Biomedical subjects

Hella Lichtenberg-Fraté

Publications and source records attributed to Hella Lichtenberg-Fraté.

11 recordsLinked to original sources

PubMed2006

Ring test assessment of the mKir2.1 growth based assay in Saccharomyces cerevisiae using parametric models and model-free fits.

Inward rectifying K+ (Kir) channels are a subfamily of the potassium channel superfamily. They mediate potassium influx into the cells, a process responding to the polarization state, a variety of intracellular messengers and specific auxiliary proteins, thereby they are involved in important physiological processes such as the pacemaker activity in the heart, insulin release, and potassium uptake in glial cells. The Saccharomyces cerevisiae mKir2.1 in vitro assay was subjected to a ring test assessment. Compound-associated mKir2.1 modulating effects were detected by growth determination of functionally complemented S. cerevisiae cells in a 96-well format within 15 h. Dose-response diagrams and EC50 value calculations were determined by parametric model and model-free fits using cubic spline interpolation. These characteristics were evaluated by statistical methods to determine reproducibility among working groups. Nonparametric bootstrap simulations of the variability of the data revealed that EC50 values of the mKir2.1 indicator strain were well-matched (81-92 microM), enabling unambiguous quantitative statements about inhibitory effects and no significant influence of the different laboratory conditions. Limitations of the assay include compounds/samples that are either insoluble under the conditions of the test or strongly cytotoxic to yeast. Thus, the described test is a sensitive and reliable tool that can be used in different laboratories and is applicable in drug discovery and development as simple and reliable prescreening procedure.

Biological Assay↗

PubMed2006

Estrogenic effects of natural and synthetic compounds including tibolone assessed in Saccharomyces cerevisiae expressing the human estrogen alpha and beta receptors.

The human estrogen receptors (hER)alpha and hERbeta, differentially expressed and localized in various tissues and cell types, mediate transcriptional activation of target genes. These encode a variety of physiological reproductive and nonreproductive functions involved in energy metabolism, salt balance, immune system, development, and differentiation. As a step toward developing a screening assay for the use in applications where significant numbers of compounds or complex matrices need to be tested for (anti) estrogenic bioactivity, hERalpha and hERbeta were expressed in a genetically modified Saccharomyces cerevisiae strain, devoid of three endogenous xenobiotic transporters (PDR5, SNQ2, and YOR1). By using receptor-mediated transcriptional activation of the green fluorescent protein optimized for expression in yeast (yEGFP) as reporter 17 natural, comprising estrogens and phytoestrogens or synthetic compounds among which tibolone with its metabolites, gestagens, and antiestrogens were investigated. The reporter assay deployed a simple and robust protocol for the rapid detection of estrogenic effects within a 96-well microplate format. Results were expressed as effective concentrations (EC50) and correlated to other yeast based and cell line assays. Tibolone and its metabolites exerted clear estrogenic effects, though considerably less potent than all other natural and synthetic compounds. For the blood serum of two volunteers, considerable higher total estrogenic bioactivity than single estradiol concentrations as determined by immunoassay was found. Visualization of a hERalpha/GFP fusion protein in yeast revealed a sub cellular cytosolic localization. This study demonstrates the versatility of (anti) estrogenic bioactivity determination using sensitized S. cerevisiae cells to assess estrogenic exposure and effects.

Cloning, Molecular↗

PubMed2006

Computational recognition of potassium channel sequences.

MOTIVATION: Potassium channels are mainly known for their role in regulating and maintaining the membrane potential. Since this is one of the key mechanisms of signal transduction, malfunction of these potassium channels leads to a wide variety of severe diseases. Thus potassium channels are priority targets of research for new drugs, despite the fact that this protein family is highly variable and closely related to other channels, which makes it very difficult to identify new types of potassium channel sequences. RESULTS: Here we present a new method for identifying potassium channel sequences (PSM, Property Signature Method), which-in contrast to the known methods for protein classification-is directly based on physicochemical properties of amino acids rather than on the amino acids themselves. A signature for the pore region including the selectivity filter has been created, representing the most common physicochemical properties of known potassium channels. This string enables genome-wide screening for sequences with similar features despite a very low degree of amino acid similarity within a protein family.

Algorithms↗

PubMed2006

Use of PMA1 as a housekeeping biomarker for assessment of toxicant-induced stress in Saccharomyces cerevisiae.

