RESUMO
In an attempt to increase the biological activity of the 1,2,4-triazolo[1,5-a]pyrimidine scaffold through complexation with essential metal ions, the complexes trans-[Cu(mptp)2Cl2] (1), [Zn(mptp)Cl2(DMSO)] (2) (mptp: 5-methyl-7-phenyl-1,2,4-triazolo[1,5-a]pyrimidine), [Cu2(dmtp)4Cl4]·2H2O (3) and [Zn(dmtp)2Cl2] (4) (dmtp: 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine), were synthesized and characterized as new antiproliferative and antimicrobial species. Both complexes (1) and (2) crystallize in the P21/n monoclinic space group, with the tetrahedral surroundings generating a square-planar stereochemistry in the Cu(II) complex and a tetrahedral stereochemistry in the Zn(II) species. The mononuclear units are interconnected in a supramolecular network through π-π interactions between the pyrimidine moiety and the phenyl ring in (1) while supramolecular chains resulting from C-Hâââπ interactions were observed in (2). All complexes exhibit an antiproliferative effect against B16 tumor cells and improved antibacterial and antifungal activities compared to the free ligands. Complex (3) displays the best antimicrobial activity against all four tested strains, both in the planktonic and biofilm-embedded states, which can be correlated to its stronger DNA-binding and nuclease-activity traits.
Assuntos
Complexos de Coordenação/farmacologia , Cobre/química , Zinco/química , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Complexos de Coordenação/química , Cobre/farmacologia , Cristalografia por Raios X , Humanos , Ligantes , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirimidinas/química , Pirimidinas/farmacocinética , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade , Zinco/farmacologiaRESUMO
In an attempt to propose new applications for the biomedical field, complexes with mixed ligands {[Cu(bpy)2(µ2OClO3)]ClO4}n (1) and [Cu(phen)2(OH2)](ClO4)2 (2) (bpy: 2,2'-biyridine; phen and 1,10-phenantroline) were evaluated for their antibacterial and cytotoxicicity features and for the elucidation of some of the mechanisms involved. Complex (2) proved to be a very potent antibacterial agent, exhibing MIC and MBEC values 2 to 54 times lower than those obtained for complex (1) against both susceptible or resistant Gram-positive and Gram-negative strains, in planktonic or biofilm growth state. In exchange, complex (1) exhibited selective cytotoxicity against melanoma tumor cells (B16), proving a promising potential for developing novel anticancer drugs. The possible mechanisms of both antimicrobial and antitumor activity of the copper(II) complexes is their DNA intercalative ability coupled with ROS generation. The obtained results recommend the two complexes for further development as multipurpose copper-containing drugs.
Assuntos
Antineoplásicos , Complexos de Coordenação , Antibacterianos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Quelantes/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Cobre/farmacologia , Cristalografia por Raios X , LigantesRESUMO
Novel complexes of type [Cu(N-N)(dmtp)2(OH2)](ClO4)2·dmtp ((1) N-N: 2,2'-bipyridine; (2) L: 1,10-phenantroline and dmtp: 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine) were designed in order to obtain biologically active compounds. Complexes were characterized as mononuclear species that crystallized in the space group P-1 of the triclinic system with a square pyramidal geometry around the copper (II). In addition to the antiproliferative effect on murine melanoma B16 cells, complex (1) exhibited low toxicity on normal BJ cells and did not affect membrane integrity. Complex (2) proved to be a more potent antimicrobial in comparison with (1), but both compounds were more active in comparison with dmtp-both against planktonic cells and biofilms. A stronger antimicrobial and antibiofilm effect was noticed against the Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA). Both electron paramagnetic resonance (EPR) and Saccharomyces cerevisiae studies indicated that the complexes were scavengers rather than reactive oxygen species promoters. Their DNA intercalating capacity was evidenced by modifications in both absorption and fluorescence spectra. Furthermore, both complexes exhibited nuclease-like activity, which increased in the presence of hydrogen peroxide.
