RESUMO
Cationic and heavy metal toxicity is involved in a substantial number of diseases in mammals and crop plants. Therefore, the understanding of tightly regulated transporter activities, as well as conceiving the interplay of regulatory mechanisms, is of substantial interest. A generalized thermodynamic description is developed for the complex interplay of the plasma membrane ion transporters, membrane potential and the consumption of energy for maintaining and restoring specific intracellular cation concentrations. This concept is applied to the homeostasis of cation concentrations in the yeast cells of S. cerevisiae. The thermodynamic approach allows to model passive ion fluxes driven by the electrochemical potential differences, but also primary or secondary active transport processes driven by the inter- play of different ions (symport, antiport) or by ATP consumption (ATPases). The model-confronted with experimental data-reproduces the experimentally observed potassium and proton fluxes induced by the external stimuli KCl and glucose. The estimated phenomenological constants combine kinetic parameters and transport coefficients. These are in good agreement with the biological understanding of the transporters thus providing a better understanding of the control exerted by the coupled fluxes. The model predicts the flux of additional ion species, like e.g. chloride, as a potential candidate for counterbalancing positive charges. Furthermore, the effect of a second KCl stimulus is simulated, predicting a reduced cellular response for cells that were first exposed to a high KCl stimulus compared to cells pretreated with a mild KCl stimulus. By describing the generalized forces that are responsible for a given flow, the model provides information and suggestions for new experiments. Furthermore, it can be extended to other systems such as e.g. Candida albicans, or selected plant cells.
Assuntos
Cátions/metabolismo , Homeostase/fisiologia , Modelos Biológicos , Saccharomyces cerevisiae/fisiologia , Algoritmos , Cátions/química , Biologia Computacional , Canais Iônicos/química , Canais Iônicos/metabolismo , TermodinâmicaRESUMO
Although considered as essential cofactors for a variety of enzymatic reactions and for important structural and functional roles in cell metabolism, metals at high concentrations are potent toxic pollutants and pose complex biochemical problems for cells. We report results of single dose acute toxicity testing in the model organism S. cerevisiae. The effects of moderate toxic concentrations of 10 different human health relevant metals, Ag(+), Al(3+), As(3+), Cd(2+), Co(2+), Hg(2+), Mn(2+), Ni(2+), V(3+), and Zn(2+), following short-term exposure were analyzed by transcription profiling to provide the identification of early-on target genes or pathways. In contrast to common acute toxicity tests where defined endpoints are monitored we focused on the entire genomic response. We provide evidence that the induction of central elements of the oxidative stress response by the majority of investigated metals is the basic detoxification process against short-term metal exposure. General detoxification mechanisms also comprised the induction of genes coding for chaperones and those for chelation of metal ions via siderophores and amino acids. Hierarchical clustering, transcription factor analyses, and gene ontology data further revealed activation of genes involved in metal-specific protein catabolism along with repression of growth-related processes such as protein synthesis. Metal ion group specific differences in the expression responses with shared transcriptional regulators for both, up-regulation and repression were also observed. Additionally, some processes unique for individual metals were evident as well. In view of current concerns regarding environmental pollution our results may support ongoing attempts to develop methods to monitor potentially hazardous areas or liquids and to establish standardized tests using suitable eukaryotic a model organism.
Assuntos
Metais/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico/efeitos dos fármacos , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Sítios de Ligação/genética , Análise por Conglomerados , Meios de Cultura/química , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Genes Fúngicos/genética , Humanos , Íons , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/genética , Testes de Toxicidade , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The fluorescent dye 3,3'-dipropylthiadicarbocyanine, diS-C(3)(3), is a suitable probe to monitor real changes of plasma membrane potential in yeast cells which are too small for direct membrane potential measurements with microelectrodes. A method presented in this paper makes it possible to convert changes of equilibrium diS-C(3)(3) fluorescence spectra, measured in yeast cell suspensions under certain defined conditions, into underlying membrane potential differences, scaled in the units of millivolts. Spectral analysis of synchronously scanned diS-C(3)(3) fluorescence allows to assess the amount of dye accumulated in cells without otherwise necessary sample taking and following separation of cells from the medium. Moreover, membrane potential changes can be quantified without demanding calibration protocols. The applicability of this approach was demonstrated on the depolarization of Rhodotorula glutinis yeast cells upon acidification of cell suspensions and/or by increasing extracellular K(+) concentration.
