J M Salmon

Publications and source records attributed to J M Salmon.

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↗

PubMed2000

Pathology of skeletal metastases.

Metastatic disease involving the skeleton is an unfortunate and common occurrence in cancer patients. Choosing the best diagnostic approach requires knowledge of the patient's clinical history, the radiologic appearance of the lesion, the differential diagnosis, and the ability of the diagnostic modality to answer the questions that must be addressed. In difficult cases, interaction between the pathologist and clinician before biopsy may make the difference between a rapid procedure serving to definitively diagnose and effectively stage a patient and a costly procedure that provides little or no information.

Algorithms↗

PubMed2000

Native fluorescence and mag-indo-1-protein interaction as tools for probing unfolding and refolding sequences of the bovine serum albumin subdomain in the presence of guanidine hydrochloride.

Changes in the fluorescence spectrum of tryptophans Trp 134 and Trp 212 in bovine serum albumin (BSA) and of Trp 214 of human serum albumin in the presence of the chaotropic agent guanidine hydrochloride (Gnd) were studied. A detailed analysis of the fluorescence spectrum of native BSA yielded the fluorescence spectrum for each tryptophan of BSA. Modifications in the binding of Mag-indo-1 to BSA, which results in a specific quenching of the fluorescence spectrum of Trp 134 associated with an energy transfer from Trp 134 to the protein-bound Mag-indo-1, were also investigated. Changes occurring when the Gnd concentration is decreased stepwise cover a larger concentration scale of Gnd than the reverse protocol, allowing one to suggest that the resulting conformational changes in the subdomain IA of BSA involve at least three different steps.

Animals↗

PubMed2000

Cultured tumor cells of murine submandibular gland origin: a model to investigate pHi regulation of salivary cells.

Intracellular pH (pHi) and several ion transport mechanisms in cultured murine salivary gland cells (SCA-9) were studied using a videomicrofluorometric method and the H+-specific probe C-SNARF-1. The aim of this study was to test SCA-9 cells' pHi regulation mechanisms and evaluate if this cell line is representative of submandibular gland cells. Resting pHi in unstimulated cells was estimated to be 7.17+/-0.07. To investigate the presence of Na+/H+ and Cl-/HCO3- antiports as well as Na+/K+/2Cl- symports in SCA-9 cells, we used different specific blockers, dimethyl-amiloride, disulfonic stilbene, bumetanide and furosemide. In order to study SCA-9 cell capacity to regulate their pHi in response to alkaline and acid loads, we applied the NH4Cl prepulse method to all these blockers. The results showed that SCA-9 cells possess both antiports and symports involved in pHi regulation, and that this cell line can be used as a convenient model to study pHi regulatory mechanisms in salivary cells.

Acid-Base Equilibrium↗

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↗

PubMed1999

Purification and some properties of an Aspergillus niger beta-apiosidase from an enzyme preparation hydrolyzing aroma precursors.

A beta-apiosidase was isolated and purified to electrophoretic homogeneity from an enzyme preparation, Klerzyme 200, through ammonium sulfate precipitation, gel filtration chromatography, ion-exchange chromatography, and HPLC on ion-exchange and size exclusion columns. The purification of the enzyme was aided by the synthesis of 4-methylumbelliferyl beta-D-apiofuranoside for the specific detection of activity on electrophoresis gels. The molecular mass estimated by SDS-PAGE was 120 kDa. The optimum activity of the beta-apiosidase was found at pH 5 and 40 degrees C. The K(m) and V(max) for p-nitrophenyl beta-D-apiofuranoside were 4.2 mM and 2460 nkat/mg of protein, respectively. The enzyme was not inhibited by glucose and ethanol. This enzyme hydrolyzed the intersugar linkages of apiofuranosylglucosides, aroma precursors from grape.

Aspergillus niger↗

PubMed1998

Arthroscopic stabilization of the shoulder for acute primary dislocations using a transglenoid suture technique.

Many studies report the results of arthroscopic stabilization for recurrent shoulder instability, with widely variable recurrence rates; however, there are very few reports of the use of these techniques in acute first-time dislocations. We report the clinical outcomes of 17 patients who had arthroscopic stabilization using a transglenoid suture technique for acute primary dislocation. The surgery took place between March 1992 and March 1994 and, to date, there has been one recurrent dislocation (6%) and no recurrent subluxation. There were no major complications, although a number of patients found the knot tied over the infraspinatus fascia to be uncomfortable until it resorbed. All patients examined had normal power and range of motion, and a clinically stable shoulder. All 16 patients without recurrence were satisfied with their result. Nine patients returned to sports at the same or higher level, including such vigorous contact sports as Australian Rules football and rugby. Three patients did not return to the same level of sporting activity because of lack of confidence in the shoulder or a fear of dislocation despite no clinical evidence of instability. Five patients reported a lack of confidence in the shoulder without clinical evidence of instability. We suggest that arthroscopic stabilization with transglenoid sutures or a suture anchor technique is a reasonable option for the athlete with an acute primary shoulder dislocation who wishes to return to sports.

Adult↗

PubMed1998

Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae.

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.

Aspergillus oryzae↗

PubMed1998

Improvement of nitrogen assimilation and fermentation kinetics under enological conditions by derepression of alternative nitrogen-assimilatory pathways in an industrial Saccharomyces cerevisiae strain.

Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways in Saccharomyces cerevisiae. One mutant carrying a recessive allele of ure2 was isolated from an industrial S. cerevisiae strain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO2 production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened. S. cerevisiae strains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts.

Crosses, Genetic↗

PubMed1997

Determination of the relative ploidy in different Saccharomyces cerevisiae strains used for fermentation and 'flor' film ageing of dry sherry-type wines.

The full chromosomal karyotype of six enological Saccharomyces cerevisiae strains used for fermentation and biological ageing of sherry-type wines was studied. A genetic method based on the analysis of segregation frequencies of auxotrophic markers, among random spore progeny of hybrids, constructed between laboratory and industrial wine strains (Bakalinsky and Snow, 1990) was used. This method was combined with the analysis of strains by pulsed-field gel electrophoresis. The results obtained clearly indicate the presence of two, three or four copies of a chromosome in the industrial strains examined, and thus confirm that aneuploidy/polyploidy is not uncommon in these strains. In all strains examined, chromosome XIII polysomy is observed. This chromosome contains the ADH2 and ADH3 loci, that code for the ADHII and ADHIII isoenzymes of alcohol dehydrogenase, which are involved in ethanol oxidative utilization during biological ageing of wines. Tetrad analysis for the 'flor formation' character suggest two possibilities: this character is either regulated by at least a digenic system, or by only one gene present on a chromosome which is, at least, disomic.

Chromosomes, Fungal↗

PubMed1997

Relationship between effect of ethanol on proton flux across plasma membrane and ethanol tolerance, in Pichia stipitis.

Pichia stipitis efficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. In Saccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. In P. stipitis the passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H(+)-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H(+)-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H(+)-extrusion. Comparison of the rates of H(+)-flux with the related in vitro H(+)-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) of P. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H(+)-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grown P. stipitis cells.

Journal Article↗

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↗

PubMed1996

Malolactic fermentation by engineered Saccharomyces cerevisiae as compared with engineered Schizosaccharomyces pombe.

The ability of yeast strains to perform both alcoholic and malolactic fermentation in winemaking was studied with a view to achieving a better control of malolactic fermentation in enology. The malolactic gene of Lactococcus lactis (mleS) was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The heterologous protein is expressed at a high level in cell extracts of a S. cerevisiae strain expressing the gene mleS under the control of the alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid. Malolactic enzyme specific activity is three times higher than in L. lactis extracts. Saccharomyces cerevisiae expressing the malolactic enzyme produces significant amounts of L-lactate during fermentation on glucose-rich medium in the presence of malic acid. Isotopic filiation was used to demonstrate that 75% of the L-lactate produced originates from endogenous L-malate and 25% from exogenous L-malate. Moreover, although a small amount of exogenous L-malate was degraded by S. cerevisiae transformed or not by mleS, all the exogenous degraded L-malate was converted into L-lactate via a malolactic reaction in the recombinant strain, providing evidence for very efficient competition of malolactic enzyme with the endogenous malic acid pathways. These results indicate that the sole limiting step for S. cerevisiae in achieving malolactic fermentation is in malate transport. This was confirmed using a different model, S. pombe, which efficiently degrades L-malate. Total malolactic fermentation was obtained in this strain, with most of the L-malate converted into L-lactate and CO2. Moreover, L-malate was used preferentially by the malolactic enzyme in this strain also.

Base Sequence↗

PubMed1996

Evidence for a selective and electroneutral K+/H(+)-exchange in Saccharomyces cerevisiae using plasma membrane vesicles.

The existence of a K+/H+ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K+/H+ transport system, which is able to catalyse both K+ influx and efflux, is mainly driven by the K+ transmembrane gradient and can function even if the plasma membrane H(+)-ATPase is not active. Using the anionic oxonol VI and the cationic DISC2(5) probes, it was shown that a membrane potential is not created during K+ fluxes. Such a dye response argues for the presence of a K+/H+ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6.8. This reversible transport system presents a high selectivity for K+ among other monovalent cations and a higher affinity for the K+ influx into the vesicles (exit from cells). The possible role of this K+/H+ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.

Biological Transport, Active↗

PubMed1996

Effects of a strain of Saccharomyces cerevisiae (Levucell SC1), a microbial additive for ruminants, on lactate metabolism in vitro.

The effect of Levucell SC, a strain of Saccharomyces cerevisiae marked as a feed additive for ruminants, was investigated in vitro on lactate metabolism by the ruminal bacteria Streptococcus bovis and Megasphaera elsdenii. The coculture between 10(7) live cells x mL(-1) of SC and a Streptococcus bovis strain in the presence of glucose reduced lactate production by the bacterial strain. Live yeast cells were able to compete with Streptococcus bovis for glucose utilization in strictly anaerobic conditions, so less glucose was available for the bacterium. SC also stimulated L-lactate utilization by a strain of M. elsdenii. The effect depended on the concentration of yeast cells added. Bacterial growth and fermentation end-product concentrations were also increased in the presence of SC. Some amino acids and vitamins, but not dicarboxylic acids, stimulated the bacterial specific activity of L-lactate uptake. SC was able to provide amino acids to M. elsdenii. In a coculture of Streptococcus bovis and M. elsdenii on glucose, the reduction of lactate concentration was improved by SC, the same trend being observed when maltose or soluble starch were used as carbon and energy source. These results indicate that SC can be a very useful tool to reduce lactate accumulation in vitro during fermentation of soluble sugars.

Acetates↗

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↗