Assuntos
Anti-Infecciosos , Quelantes , Complexos de Coordenação , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Pirimidinas , Saccharomyces cerevisiae/crescimento & desenvolvimento , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Linhagem Celular Tumoral , Quelantes/síntese química , Quelantes/química , Quelantes/farmacologia , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Humanos , Camundongos , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologiaRESUMO
Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na+/K+-ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca2+-sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca2+ influx into the cytosol and that failing to pump Ca2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca2+ or Mn2+ uptake may be a way of controlling oleandrin toxicity.
Assuntos
Cálcio/metabolismo , Cardenolídeos/química , Glicosídeos Cardíacos/química , Glicosídeos Cardíacos/metabolismo , Manganês/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Cardenolídeos/farmacologia , Glicosídeos Cardíacos/farmacologia , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Citosol/metabolismo , Citosol/ultraestrutura , Inibidores Enzimáticos/metabolismo , Humanos , ATPase Trocadora de Sódio-Potássio/metabolismo , Espectrometria de FluorescênciaRESUMO
Complexes with mixed ligands [Cu(N-N)2(pmtp)](ClO4)2 ((1) N-N: 2,2'-bipyridine; (2) L: 1,10-phenanthroline and pmpt: 5-phenyl-7-methyl-1,2,4-triazolo[1,5-a]pyrimidine) were synthesized and structurally and biologically characterized. Compound (1) crystallizes into space group Pa and (2) in P-1. Both complexes display an intermediate stereochemistry between the two five-coordinated ones. The biological tests indicated that the two compounds exhibited superoxide scavenging capacity, intercalative DNA properties, and metallonuclease activity. Tests on various cell systems indicated that the two complexes neither interfere with the proliferation of Saccharomyces cerevisiae or BJ healthy skin cells, nor cause hemolysis in the active concentration range. Nevertheless, the compounds showed antibacterial potential, with complex (2) being significantly more active than complex (1) against all tested bacterial strains, both in planktonic and biofilm growth state. Both complexes exhibited a very good activity against B16 melanoma cells, with a higher specificity being displayed by compound (1). Taken together, the results indicate that complexes (1) and (2) have specific biological relevance, with potential for the development of antitumor or antimicrobial drugs.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Bactérias/efeitos dos fármacos , Complexos de Coordenação/química , Cobre/química , Compostos Heterocíclicos/química , Melanoma Experimental/tratamento farmacológico , Antibacterianos/química , Antineoplásicos/química , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , LigantesRESUMO
In this study we describe the synthesis and characterisation of a new hydrazone-based fluorescent compound that is able to selectively label the endoplasmic reticulum (ER) in yeast and mammalian living cells. The fluorescence properties of the compound depended on the DMSO/water ratio and on the pH. NMR experiments allowed determination of the conformation adopted in various environments. Apart from the convenient synthetic procedure, our compound displays low cell toxicity and blue emission compatible with filters routinely used in fluorescence microscopy.
Assuntos
Corantes Fluorescentes/química , Hidrazonas/química , Saccharomyces cerevisiae/citologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Retículo Endoplasmático/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Células HeLa , Humanos , Hidrazonas/síntese química , Hidrazonas/farmacologia , Microscopia de Fluorescência , Estrutura Molecular , Saccharomyces cerevisiae/química , Relação Estrutura-AtividadeRESUMO
Lanthanides are a group of non-essential elements with important imaging and therapeutic applications. Although trivalent lanthanide ions (Ln³âº) are used as potent blockers of Ca²âº channels, the systematic studies correlating Ln³âº accumulation and toxicity to Ca²âº channel blocking activity are scarce. In this study, we made use of the eukaryotic model Saccharomyces cerevisiae to investigate the correlation between Ln³âº accumulation, their toxicity and their capacity to block the exogenous stress-induced Ca²âº influx into the cytosol. It was found that the Ln³âº blocked the Ca²âº entry into the yeast cells only when present at concentration high enough to allow rapid binding to cell surface. At lower concentrations, Ln³âº were taken up by the cell, but Ca²âº blockage was no longer achieved. At 1 mM concentration, all ions from the Ln³âº series could block Ca²âº entry into cytosol with the exception of La³âº, and to a lesser extent, Pr³âº and Nd³âº. The plasma membrane Ca²âº-channel Cch1/Mid1 contributed to La³âº and Gd³âº entry into the cells, with a significant preference for La³âº. The results open the possibility to obtain cells loaded with controlled amounts and ratios of Ln³âº.