Assuntos
Carbocianinas/química , Corantes Fluorescentes/química , Potenciais da Membrana/fisiologia , Rhodotorula/fisiologia , Rhodotorula/citologiaRESUMO
Postmenopausal women with elevated serum sex steroids have an increased risk of breast cancer. Most of this risk is believed to be exerted through binding of the sex steroids to their receptors. For the first time, we investigate the association of estrogen receptor (ER) and androgen receptor (AR) serum bioactivity (SB) in addition to hormone levels in samples from women with breast cancer collected before diagnosis. Two hundred postmenopausal women participating in the UK Collaborative Trial of Ovarian Cancer Screening who developed ER-positive breast cancer 0.6-5 years after sample donation were identified and matched to 400 controls. ER and AR bioassays were used to measure ERα, ERß, and AR SB. Androgen and estrogen levels were measured with immunoassays. Subjects were classified according to quintiles of the respective marker among controls and the associations between SB and hormones with breast cancer risk were determined by logistic regression analysis. ERα and ERß SB were significantly higher before diagnosis compared with controls, while estrogens showed no difference. Women had a twofold increased breast cancer risk if ERα SB (odds ratio (OR), 2.114; 95% confidence interval (CI), 1.050-4.425; P=0.040) was in the top quintile >2 years before diagnosis or estrone (OR, 2.205; 95% CI, 1.104-4.586; P=0.029) was in the top quintile <2 years before diagnosis. AR showed no significant association with breast cancer while androstenedione (OR, 3.187; 95% CI, 1.738-6.044; P=0.0003) and testosterone (OR, 2.145; 95% CI, 1.256-3.712; P=0.006) were significantly higher compared with controls and showed a strong association with an almost threefold increased breast cancer risk independent of time to diagnosis. This study provides further evidence on the association of androgens and estrogens with breast cancer. In addition, it reports that high ER but not AR SB is associated with increased breast risk >2 years before diagnosis.
Assuntos
Neoplasias da Mama/sangue , Receptor alfa de Estrogênio/sangue , Receptor beta de Estrogênio/sangue , Hormônios Esteroides Gonadais/sangue , Pós-Menopausa/sangue , Receptores Androgênicos/sangue , Idoso , Androstenodiona/sangue , Estudos de Casos e Controles , Estudos de Coortes , Sulfato de Desidroepiandrosterona/sangue , Estradiol/sangue , Estrona/sangue , Feminino , Humanos , Imunoensaio , Pessoa de Meia-Idade , Medição de Risco , Globulina de Ligação a Hormônio Sexual/metabolismo , Testosterona/sangueRESUMO
We designed a simple graphical presentation for the results of a transcription factor (TF) pattern matching analysis. The TF analysis algorithm utilized known sequence signature motifs from several databases. The graphical presentation enabled a quick overview of potential TF binding sites, their frequency and spacing on both DNA strands and thus straight forward identification of promising candidates for further experimental investigations. The developed tool was applied on in total four Saccharomyces cerevisiae gene promoter regions. The selected differentially expressed genes belong to functionally different families and encode duplicate functions, TRK1 and TRK2 as ion transporters and BMH1 and BMH2 as multiple regulators. Output evaluation revealed a number of TFs with promising differences in the promoter regions of each gene pair. Experimental investigations were performed by using corresponding TF yeast mutants for either phenotypic analysis of ion transport mediated growth or expression analysis of BMH1,2 genes. Upon phenotypic testing one TF mutant exhibited severely impaired growth under non-permissive conditions. This TF, Mot3p was identified as of most abundant potential binding sites and distinctive patterns among the TRK promoter regions.
Assuntos
Proteínas 14-3-3/genética , Proteínas de Transporte de Cátions/genética , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Proteínas 14-3-3/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte de Cátions/metabolismo , Biologia Computacional , Gráficos por Computador , DNA Fúngico/genética , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismoRESUMO
It has been shown previously that heterologous expression of inwardly rectifying potassium channels (K+-channels) from plants and mammals in K+-transport defective yeast mutants can restore the ability of growth in media with low [K+]. In this study, the functional expression of an outward rectifying mammalian K+-channel in yeast is presented for the first time. The outward-rectifying mammalian neuronal K+-channel rat ether à go-go channel 1 (rEAG1, Kv 10.1) was expressed in yeast (Saccharomyces cerevisiae) strains lacking the endogenous K+-uptake systems and/or alkali-metal-cation efflux systems. It was found that a truncated channel version, lacking almost the complete intracellular N-terminus (rEAG1 Delta 190) but not the full-length rEAG1, partially complemented the growth defect of K+-uptake mutant cells (trk1,2 Delta tok1 Delta) in media containing low K+ concentrations. The expression of rEAG1 Delta 190 in a strain lacking the cation efflux systems (nha1 Delta ena1-4 Delta) increased the sensitivity to high monovalent cation concentrations. Both phenotypes were observed, when rEAG1 Delta 190 was expressed in a trk1,2 Delta and nha1, ena1-4 Delta mutant strain. In the presence of K+-channel blockers (Cs+, Ba2+ and quinidine), the growth advantage of rEAG1 Delta 190 expressing trk1,2 tok1 Delta cells disappeared, indicating its dependence on functional rEAG1 channels. The results demonstrate that S. cerevisiae is a suitable expression system even for voltage-gated outward-rectifying mammalian K+-channels.