The brewer's yeast Saccharomyces cerevisiae has emerged as a versatile and robust model system for laboratory use to study toxic effects of various substances. In this study, toxicant-induced stresses of pure compounds were investigated in Saccharomyces cerevisiae utilizing a destabilized version of the green fluorescent protein optimized for expression in yeast (yEGFP3) under control of the promoter of the housekeeping plasma membrane ATPase gene PMA1. The responses of the biomarker upon increasing test compound concentrations were monitored by determining the decrease in fluorescence. The reporter assay deployed a simple and robust protocol for the rapid detection of toxic effects within a 96-well microplate format. Fluorescence emissions were normalized to cell growth determined by absorption and were correlated to internal reference standards. The results were expressed as effective concentrations (EC20). Dose-response experiments were conducted in which yeast cells were exposed in minimal medium and in the presence of 20% fetal calf serum to sublethal concentrations of an array of heavy metals, salt, and a number of stress-inducing compounds (Diclofenac, Lindane, methyl-N-nitro-N-nitrosoguanidine [MNNG], hydroxyurea, and caffeine). Long-term exposure (7 h) played a considerable role in the adaptive response to intoxication compared to early responses at 4 h exposure. The data obtained after 4 h of exposure and expressed as EC20 were compared to 50% inhibitory concentration values derived from cell line and ecotoxicological tests. This study demonstrates the versatility of the novel biomarker to complement existing test batteries to assess contaminant exposure and effects.

Animals↗

PubMed2005

Analysis of the mKir2.1 channel activity in potassium influx defective Saccharomyces cerevisiae strains determined as changes in growth characteristics.

Potassium uptake defective Saccharomyces cerevisiae strains (Deltatrk1,2 and Deltatrk1,2 Deltatok1) were used for the phenotypic analysis of the mouse inward rectifying Kir2.1 channel by growth analysis. Functional expression of both, multi-copy plasmid and chromosomally expressed GFP-mKir2.1 fusion constructs complemented the potassium uptake deficient phenotype in a pHout dependent manner. Upon application of Hygromycin B to chromosomally mKir2.1 expressing cells, significantly lower toxin sensitivity (EC50 15.4 microM) compared to Deltatrk1,2 Deltatok1 cells (EC50 2.6 microM) was observed. Growth determination of mKir2.1 expressing strains upon application of Ag+, Cs+ and Ba2+ as known blockers of mKir2.1 channels revealed significantly decreased channel function. Cells with mKir2.1 were about double sensitive to AgNO3, 350-fold more sensitive to CsCl and 1500-fold more sensitive to BaCl2 in comparison to the respective controls indicating functional expression and correct pharmacology.

Animals↗

PubMed2005

New phenotypes of functional expression of the mKir2.1 channel in potassium efflux-deficient Saccharomyces cerevisiae strains.

The functional expression of the mouse Kir2.1 potassium channel in yeast cells lacking transport systems for potassium and sodium efflux (ena1-4delta nha1delta) resulted in increased cell sensitivity to high external concentrations of potassium. The phenotype depended on the level of Kir2.1 expression and on the external pH. The activity of Kir2.1p in the yeast cells was almost negligible at pH 3.0 and the highest at pH 7.0. Kir2.1p was permeable for both potassium and rubidium cations, but neither sodium nor lithium were transported via the channel. Measurements of the cation contents in cells confirmed the higher concentration of potassium in cells with Kir2.1p. Specific inhibition of the mKir2.1 channel activity by Ba2+ cations was observed. The use of a mutant strain lacking both potassium efflux and uptake transporters (ena1-4delta nha1delta trk1delta trk2delta) enabled the monitoring of channel activity on two levels--the provision of the necessary amount of intracellular K+ in media with low potassium concentrations, and simultaneously, the channel's contribution to cell potassium sensitivity in the presence of high external K+. This combination of mutations proved to be a new, sensitive and practical tool for characterizing the properties of heterologously expressed transporters mediating both the efflux and influx of alkali-metal-cations.

Barium↗

PubMed2005

The toxic potential of an industrial effluent determined with the Saccharomyces cerevisiae-based assay.

Increasing levels of environmental pollution and the continuous monitoring of water quality both request specific and sensitive methods for the detection of detrimental water contents. On a regulatory basis genotoxicity is assessed by the standard umu-test (ISO 13829) that responds to DNA damage induced by chemicals. The focus of this study was the examination of the toxic potential of samples taken from the wastewater treatment plant of a refinery factory to explore the applicability of the Saccharomyces cerevisiae (bakers yeast) test for the detection of bio-available genotoxic activity in complex matrices. The toxic potential of samples without pre-treatment and following centrifugation was determined with the eukaryotic Saccharomyces cerevisiae bioassay based on the transcriptional activation of the green fluorescent protein (gfp) fused to the DNA damage inducible RAD54 promoter and general growth inhibition. Primary effluent samples were taken as qualified sterile spot samples from the final effluent of the purification plant. The Saccharomyces cerevisiae assay yielded geno- and cytotoxic responses in all complex untreated and centrifuged samples with high reproducibility. The obtained results suggest that the yeast assay is suited as a screening tool to monitor genotoxic potential of wastewater.

Biological Assay↗

PubMed2004

In vitro bioactivity of 17alpha-estradiol.