Assuntos
Cálcio/química , Elementos da Série dos Lantanídeos/química , Saccharomyces cerevisiae/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Íons/farmacologia , Elementos da Série dos Lantanídeos/toxicidade , Modelos Biológicos , Saccharomyces cerevisiae/químicaRESUMO
Reaction between ortho-phthalaldehyde and various aroylhydrazines unexpectedly yields N-(1-(2-aryl-hydrazono)isoindolin-2-yl)benzamides as major products along with the predictable 1,2-bis-aroylhydrazones. NMR investigation of the major reaction products indicate the presence of a mixture of geometrical isomers, in various ratios. Single crystal X-ray diffraction confirms the proposed structure and indicates a Z configuration of the CâN double bond substitutents. Optimization of the condensation reaction conditions enabled quantitative isolation of the cyclic isomer. Oxidation of the isomers with bis(trifluoroacetoxy)iodobenzene (PIFA) leads to rapid formation of new highly fluorescent 1,2-bis(5-aryl-1,3,4-oxadiazol-2-yl)benzenes.
Assuntos
Benzamidas/química , Benzamidas/síntese química , Hidrazinas/química , Iodobenzenos/química , Oxidiazóis/química , Oxidiazóis/síntese química , o-Ftalaldeído/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Superoxide dismutases (SODs) stand in the prime line of enzymatic antioxidant defense in nearly all eukaryotic cells exposed to oxygen, catalyzing the breakdown of the superoxide anionic radical to O(2) and H(2)O(2). Overproduction of superoxide correlates with numerous pathophysiological conditions, and although the native enzyme can be used as a therapeutic agent in superoxide-associated conditions, synthetic low molecular weight mimetics are preferred in terms of cost, administration mode, and bioavailability. In this study we make use of the model eukaryote Saccharomyces cerevisiae to investigate the SOD-mimetic action of a mononuclear mixed-ligand copper(II) complex, [CuCl(acac)(tmed)] (where acac is acetylacetonate anion and tmed is N,N,N',N'-tetramethylethylenediamine). Taking advantage of an easily reproducible phenotype of yeast cells which lack Cu-Zn SOD (Sod1p), we found that the compound could act either as a superoxide scavenger in the absence of native Sod1p or as a Sod1p modulator which behaved differently under various genetic backgrounds.
Assuntos
Complexos de Coordenação/farmacologia , Chaperonas Moleculares/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ligantes , Chaperonas Moleculares/metabolismo , Estrutura Molecular , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
Green tea extracts (GTEs) as well as their main component, the polyphenol epigallocatechin gallate (EGCG), are known for their versatile antioxidant, antimicrobial, antitumoral or anti-inflammatory effects. In spite of the huge beneficial action, there is increasing evidence that under certain conditions green tea and its components can be detrimental to living organisms. Using Saccharomyces cerevisiae strains with various defects in the response to oxidative stress, we found that GTEs or EGCG act in synergy with visible light, exhibiting either deleterious or protective effects depending on the solvent employed. Similar synergistic effects could be observed under singlet oxygen-generating conditions, such as light exposure in the presence of photosensitizers or UV-A irradiation, therefore solvent variance may represent a powerful tool to modulate the preparation of green tea extracts, depending on the intended target.
