ABCC Subfamily Vacuolar Transporters are Involved in Pb (Lead) Detoxification in Saccharomyces cerevisiae

The present work has as objective to contribute for the elucidation of the mechanism associated with Pb detoxification, using the yeast Saccharomyces cerevisiae as a model organism. The deletion of GTT1 or GTT2 genes, coding for functional glutathione transferases (GST) enzymes in S. cerevisiae, caused an increased susceptibility to high Pb concentrations (500-1000 μmol L(-1)). These results suggest that the formation of glutathione-Pb conjugate (GS-Pb), dependent of GSTs, is important in Pb detoxification. The involvement of ATP-binding cassette (ABC) vacuolar transporters, belonging to class C subfamily (ABCC) in vacuolar compartmentalization of Pb, was evaluated. For this purpose, mutant strains disrupted in YCF1, VMR1, YBT1 or BPT 1 genes were used. All mutants tested, without vacuolar ABCC transporters, presented an increased sensitivity to 500-1000 μmol L(-1) Pb comparative to wild-type strain. Taken together, the obtained results suggest that Pb detoxification, by vacuolar compartmentalization, can occur as a result of the concerted action of GSTs and vacuolar ABCC transporters. Pb is conjugated with glutathione, catalysed by glutathione transferases and followed to the transport of GS-Pb conjugate to the vacuole by ABCC transporters.

Estudo da Toxicidade do Chumbo na Levedura Saccharomyces cerevisiae

O chumbo é um importante poluente ambiental. A levedura Saccharomyces cerevisiae constitui um modelo útil para o estudo dos efeitos tóxicos do chumbo. O conhecimento dos mecanismos de defesa e resistência à presença de metais pesados poderá ser útil em tecnologias de proteção ambiental, nomeadamente no desenvolvimento de novas metodologias para a biorremediação de metais pesados. O presente trabalho teve como objetivo avaliar o impacto do Pb na capacidade proliferativa, na integridade membranar e na produção intracelular de espécies reativas de oxigénio (ROS), na estirpe laboratorial da levedura Saccharomyces cerevisiae BY4741 (estirpe selvagem, WT). Foi também estudado o papel das mitocôndrias, como fonte de ROS induzida por Pb, e o envolvimento da H+-ATPase vacuolar (V-ATPase) e de transportadores vacuolares pertencentes à superfamília ABC (de ATP-binding cassette) na defesa contra a toxicidade do Pb. O estudo cinético do impacto de duas concentrações de Pb na viabilidade das leveduras (avaliado através de um ensaio clonogénico), na integridade da membrana celular (determinada com iodeto de propídio) e na produção intracelular de ROS (o anião superóxido foi detetado com dihidroetídio e o peróxido de hidrogénio com 2’,7’- diclorodihidrofluoresceína), revelou uma perda progressiva da capacidade proliferativa (53 e 17% de células viáveis, após a exposição durante 3h a 250 ou 1000 µmol/l de chumbo, respetivamente), coincidente com a acumulação intracelular de anião superóxido e de peróxido de hidrogénio, na ausência de perda da integridade membranar. A importância das mitocôndrias na produção de ROS, induzida por chumbo, foi levada a cabo usando um mutante deficiente respiratório desprovido de ADN mitocondrial (ƿ0). Quando comparado com a respetiva estirpe parental, o mutante ƿ0 apresentou uma maior resistência ao Pb e uma menor produção de ROS induzida por Pb. A exposição das células da estirpe BY4741 a 250 e 1000 µmol/l de chumbo originou a formação de 49 e 58% de células deficientes respiratórias, respetivamente. A função da V-ATPase, na desintoxicação de chumbo, foi avaliada utilizando mutantes com uma estrutura vacuolar normal mas defetivos em subunidades da VATPase (vma1Δ, vma2Δ, vma3Δ e vph1Δ). Comparativamente às células da estirpe WT, todos os mutantes testados, sem V-ATPase funcional, apresentaram uma maior suscetibilidade ao Pb. O papel dos transportadores vacuolares pertencentes à superfamília ABC, na defesa contra a toxicidade induzida por chumbo, foi levada a cabo utilizando mutantes sem os transportadores Ycf1p ou Vmr1p. Os resultados preliminares mostraram que quando comparadas com as células da estirpe WT, as células das estirpes ycf1Δ ou vmr1Δ não apresentavam uma maior perda da viabilidade. A modificação da morfologia vacuolar, em células expostas a chumbo, foi visualizada utilizando a estirpe Vma2p-GFP. O tratamento das células com Pb originou a fusão dos vacúolos de tamanho médio num único vacúolo de grande dimensão. Em conclusão, os estudos desenvolvidos no presente trabalho, utilizando a estirpe laboratorial BY4741, mostraram que a perda da capacidade proliferativa das leveduras, induzida pelo chumbo, pode ser atribuída à acumulação intracelular do anião superóxido e de peróxido de hidrogénio. As mitocôndrias parecem ser uma das principais fontes de ROS induzido por Pb e, simultaneamente, um dos principais alvos da sua toxicidade. Em S. cerevisiae, o vacúolo desempenha um papel importante na desintoxicação do Pb. A modificação da morfologia vacuolar após exposição ao chumbo poderá ser a consequência da acumulação de Pb no vacúolo. Enquanto os transportadores da superfamília ABC parecem não estar envolvidos na sequestração vacuolar de Pb, é necessária a presença, num estado funcional, da V-ATPase para que ocorra a compartimentação do Pb. Muito provavelmente, a compartimentação do Pb no vacúolo previne a sua acumulação no citosol e o desencadear dos respetivos efeitos tóxicos.

