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K Sigler

Publications and source records attributed to K Sigler.

At least 55 records · Page 3Linked to original sources
PubMed1996

Possible nystatin-protein interaction in yeast plasma membrane vesicles in the presence of ergosterol. A Förster energy transfer study.

The Förster energy transfer from tryptophan residues of membrane proteins to nystatin was measured in reconstituted yeast plasma membrane vesicles free of, or doped with, ergosterol. We wanted to elucidate whether the functional change of membrane transport proteins from H+ symporters to facilitators, observed in ergosterol-containing plasma membrane vesicles on addition of nystatin [Opekarová and Tanner (1994) FEBS Lett. 350, 46-50], is reflected in altered protein-nystatin relations within the membrane. Both frequency-domain and time-domain time-resolved fluorescence spectroscopy showed that in the presence of ergosterol nystatin is located much closer to membrane proteins than in its absence.

Cell Membrane↗
PubMed1996

Three fluorescent probes for the flow-cytometric assessment of membrane potential in Saccharomyces cerevisiae.

Three fluorescent probes, tetramethyl rhodamine ethyl ester (TMRE), 3,3'-dipropylthiacarbocyanine iodide (diS-C3(3)) and 3,3'-dipropyloxacarbocyanine iodide (diO-C3(3)), were tested for their suitability as fluorescent indicators of membrane potential in Saccharomyces cerevisiae in studies performed by flow cytometry. For all these dyes the intensity of fluorescence of stained cells increased with probe concentration in the range of 60-3000 nmol/L. The optimum staining period was 15-20 min for diS-C3(3). Depolarization of cells by increased extracellular potassium level and by valinomycin elicited with all probes a drop in fluorescence intensity. In some yeast batches this depolarization was accompanied by a separation of subpopulations with different fluorescence properties.

Artifacts↗
PubMed1996

Cd2+-induced damage to yeast plasma membrane and its alleviation by Zn2+: studies on Schizosaccharomyces pombe cells and reconstituted plasma membrane vesicles

In Schizosaccharomyces pombe, Cd2+ shares the same uphill uptake system with Zn2+. Both heavy metals inhibited growth, respiration, H+/glucose uptake, and glucose-induced proton extrusion, Cd2+ being a 10-15-fold stronger inhibitor. In contrast, both had a similar effect on the plasma membrane H+-ATPase, enhancing its affinity for ATP and reducing the rate of ATP splitting. Cd2+ caused protracted strong fluidization of the plasma membrane of energized cells, whereas deenergized cells, phosphatidylcholine liposomes, and plasma membrane fragments, either purified or incorporated into the liposomes, exhibited only a short initial fluidization. Zn2+, which caused only a marginal membrane fluidization, suppressed the fluidizing action of Cd2+. The fluidizing effect of both heavy metals on liposomes was reduced by the presence of plasma membrane fragments in the liposome membrane. At 50 &mgr;M, Cd2+ brought about loss K+ (18 K+/1 Cd2+) from energized, but not from deenergized cells since Cd2+ must first accumulate in the cells before causing a detectable effect. A simple membrane disruption by external Cd2+ is, therefore, unlikely to be the main mechanism of cadmium-induced potassium loss in intact cells. Zn2+ had virtually no effect below 1 mM concentration, and it again weakened the K+-releasing effect of Cd2+. Cd2+ caused a strong loss of K+ also from K+-containing liposomes, probably because of a direct interaction with liposome phospholipids. Incorporation of plasma membrane fragments into the liposomes reduced the K+ loss sixfold.

Journal Article↗
PubMed1996

Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis.