Assuntos
Catequina/análogos & derivados , Luz , Saccharomyces cerevisiae/efeitos dos fármacos , Oxigênio Singlete/metabolismo , Raios Ultravioleta/efeitos adversos , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Catequina/química , Catequina/farmacologia , Luz/efeitos adversos , Estresse Oxidativo , Fármacos Fotossensibilizantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Solventes , CháRESUMO
Three enantiomeric pairs consisting of copper(II) complexes with tridentate Schiff bases have been synthesized for employing in biological assessments: 1∞[Cu2(R/S-salmet)2(H2O)] (1-R/S·H2O), 1∞[Cu(R/S-3-HOMe-5-Me-salmet)] (2-R/S), and 1∞[Cu(R/S-3-MeO-salmet)] (3-R/S) (where R/S-salmetH2, R/S-3-HOMe-5-Me-salmetH2, and R/S-3-MeO-salmetH2 result from the condensation of R/S-methionine with salicylaldehyde, 2-hydroxy-3-(hydroxymethyl)-5-methylbenzaldehyde, and 3-methoxy-salicylaldehyde, respectively, in a 1 : 1 molar ratio). The crystal structures of 1-R·H2O and 2-R/S are reported. Moreover, the 1-R/S·H2O enantiomers have been subjected to a single-crystal-to-single-crystal (SC-SC) transformation by heating at 160 °C to afford their dehydrated forms, 1∞[Cu2(R/S-salmet)2] (1-R/S), whose structures have also been crystallographically determined. The coordination polyhedra of the metal centers, the binding modes of the ligands, and the 1-D double chain assemblies generated by the chiral mononuclear units are comparatively described. The diffuse reflectance UV-Vis and circular dichroism (CD) spectra of compounds 1-R/S·H2O, 1-R/S, and 2-R/S are analysed with respect to their structural peculiarities and compared to those of 3-R/S. The UV-Vis and CD spectra of solutions of 1-R/S, 2-R/S, and 3-R/S point to the collapse of the double chains via dissolution. Biological tests performed on the model eukaryote Saccharomyces cerevisiae indicated low toxicity for 1-R/S, 2-R/S, and 3-R, and moderate toxicity for 3-S. The S-type complexes were accumulated by cells in higher quantity compared to their R-type counterparts due to selective transport via the high-affinity S-methionine transporter, Mup1. A chemogenomic analysis of 3-S toxicity performed on a collection of yeast knockout mutants revealed that most of the deleted genes identified in the screen were involved in the cell response to oxidative stress, calcium-mediated response, or metal homeostasis. Altogether, it was concluded that 3-S accumulation may perturb the redox state of the cell, also interfering with the calcium-mediated response to oxidative stress or metal-related oxidative stress.
Assuntos
Cobre , Metionina , Cobre/farmacologia , Cobre/química , Ligantes , Cálcio , Estrutura Molecular , Bases de Schiff/químicaRESUMO
Copper is essential for life, but it can be deleterious in concentrations that surpass the physiological limits. Copper pollution is related to widespread human activities, such as viticulture and wine production. To unravel aspects of how organisms cope with copper insults, we used Saccharomyces cerevisiae as a model for adaptation to high but subtoxic concentrations of copper. We found that S. cerevisiae cells could tolerate high copper concentration by forming deposits on the cell wall and that the copper-containing deposits accumulated predominantly when cells were grown statically on media prepared with reducing sugars (glucose, galactose) as sole carbon source, but not on media containing nonreducing carbon sources, such as glycerol or lactate. Exposing cells to copper in liquid media under strong agitation prevented the formation of copper-containing deposits at the cell wall. Disruption of low-affinity copper intake through the plasma membrane increased the potential of the cell to form copper deposits on the cell surface. These results imply that biotechnology problems caused by high copper concentration can be tackled by selecting yeast strains and conditions to allow the removal of excess copper from various contaminated sites in the forms of solid deposits which do not penetrate the cell.
RESUMO
Transient potential receptor (TRP) channels are conserved cation channels found in most eukaryotes, known to sense a variety of chemical, thermal or mechanical stimuli. The Saccharomyces cerevisiae TRPY1 is a TRP channel with vacuolar localization involved in the cellular response to hyperosmotic shock and oxidative stress. In this study, we found that S. cerevisiae diploid cells with heterozygous deletion in TRPY1 gene are haploinsufficient when grown in synthetic media deficient in essential metal ions and that this growth defect is alleviated by non-toxic Mn2+ surplus. Using cells expressing the Ca2+-sensitive photoprotein aequorin we found that Mn2+ augmented the Ca2+ flux into the cytosol under oxidative stress, but not under hyperosmotic shock, a trait that was absent in the diploid cells with homozygous deletion of TRPY1 gene. TRPY1 activation under oxidative stress was diminished in cells devoid of Smf1 (the Mn2+-high-affinity plasma membrane transporter) but it was clearly augmented in cells lacking Pmr1 (the endoplasmic reticulum (ER)/Golgi located ATPase responsible for Mn2+ detoxification via excretory pathway). Taken together, these observations lead to the conclusion that increased levels of intracytosolic Mn2+ activate TRPY1 in the response to oxidative stress.