Novel potentiometric sensors of molecular imprinted polymers for specific binding of chlormequat

Molecularly imprinted polymers (MIP) were used as potentiometric sensors for the selective recognition and determination of chlormequat (CMQ). They were produced after radical polymerization of 4-vinyl pyridine (4-VP) or methacrylic acid (MAA) monomers in the presence of a cross-linker. CMQwas used as template. Similar nonimprinted (NI) polymers (NIP) were produced by removing the template from reaction media. The effect of kind and amount of MIP or NIP sensors on the potentiometric behavior was investigated. Main analytical features were evaluated in steady and flow modes of operation. The sensor MIP/4-VP exhibited the best performance, presenting fast near-Nernstian response for CMQover the concentration range 6.2×10-6 – 1.0×10-2 mol L-1 with detection limits of 4.1×10-6 mol L-1. The sensor was independent from the pH of test solutions in the range 5 – 10. Potentiometric selectivity coefficients of the proposed sensors were evaluated over several inorganic and organic cations. Results pointed out a good selectivity to CMQ. The sensor was applied to the potentiometric determination of CMQin commercial phytopharmaceuticals and spiked water samples. Recoveries ranged 96 to 108.5%.

Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Ligand-Induced structural changes in TEM‑1 probed by molecular dynamics and relative binding free energy calculations

The TEM family of enzymes has had a crucial impact on the pharmaceutical industry due to their important role in antibiotic resistance. Even with the latest technologies in structural biology and genomics, no 3D structure of a TEM- 1/antibiotic complex is known previous to acylation. Therefore, the comprehension of their capability in acylate antibiotics is based on the protein macromolecular structure uncomplexed. In this work, molecular docking, molecular dynamic simulations, and relative free energy calculations were applied in order to get a comprehensive and thorough analysis of TEM-1/ampicillin and TEM-1/amoxicillin complexes. We described the complexes and analyzed the effect of ligand binding on the overall structure. We clearly demonstrate that the key residues involved in the stability of the ligand (hot-spots) vary with the nature of the ligand. Structural effects such as (i) the distances between interfacial residues (Ser70−Oγ and Lys73−Nζ, Lys73−Nζ and Ser130−Oγ, and Ser70−Oγ−Ser130−Oγ), (ii) side chain rotamer variation (Tyr105 and Glu240), and (iii) the presence of conserved waters can be also influenced by ligand binding. This study supports the hypothesis that TEM-1 suffers structural modifications upon ligand binding.