A miniaturised short-term in vitro assay based on the activation of the human estrogen receptor alpha and genetically modified yeast (Saccharomyces cerevisiae) cells was performed to explore the capacity of this system to monitor the bioactivity of estrogenic compounds, particularly 17alpha- and 17beta-estradiol. Together with the human estrogen receptor (hER)-alpha plasmid, the reporter plasmid containing a yeast-optimised version of the green fluorescent protein (yEGFP) linked to three repeats of the cis-acting estrogen hormone-responsive element (ERE) were expressed in a strain being deleted in the pleiotropic drug resistance transporters Pdr5, Snq2 and Yor1, known to facilitate efflux of organic compounds including steroids and chemotherapeutics. Agonists that bind to hER in vitro trigger estrogen receptor-mediated transcriptional activation of the GFP reporter gene monitored by fluorescence emission at 535 nm. The sensitivity of the assay was tested with various 17alpha- and 17beta-estradiol concentrations, yielding a detection limit of 5 pg/ml (0.018 nM) for the agonist 17beta-E2 in solvent and in human charcoal-stripped serum using a S. cerevisiae pdr5, snq2 and yor1 mutant strain. For 17alpha-estradiol only, at approximately 1500 pg/ml a similar fluorescence response compared to 100 pg/ml 17beta-E2 was observed implicating a much weaker potency of this stereoisomer. The specificity of the system was tested by expression of a truncated hER lacking the ligand-binding domain E and by administration of the androgen, 4-androsten 3,17 dione. Both controls did not yield an increase in fluorescence emission. This fluorescence emission assay enables detection of estrogenic biological activity induced by direct agonists, such as 17beta-E2 at concentrations similar to those found in human sera or by estrogen-like chemicals.

Biological Assay↗

PubMed2004

Phenotypic yeast growth analysis for chronic toxicity testing.

In Saccharomyces cerevisiae the pH-dependent growth inhibition of the heavy metals Cu(2+), Cr(6+), Zn(2+), Co(2+), and Cd(2+) was examined in comparison to that of organic solvents and pure compounds DMSO, MNNG, 4-NQO, MTBE, ethanol, and 2-AA. The assay was based on both S. cerevisiae wild-type and genetically modified cells deleted in the transporters Pdr5, Snq2, and Yor1 that facilitate pleiotropic drug resistance to explore the potential for short-term chronic aquatic toxicity tests. The strain deleted in the proteins that mediate the efflux of structurally diverse hydrophobic compounds exhibited high sensitive growth inhibition at low (0.04 mg/L 4-NQO) to moderate (5.5 mg/L DMSO) organic compound exposure. At pH 6.4 the EC(50)'s, for all tested heavy metals were significantly low, in contrast to acidic pH conditions, in which both strains were able to grow in the presence of high concentrations of the transition metals Cu(2+), Zn(2+), and Co(2+), with the pdr5 yor1 snq2 mutant being more tolerant. Cd(2+) exerted the highest toxicity, with an EC(50) of 0.49 mg/L. Obtained results were compared with data determined from growth-inhibition tests involving other unicellular species. The comparison provided evidence that yeast is a sensitive and practical model system for toxicological risk assessment.

Dose-Response Relationship, Drug↗

PubMed2003

Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations.

Saccharomyces cerevisiae cells express three defined potassium-specific transport systems en-coded by TRK1, TRK2 and TOK1. To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of trk1delta, trk2delta and tok1delta null mutations. The in vivo K+ transport characteristics of each strain have been documented using growth-based assays, and the in vitro biochemical and electrophysiological properties associated with K+ transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high-affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of TRK1 and TRK2, strains lack the ability specifically to take up K+, and trk1deltatrk2delta double mutant cells depend upon poorly understood non-specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non-specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs+. Based on this finding, we have established a growth-based assay that monitors the in vivo activity of Tok1p.

Biological Transport↗

PubMedDate not supplied

A yeast-based method for the detection of cyto and genotoxicity.

A miniaturized short-term in vivo genotoxicity screening assay based on genetically modified yeast (Saccharomyces cerevisiae) cells was performed to explore the capacity of this eukaryotic organism to detect the presence of genotoxic compounds. An increased general sensitivity of yeast cells to toxic compounds was obtained by using a strain being deleted in the prominent pleiotropic drug resistance mediating efflux transporters PDR5, SNQ2 and YOR1. In order to detect genotoxic effects, a yeast optimized version of the green fluorescent protein (GFP) was fused to the RAD54 promoter that is activated upon DNA damage. Various model substances including the oxygenated fuel additive methyl tertiary-butyl ether (MTBE) and the direct acting genotoxins methyl-N-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline-1-oxide (4-NQO) were tested. All model substances were in parallel examined for chronic cytotoxicity. The results point out the sufficiency of both the sensitivity of the yeast cells to detect chronic cytotoxicity and the intensity of the fluorescence signal for the assessment of genotoxic effects. Thus, the test enables simultaneous detection of cytotoxic and genotoxic effects. By partial automation and implementation of the test in the microtitre scale this bioassay allows parallel sensitive pre-screening of numerous samples.

Cell Division↗