Assuntos
Cálcio/metabolismo , Haploinsuficiência , Peróxido de Hidrogênio/toxicidade , Manganês/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Canais de Cátion TRPC/genética , Vacúolos/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Diploide , Haploinsuficiência/efeitos dos fármacos , Heterozigoto , Mutação/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Vacúolos/efeitos dos fármacosRESUMO
Cerebrovascular accidents are currently the second major cause of death and the third leading cause of disability in the world, according to the World Health Organization (WHO), which has provided protocols for stroke prevention. Although there is a multitude of studies on the health benefits associated with anthocyanin (ACN) consumption, there is no a rigorous systematization of the data linking dietary ACN with stroke prevention. This review is intended to present data from epidemiological, in vitro, in vivo, and clinical studies dealing with the stroke related to ACN-rich diets or ACN supplements, along with possible mechanisms of action revealed by pharmacokinetic studies, including ACN passage through the blood-brain barrier (BBB).
Assuntos
Antocianinas/farmacocinética , Dieta Saudável , Suplementos Nutricionais , Acidente Vascular Cerebral/prevenção & controle , Animais , Antocianinas/administração & dosagem , Disponibilidade Biológica , Biotransformação , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Suplementos Nutricionais/efeitos adversos , Humanos , Absorção Intestinal , Valor Nutritivo , Fatores de Risco , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologiaRESUMO
Epigallocatechin-3-O-gallate (EGCG), the main green tea component, is intensively studied for its anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer effects. In the present study, a screen on a Saccharomyces cerevisiae gene deletion library was performed to identify conditions under which EGCG had deleterious rather than beneficial effects. Two genes were identified whose deletion resulted in sensitivity to EGCG: FET3 and FTR1, encoding the components of the Fet3/Ftr1 high-affinity iron uptake system, also involved in Cu(I)/Cu(II) balance on the surface of yeast cells. The presence of EGCG in the growth medium induced the production of Cu(I), with deleterious effects on fet3Δ and ftr1Δ cells. Additionally, when combined, physiological surpluses of Cu(II) and EGCG acted in synergy not only against fet3Δ and ftr1Δ, but also against wild type cells, by generating surplus Cu(I) in the growth medium. The results imply that caution should be taken when combining EGCG-rich beverages/nutraceuticals with copper-rich foods.
Assuntos
Catequina/análogos & derivados , Ceruloplasmina/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Chá/química , Catequina/química , Catequina/isolamento & purificação , Catequina/farmacologia , Ceruloplasmina/deficiência , Cobre/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Saccharomyces cerevisiae/genética , Chá/metabolismoRESUMO
Novel microarray platform for single nucleotide polymorphisms (SNPs) detection has been developed using silicon nanowires (SiNWs) as support and two different surface modification methods for attaining the necessary functional groups. Accordingly, we compared the detection specificity and stability over time of the probes printed on SiNWs modified with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GAD), or coated with a simpler procedure using epoxy-based SU-8 photoresist. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) were used for comparative characterization of the unmodified and coated SiNWs. The hybridization efficiency was assessed by comprehensive statistical analysis of the acquired data from confocal fluorescence scanning of the manufactured biochips. The high detection specificity between the hybridized probes containing different mismatch types was demonstrated on SU-8 coating by one way ANOVA test (adjusted p value *** < .0001). The stability over time of the probes tethered on SiNWs coated with SU-8 was evaluated after 1, 4, 8 and 21 days of probe incubation, revealing values for coefficient of variation (CV) between 2.4% and 5.6%. The signal-to-both-standard-deviations ratio measured for SU-8 coated SiNWs platform was similar to the commercial support, while the APTES-GAD coated SiNWs exhibited the highest values.