Basic tenets related to the use of three main classes of potentiometric redistribution fluorescent dyes (carbocyanines, oxonols, and rhodamines) are discussed in detail. They include the structure/function relationship, formation of nonfluorescent (H-type) and fluorescent (J-type) dimers and higher aggregates, probe partitioning between membranes and medium and binding to membranes and intracellular components (with attendant changes in absorption and emission spectra, fluorescence quantum yield and lifetime). The crucial importance of suitable probe-to-cell concentration ratio and selection of optimum monitored fluorescence wavelength is illustrated in schematic diagrams and possible artifacts or puzzling results stemming from faulty experimental protocol are pointed out. Special attention is paid to procedures used for probe-response calibration (potential clamping by potassium in the presence of valinomycin, use of gramicidin D in combination with N-methylglucamine, activation of Ca-dependent K-channels by A23187, the null-point technique). Among other problems treated are dye toxicity, interaction with mitochondria and other organelles, and possible effects of intracellular pH and the quantity of cytosolic proteins and/or RNA on probe response. Individual techniques using redistribution dyes (fluorescence measurements in cuvettes, flow cytometry and microfluorimetry of individual cells including fluorescence confocal microscopy) are discussed in terms of reliability, limitations and drawbacks, and selection of suitable probes. Up-to-date examples of application of slow dyes illustrate the broad range of problems in which these probes can be used.

Animals↗
PubMed1996

Ba2+ ions hyperpolarize algal cell membrane and enhance plasmalemma resistance without affecting ion and water contents.

Culturing the fresh-water alga Hydrodictyon reticulatum for 9 days in the presence of 0.2 and 0.4 mmol/l Ba2+ increases the membrane potential about twice and the plasmalemma resistance 5-10 times without having any significant effect on the cell contents of water and Na+, K+ and Cl- ions. While in control cells the membrane potential is determined by the diffusion potential of K+, in Ba(2+)-treated cells the factor determining membrane potential and resistance is the unequal distribution of protons across the membrane.

Barium↗
PubMed1995

Comparison of three different methods for determining yeast killer toxin K1 activity and standardisation of units.

A new rapid assay of the yeast killer toxin K1 activity in Saccharomyces cerevisiae, the BCP (bromocresol purple) fluorescence test, was compared with two standard methods, the plating test and the well test. The BCP test is as precise as the plating test and more sensitive than the well test. Under suitable conditions, the test is universally applicable for determining the proportion of cells with a damaged plasma membrane in a population. A procedure was developed for calculating the results of all three tests in terms of a single well-defined unit (lethal or killer unit, LU).

Killer Factors, Yeast↗
PubMed1995

Inhibition of gap junctional intercellular communication and enhancement of growth in BALB/c 3T3 cells treated with connexin43 antisense oligonucleotides.

Many studies have correlated reductions in gap junctional intercellular communication (GJIC) with altered cellular growth, tumor promotion, and neoplastic transformation. To test directly whether reduced GJIC affects cellular growth, GJIC was inhibited in murine BALB/c 3T3 fibroblasts by treatment with a phosphorothioate-modified antisense oligonucleotide targeted against the connexin43 translation start codon, and in vitro cell growth was monitored. The cells were incubated with the oligonucleotide (0.1-0.5 microM) in liposomes in serumless culture medium for 16 h; washed and refed with serum-containing medium; and analyzed for dye-coupling, connexin43 protein and mRNA levels, and cell growth over the next 5 d. The antisense oligonucleotide inhibited dye-coupling and reduced connexin43 protein levels in a concentration-dependent manner but had no effect on connexin43 mRNA levels. Cell growth rate was not affected, but saturation density was increased approximately threefold by the oligonucleotide. These data support a role for GJIC in the establishment of contact inhibition of in vitro cell growth.

3T3 Cells↗
PubMed1995

Oxidative stress and living cells.

The review deals with the effects of reactive oxygen species, both radical and nonradical (e.g. hydrogen peroxide), on cells and organisms. The chemical and biochemical aspects include description of individual reactive oxygen species, chemical reactions giving rise to them, their interconversions and interactions with metals (Fe2+, Cu2+, Cu+) and other substances (scavengers, antioxidants). The biological aspects concern the specific features and locations of cellular enzyme systems involved in radical production and/or removal. Major harmful effects of the species on the molecular (protein oxidation, lipid peroxidation, damage to DNA) and cellular level (effect on signal transduction, on cell membrane functions and on gene expression) are surveyed. Methods whereby cells and organisms cope with the onslaught of these reactive species are reviewed as well as implications for plant, animal and human health.

Animals↗
PubMed1995

Significance of the lag phase in K1 killer toxin action on sensitive yeast cells.