Assuntos
Canais de Potássio Éter-A-Go-Go/fisiologia , Saccharomyces cerevisiae/metabolismo , Animais , Compostos de Bário/farmacologia , Cloretos/farmacologia , Canais de Potássio Éter-A-Go-Go/genética , Concentração de Íons de Hidrogênio , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Proteínas Recombinantes/metabolismo , Rubídio/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Bioensaio , Avaliação Pré-Clínica de Medicamentos/métodos , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Biomassa , Densitometria , Relação Dose-Resposta a DrogaRESUMO
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.
Assuntos
Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Estrogênios/farmacologia , Norpregnenos/farmacologia , Saccharomyces cerevisiae/fisiologia , Clonagem Molecular , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/efeitos dos fármacos , Genes Reporter , Humanos , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
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.
Assuntos
Biologia Computacional/métodos , Canais de Potássio/química , Algoritmos , Sequência de Aminoácidos , Animais , Genoma , Genoma Fúngico , Humanos , Dados de Sequência Molecular , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Transdução de Sinais , SoftwareRESUMO
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.
Assuntos
ATPases Translocadoras de Prótons/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Animais , Sequência de Bases , Biomarcadores , Bovinos , DNA Fúngico/genética , Genes Fúngicos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Metais Pesados/toxicidade , Metilnitronitrosoguanidina/toxicidade , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimologiaRESUMO
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.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Potássio/metabolismo , Saccharomyces cerevisiae/metabolismo , Bário/farmacologia , Transporte de Íons , Metais Alcalinos/metabolismo , Fenótipo , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/biossíntese , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação GenéticaRESUMO
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.
Assuntos
Bioensaio , Resíduos Industriais/análise , Mutagênicos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Eliminação de Resíduos Líquidos , Poluentes Químicos da Água/toxicidade , Biomarcadores/análise , Dano ao DNA , Testes de Mutagenicidade , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Sensibilidade e Especificidade , Água/química , Poluentes Químicos da Água/análiseRESUMO
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.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Potássio/metabolismo , Saccharomyces cerevisiae/genética , Animais , Proteínas de Transporte de Cátions/genética , Teste de Complementação Genética , Higromicina B/farmacologia , Transporte de Íons/genética , Transporte de Íons/fisiologia , Camundongos , Mutação/genética , Potássio/análise , Canais de Potássio Corretores do Fluxo de Internalização/análise , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Metais Pesados/toxicidade , Fenótipo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Testes de Toxicidade Crônica/métodos , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Compostos Orgânicos/toxicidadeRESUMO
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.
Assuntos
Estradiol/farmacologia , Bioensaio , Relação Dose-Resposta a Droga , Humanos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Testes de Mutagenicidade , Mutagênicos/toxicidade , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia , Xenobióticos/toxicidade , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Dano ao DNA , DNA Helicases , Enzimas Reparadoras do DNA , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Transporte Biológico , Mutação , Potássio/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cátions/metabolismo , Meios de Cultura , Eletrofisiologia , Genes Fúngicos , Canais de Potássio/genética , Canais de Potássio/metabolismo , Rubídio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
A hallmark of astrocytic tumors is their infiltrative nature. Although their aggressive and typically widespread dispersal in the adult brain differs fundamentally from that of other brain tumors, little is known about their cellular basis. Astrocytic tumors express the gap junction protein connexin 43 (Cx43), and we show here that Cx43 expression induced the morphological transformation of glioma cells into an epithelial phenotype. In a short-term aggregation assay, Cx43 expression was associated with a several-fold increase in the competence of glioma cells to aggregate. Antibodies directed against the extracellular domain of Cx43 restored the connexin-deficient phenotype, as manifested by a dose-dependent reduction in aggregation. Apart from their role in gap junction formation, connexins may therefore be considered a distinct class of membrane proteins with adhesive properties. Moreover, implanted Cx43-expressing glioma cells established functional gap junction channels with host astrocytes and dispersed through a substantially greater volume of brain parenchyma than mock- and mutant Cx43-transfected sister cells. Cx43 expression therefore may modulate not only the adhesion of astrocytes to one another, but the spread of glial tumor cells throughout astrocytic syncytia. These observations widen our concept of the potential interactions between tumor cells and their surroundings and suggest that both connexin proteins and their derived gap junctions are critical determinants of the invasiveness of central gliomas.