Aislamiento y selección de cepas psicrotolerantes de bacterias lácticas enológicas de la región patagónica

La vinificación de varietales tintos patagónicos ocurre en abril y mayo, con bajas temperaturas ambientales que inhiben el progreso de la fermentación maloláctica (FML), por ser un factor de estrés para las bacterias lácticas (BAL) que conducen el proceso. Las bodegas deben calentar los tanques de fermentación, incrementando los costos de producción. El desarrollo de cultivos malolácticos nativos, tolerantes a bajas temperaturas, constituye una herramienta estratégica para resolver el problema y potenciar el carácter regional de los vinos. El objetivo del trabajo consistió en obtener cepas enológicas patagónicas de BAL, capaces de sobrevivir y consumir ácido L-málico en vinificaciones a bajas temperaturas (4 y 10 ºC). Los aislamientos psicrotolerantes procedieron de vino Pinot noir (bodega comercial de General Roca, Río Negro). Su identificación se realizó por secuenciación del gen 16S rRNA y la similitud genética se analizó por RAPD-PCR con el primer M13. Como primer criterio de selección se evaluó su supervivencia en vino estéril incubado a bajas temperaturas. Posteriormente, las cepas que exhibieron mejor tolerancia, UNQoE19 (O. oeni) y UNQLh1.1 (Lb. hilgardii), se inocularon en vino, a escala de laboratorio, y se evaluaron su supervivencia y sus capacidades de implantación y de consumo de ácido L-málico, con resultados promisorios

Cytotoxicity and intracellular calcium fluxes after long-term exposure to flame retardants (Pbdes) in primary cultures of cortical neurons

Trabajo presentado al 8th Forum of Neuroscience (FENS) celebrado en Barcelona del 14 al 18 de julio de 2012.Polybrominated diphenyl ethers (PBDEs) are hydrophobic and persistent additive flame retardants that seemingly transfer into environmental compartments where they bioaccumulate in the adipose tissue of animals. Of particular concern are the reported increasingly high levels of PBDEs in human blood, adipose tissue, and breast milk as detected in several countries. In spite of their widespread occurrence in the environment, only limited information is available on the neurotoxicology of PBDEs. Animal studies suggest that PBDEs may exert developmental neurotoxicity, via mechanisms that are still elusive. It has been shown that congener substitution plays a role in the distribution, metabolism, and excretion of PBDEs in mice. In brain, PBDE-153 seems to be accumulated in higher concentration than other congeners and thus, suggesting a differential risk to human health. Moreover, PBDEs have been reported to cause oxidative stress and apoptotic cell death in neurons in vitro. However, little is known about the effects of low chronic exposure to these pollutants. In the present work, primary cultures of cortical neurons were used to study the mechanisms involved in the neurotoxic effects of three PBDE congeners (PBDE-47, -153 and -183). Cell viability was determined by measuring the reduction of MTT after 7 days in vitro (DIV) of PBDEs exposure. Intracellular calcium concentration ([Ca+2]i) was determined with Fluo-3 AM after 8 DIV of exposure. Different concentrations of PBDEs were used to analyze the effect of these compounds on glutamate-, NMDA-, and kainate-induced increase of [Ca+2]i. Furthermore, the effect of long-term exposure to PBDEs on glutamate-, NMDA-, and kainate-induced excitotoxicity was also evaluated.Peer Reviewe

Toxicity evaluation of new agricultural fungicides in primary cultured cortical neurons

© 2015 Elsevier Inc. Fungicides are crucial for food protection as well as for the production of crops of suitable quality and quantity to provide a viable economic return. Like other pesticides, fungicides are widely sprayed on agricultural land, especially in wine-growing areas, from where they can move-off after application. Furthermore, residues of these agrochemicals can remain on crops after harvest and even after some food processing operations, being a major exposure pathway. Although a relatively low toxicity has been claimed for this kind of compounds, information about their neurotoxicity is still scarce.In the present study, nine fungicides recently approved for agricultural uses in the EU - ametoctradin, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone and pyraclostrobin - have been evaluated for their toxicity in primary cultured mouse cortical neurons. Exposure to 0.1-100μM for 7 days in vitro resulted in a dose-dependent toxicity in the MTT cell viability assay. Strobilurin fungicides kresoxim-methyl (KR) and pyraclostrobin (PY) were the most neurotoxic compounds (lethal concentration 50 were in the low micromolar and nanomolar levels, respectively) causing a rapid raise in intracellular calcium [Ca2+]i and strong depolarization of mitochondrial membrane potential. KR- and PY-induced cell death was reversed by the calcium channels blockers MK-801 and verapamil, suggesting that calcium entry through NMDA receptors and voltage-operated calcium channels are involved in KR- and PY-induced neurotoxicity. These results highlight the need for further evaluation of their neurotoxic effects in vivo.This research was supported by the projects PI10/00453 and PI13/01252 from the Instituto de Salud Carlos III cofinanced with European Social Funds “A Way to build Europe” and AGL2011-30378-C03-01 of the Spanish Ministries of Health, and Science and Innovation, respectively. J. R. would like to acknowledge the Ministry of Science and Innovation of Spain for his Juan de la Cierva contract. N.O. is recipient of a CSIC JAE-Doc contract from the “Junta para la Ampliación de Estudios” Program cofinanced with European Social FundsPeer Reviewe