The minimum period between the addition of killer toxin K1 to sensitive yeast cells and the appearance of first cells stained with bromocresol purple indicating membrane damage, is approximately 20 min. The length of this lag phase depends strongly on toxin concentration, extending sharply at toxin levels lower than 60 lethal units (LU) per cell (about one-tenth of the toxin concentration necessary for saturating all surface receptors). As the binding of the toxin to the cell is virtually complete within 1 min, the rest of the lag phase reflects processes different from actual binding, e.g. combination of several toxin molecules to form a membrane ion channel or pore.

Cell Membrane↗
PubMed1994

Transmembrane potentials in cells: a diS-C3(3) assay for relative potentials as an indicator of real changes.

The mechanism by which the fluorescent cationic dye diS-C3(3) reports on cellular transmembrane potential has been investigated in murine haemopoietic cells. Due to the large molar absorbance of diS-C3(3) and its high quantum yield of fluorescence in cells, this dye can be used at very low labelling concentrations (5 x 10(-8) to 2 x 10(-7) M). In contrast to the quenching of fluorescence observed for the most commonly used voltage-sensitive dyes of the carbocyanine class, the fluorescence intensity of diS-C3(3) increases when the dye accumulates in the cells. The method of synchronous emission spectroscopy was used to resolve the intracellular and extracellular components of the diS-C3(3) fluorescence of suspensions of labelled cells. In comparing changes in these signals consequent on changes in transmembrane potential induced by varying the extracellular concentration of potassium ions in the presence of valinomycin, the logarithm of the ratio of intensities of these two components, as predicted theoretically, was found to be a good linear measure of transmembrane potential under these conditions. The dye was also demonstrated to be suitable for flow-cytofluorimetric analysis, the logarithm of the mean population signal similarly being found to provide a good linear measure of the transmembrane potential. The conditions under which such linearity may be expected with respect to possible effects due to changes in the capacity for binding of the dye to proteins and various cytosolic structures are delineated and their validity with respect to the possibly contentious role of mitochondria in such measurements examined in particular. The use of the method in indicating changes in the transmembrane potential and/or changes in the transport numbers of the major ions determining transmembrane potential between different physiological states, the possible extension to determinations of absolute differences in potential between different cell states without calibration or comparison with potassium-ion potentials, and the conditions for validity and limitations of these partially complementary measurements, are discussed.

Animals↗
PubMed1994

Kinetic studies of killer toxin K1 binding to yeast cells indicate two receptor populations.

A recently described new method for determination of killer toxin activity was used for kinetic measurements of K1 toxin binding. The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates. The low-affinity and high-velocity binding component (KT1 = 2.6 x 10(9) L.U./ml, Vmax1 = 0.19 s-1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (KT2 = 3.2 x 10(7) L.U./ml, Vmax2 = 0.03 s-1) presumably indicates the binding of the toxin to plasma membrane receptors. Adsorption of most of the killer toxin K1 to the surface of sensitive cells occurred within 1 min and was virtually complete within 5 min. The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S. cerevisiae S6.

Adsorption↗
PubMed1994

Loss of gap junctions from DDT-treated rat liver epithelial cells.

The mechanism by which the liver tumor promoter 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT) inhibits gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells could involve gap junction loss and/or decreased gap junction channel permeability. We examined these two possibilities in the present study. Immunohistochemical studies using antibodies specific to connexin43, the major gap junction protein expressed by these cells, revealed that gap junction number and size were reduced during exposure to DDT. The reductions in gap junctions (33-91%) correlated with dose-dependent (1-10 microM) and time-dependent (0.5-4 h) decreases in cell-to-cell fluorescent dye-coupling (64-85%), as well as cellular levels of phosphorylated connexin43. These effects were reversible following removal of the tumor promoter from the culture medium, although cycloheximide reduced the level of gap junction reformation. The losses in gap junctions were not due to decreased connexin43 gene expression since steady-state levels of connexin43 mRNA were not similarly affected by DDT. Fenarimol (10 microM), a structural analog of DDT, did not inhibit GJIC and had no effect on gap junction structure or connexin43 expression. These data suggest that the inhibition of GJIC by DDT resulted from the removal of gap junctions from the plasma membrane and their degradation rather than simply a decrease in their permeability.