The transport of carboxylic acids and important role of the Jen1p transporter during the development of yeast colonies

On solid substrates, yeast colonies pass through distinct developmental phases characterized by the changes in pH of their surroundings from acidic to nearly alkaline and vice versa. At the beginning of the alkali phase colonies start to produce ammonia, which functions as a quorum-sensing molecule inducing the reprogramming of cell metabolism. Such reprogramming includes, among others, the activation of several plasma membrane transporters and is connected with colony differentiation. In the present study, we show that colony cells can use two transport mechanisms to import lactic acid: a ‘saturable’ component of the transport, which requires the presence of a functional Jen1p transporter, and a ‘non-saturable’ component (diffusion) that is independent of Jen1p. During colony development, the efficiency of both transport components changes similarly in central and outer colonial cells. Although the lactate uptake capacity of central cells gradually decreases during colony development, the lactate uptake capacity of outer cells peaks during the alkali phase and is also kept relatively high in the second acidic phase. This lactate uptake profile correlates with the localization of the Jen1p transporter to the plasma membrane of colony cells. Both lactic acid uptake mechanisms are diminished in sok2 colonies where JEN1 expression is decreased. The Sok2p transcription factor may therefore be involved in the regulation of non-saturable lactic acid uptake in yeast colonies.

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

Haemoglobin smart plastic antibody material tailored with charged binding sites on silica nanoparticles: its application as an ionophore in potentiometric transduction

This work uses surface imprinting to design a novel smart plastic antibodymaterial (SPAM) for Haemoglobin (Hb). Charged binding sites are described here for the first time to tailor plastic antibody nanostructures for a large size protein such as Hb. Its application to design small, portable and low cost potentiometric devices is presented. The SPAM material was obtained by linking Hb to silica nanoparticles and allowing its ionic interaction with charged vinyl monomers. A neutral polymeric matrix was created around these and the imprinted protein removed. Additional materials were designed in parallel acting as a control: a neutral imprinted material (NSPAM), obtained by removing the charged monomers from the procedure, and the Non-Imprinted (NI) versions of SPAM and NSPAM by removing the template. SEM analysis confirmed the surface modification of the silica nanoparticles. All materials were mixed with PVC/plasticizer and applied as selective membranes in potentiometric transduction. Electromotive force (emf) variations were detected only for selective membranes having a lipophilic anionic additive in the membrane. The presence of Hb inside these membranes was evident and confirmed by FTIR, optical microscopy and Raman spectroscopy. The best performance was found for SPAM-based selective membranes with an anionic lipophilic additive, at pH 5. The limits of detection were 43.8 mg mL 1 and linear responses were obtained down to 83.8 mg mL 1, with an average cationic slope of +40 mV per decade. Good selectivity was also observed against other coexisting biomolecules. The analytical application was conducted successfully, showing accurate and precise results.

SPR based Studies for Pentagalloyl Glucose Binding to α-Amylase

Astringency is an organoleptic property resulting mostly from the interaction of salivary proteins with dietary polyphenols. It is of great importance to consumers but being typically measured by sensorial panels it turns out subjective and expensive. The main goal of the present work is to develop a sensory system to estimate astringency relying on protein/polyphenol interactions. For this purpose, a model protein was immobilized on a sensory gold surface and its subsequent interaction with polyphenols was measured by Surface Plasma Resonance (SPR). α-amylase and pentagalloyl glucose (PGG) were selected as model protein and polyphenol, respectively. To ensure specific binding between these, various surface chemistries were tested. Carboxylic terminated thiol decreased the binding ability of PGG and allowed covalent attachment of α-amylase to the surface. The pH 5 was the optimal condition for α-amylase immobilization on the surface. Further studies focus on Localized SPR sensor and application to wine samples, providing objectivity when compared to a trained panel.

Smart Plastic Antibody Material for Hemoglobin Tailored by Silica Surface Imprinting and with Charged Binding Sites: Its use as Ionophore in Potentiometric Transduction

JORNADAS DE ELECTROQUÍMICA E INOVAÇÃO 2013