Toxicity evaluation of new agricultural fungicides in primary cultured cortical neurons

Trabajo presentado al 8th Forum of Neuroscience (FENS) celebrado en Barcelona del 14 al 18 de julio de 2012.The research was supported by the projects PI 10/00453 and AGL2011-30378-C03-01 of the Spanish Ministries of Health, and Science and Innovation, respectively.Peer Reviewe

GABAergic pharmacological activity of propofol related compounds as possible enhancers of general anesthetics and interaction with membranes

El pdf del artículo es el manuscrito revisado de autor.Phenol compounds, such as propofol and thymol, have been shown to act on the GABAA receptor through interaction with specific sites of this receptor. In addition, considering the high lipophilicity of phenols, it is possible that their pharmacological activity may also be the result of the interaction of phenol molecules with the surrounding lipid molecules, modulating the supramolecular organization of the receptor environment. Thus, in the present study, we study the pharmacological activity of some propofol- and thymol-related phenols on the native GABAA receptor using primary cultures of cortical neurons and investigate the effects of these compounds on the micro viscosity of artificial membranes by means of fluorescence anisotropy. The phenol compounds analyzed in this article are carvacrol, chlorothymol, and eugenol. All compounds were able to enhance the binding of [3H]flunitrazepam with EC50 values in the micromolar range and to increase the GABA-evoked Cl- influx in a concentration-dependent manner, both effects being inhibited by the competitive GABAA antagonist bicuculline. These results strongly suggest that the phenols studied are positive allosteric modulators of this receptor. Chlorothymol showed a bell-type effect, reducing its positive effect at concentrations >100 μM. The concentrations necessary to induce positive allosteric modulation of GABAA receptor were not cytotoxic. Although all compounds were able to decrease the micro viscosity of artificial membranes, chlorothymol displayed a larger effect which could explain its effects on [3H]flunitrazepam binding and on cell viability at high concentrations. Finally, it is suggested that these compounds may exert depressant activity on the central nervous system and potentiate the effects of general anesthetics. © 2013 Springer Science+Business Media New York.This study was supported by Argentinian Grants FONCyT, CONICET, SECyT-UNC, Mincyt-Cba, IBRO, and by Spanish Grants from the Ministry of Science and Innovation (FIS 10/0453) and from the Generalitat de Catalunya (2009/SGR/214). D.A.G. and M.S.B. are members of CONICET (Argentina), G.N.R is a fellowship holder from the same institution, and L.D.M. is a fellowship holder from SECyT-UNC. N.O. is recipient of a CSIC contract in the JAE-Doc program cofinanced with European Social Funds.Peer Reviewe

Transcriptomic and proteomic analysis of [i]Oenococcus oeni[/i] PSU-1 response to ethanol shock

The correct development of malolactic fermentation depends on the capacity of [i]Oenococcus oeni[/i] tosurvive under harsh wine conditions. The presence of ethanol is one of the most stressful factors affecting O. oeni performance. In this study, the effect of ethanol addition (12% vol/vol) on [i]O. oeni[/i] PSU-1 has been evaluated using a transcriptomic and proteomic approach. Transcriptomic analysis revealed that the main functional categories of the genes affected by ethanol were metabolite transport and cell wall and membrane biogenesis. It was also observed that some genes were over-expressed in response to ethanol stress (for example, the heat shock protein Hsp20 and a dipeptidase). Proteomic analysis showed that several proteins are affected by the presence of ethanol. Functions related to protein synthesis and stability are the main target of ethanol damage. In some cases the decrease in protein concentration could be due to the relocation of cytosolic proteins in the membrane, as a protective mechanism. The omic approach used to study the response of [i]O. oeni[/i] to ethanol highlights the importance of the cell membrane in the global stress response and opens the door to future studies on this issue