Animals↗
PubMed1994

Growth inhibition of rat liver epithelial tumor cells by monoterpenes does not involve Ras plasma membrane association.

The role of altered ras oncoprotein (Ras) farnesylation and membrane association in the growth inhibitory effects of several monoterpenes (limonene, perillic acid, perillyl alcohol, menthol, pinene and cineole) was investigated in rat liver epithelial cells. All of the above compounds except cineole inhibited the growth of viral Ha-ras-transformed rat liver epithelial cells (WB-ras cells) at concentrations of 0.25-2.5 mM. These cells, however, were not necessarily more sensitive to these compounds compared to non-transformed and viral raf-transformed rat liver epithelial cells. Growth inhibition by limonene, perillic acid and pinene was only partially restored (20-50%) by supplementing the culture medium with 2 mM mevalonic acid. Western blot analyses of cytosolic and membranous fractions of WB-ras cells treated with monoterpenes indicated no change in Ras distribution. In contrast, lovastatin, a potent inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase and Ras farnesylation, specifically reduced WB-ras cell growth and increased cytosolic levels of Ras. Thus, monoterpene-induced growth inhibition of rat liver epithelial cells was dissimilar to lovastatin and did not appear to involve altered Ras plasma membrane association.

Animals↗
PubMed1993

Gap junctional intercellular communication in mouse lung epithelial cell lines: effects of cell transformation and tumor promoters.

Gap junctional intercellular communication (GJIC) is reduced by neoplastic transformation and treatment with tumor promoters in many types of cells but few data exist for the lung. GJIC was therefore evaluated in non-transformed (C10) and transformed (E9, 82-132, and PCC4) mouse lung epithelial cell lines and in C10 cells treated with tumor promoters. GJIC was assessed by fluorescent dye microinjection (dye-coupling). Dye-coupling levels were highest in C10 cells (85-90% communicating cells) followed by 82-132 cells (40-50%), E9 cells (15-20%), and PCC4 cells (3-10%). Indirect immunofluorescent staining with anti-gap junction protein (connexin) antibodies revealed that C10 cells expressed gap junctions comprised of connexin43, but not connexin32 or connexin26. The tumor promoters, butylated hydroxytoluene (BHT), 12-O-tetradecanoylphorbol-13-acetate (TPA), and p,p'-dichlorodiphenyltrichloroethane (DDT), inhibited dye-coupling in C10 cells but phenobarbital (PB) did not. BHT promotes mouse lung tumor formation, PB does not, while the effects of TPA and DDT on lung tumor development have not been reported. These data indicate that cell transformation and certain tumor promoters reduce GJIC in mouse lung epithelial cells and demonstrate correlations between the in vitro inhibition of GJIC and lung tumor promotion.

Animals↗
PubMed1993

Enhancement of gap junctional intercellular communication in tumor promoter-treated cells by components of green tea.

Green tea (Camellia sinensis) has been reported to inhibit tumor promotion in vivo and in vitro. Many tumor promoters inhibit gap junctional intercellular communication (GJIC) which may be an important mechanism of promotion. In the present study, we hypothesized that green tea would enhance GJIC in promoter-treated cells. An aqueous extract of green tea (GTE) and several of its constituents were tested for their effects on GJIC in p,p'-dichlorodiphenyltrichloroethane (DDT)-, 12-O-tetradecanoylphorbol-13-acetate (TPA)- and dieldrin-treated WB-F344 rat liver epithelial cells. All three promoters inhibited GJIC in a dose-responsive manner at non-cytolethal concentrations. (GTE (10-80 gamma/ml) enhanced GJIC 20-80% in promoter-treated cells. (-)-Epigallocatechin gallate and (-)-epicatechin gallate also enhanced GJIC in DDT-treated cells, but no effects were seen with (+)-catechin, (-)-epicatechin, (-)-epigallocatechin, caffeine, or theobromine. These data suggest GTE may inhibit tumor promotion by enhancing GJIC and that the most active components are the catechin gallates.

Animals↗