Assuntos
Técnicas Biossensoriais , Análise Mutacional de DNA , DNA/química , Compostos de Epóxi/química , Nanofios/química , Polímeros/química , Silício/química , Pareamento Incorreto de Bases/genética , DNA/genéticaRESUMO
The various applications of Ag(I) generated the necessity to obtain Ag(I)-accumulating organisms for the removal of surplus Ag(I) from contaminated sites or for the concentration of Ag(I) from Ag(I)-poor environments. In this study we obtained Ag(I)-accumulating cells by expressing plant metallothioneins (MTs) in the model Saccharomyces cerevisiae. The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) fused to myrGFP displaying an N-terminal myristoylation sequence for plasma membrane targeting were expressed in S. cerevisiae and checked for Ag(I)-related phenotype. The transgenic yeast cells were grown in copper-deficient media to ensure the expression of the plasma membrane high-affinity Cu(I) transporter Ctr1, and also to elude the copper-related inhibition of Ag(I) transport into the cell. All plant MTs expressed in S. cerevisiae conferred Ag(I) tolerance to the yeast cells. Among them, myrGFP-NcMT3 afforded Ag(I) accumulation under high concentration (10â»50 µM), while myrGFP-AtMT1a conferred increased accumulation capacity under low (1 µM) or even trace Ag(I) (0.02â»0.05 µM). The ability to tolerate high concentrations of Ag(I) coupled with accumulative characteristics and robust growth showed by some of the transgenic yeasts highlighted the potential of these strains for biotechnology applications.
RESUMO
In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP70/genética , Mutação , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/fisiologia , Regulação para CimaRESUMO
To respond to metal surpluses, cells have developed intricate ways of defense against the excessive metallic ions. To understand the ways in which cells sense the presence of toxic concentration in the environment, the role of Ca2+ in mediating the cell response to high Cu2+ was investigated in Saccharomyces cerevisiae cells. It was found that the cell exposure to high Cu2+ was accompanied by elevations in cytosolic Ca2+ with patterns that were influenced not only by Cu2+ concentration but also by the oxidative state of the cell. When Ca2+ channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca2+ pool under Cu2+ stress was the vacuolar Ca2+ channel, Yvc1, also activated by the Cch1-mediated Ca2+ influx. Using yeast mutants defective in the Cu2+ transport across the plasma membrane, it was found that the Cu2+-dependent Ca2+ elevation could correlate not only with the accumulated metal, but also with the overall oxidative status. Moreover, it was revealed that Cu2+ and H2O2 acted in synergy to induce Ca2+-mediated responses to external stress.
Assuntos
Sinalização do Cálcio , Cobre/metabolismo , Saccharomyces cerevisiae/metabolismo , Cálcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Cobre/toxicidade , Citosol/metabolismo , Peróxido de Hidrogênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
INTRODUCTION: Leukotriene C4 synthase (LTC4S) gene -444A/C polymorphism has been implicated in susceptibility to asthma, but a large number of studies have reported inconclusive results. The aim of this study was to investigate the association between the -444A/C polymorphism of LTC4S gene and asthma, asthma phenotypes (aspirin intolerant/tolerant asthma) and different characteristics of the patients. MATERIAL AND METHODS: We included 106 patients with asthma (60 with aspirin tolerant asthma - ATA, 46 with aspirin intolerant asthma - AIA) and 103 controls. All the subjects were genotyped for LTC4S-444 A/C by Real-Time PCR. We assessed the association of LTC4S promoter polymorphism with asthma and its phenotypes and with clinical and biological characteristics of asthmatic patients. RESULTS: We did not find a significant association between the studied polymorphism and asthma, but the minor allele tended to be more frequent in AIA patients. We found a significant association between the minor allele C and lower levels of serum total immunoglobulin E and eosinophils, suggesting a possible role of -444A/C LTC4S polymorphism as modulating factor of allergic inflammation in asthma. CONCLUSION: The results show that LTC4S -444A/C SNP is not associated with susceptibility to asthma in Romanian patients, but could influence asthma phenotype, namely aspirin intolerant asthma.