RESUMEN
Pulsed electric field (PEF) treatment can be used to increase intracellular small molecule concentrations in bacteria, which can lead to enhanced robustness of these cells during further processing. In this study we investigated the effects of the PEF treatment temperature and the presence of 8% (v/v) ethanol in the PEF medium on cell survival, membrane fluidity and intracellular trehalose concentrations of Lactobacillus plantarum WCFS1. A moderate PEF treatment temperature of 21 °C resulted in a high cell survival combined with higher intracellular trehalose concentrations compared to a treatment at 10 and 35 °C. Interestingly, highest intracellular trehalose concentrations were observed upon supplementing the PEF medium with 8% ethanol, which resulted in more than a doubling in intracellular trehalose concentrations, while culture survival was retained. Overall, this study shows that treatment temperature and PEF medium optimization are important directions for improving molecule uptake upon PEF processing.
Asunto(s)
Electricidad , Etanol/farmacología , Lactobacillus plantarum/metabolismo , Trehalosa/metabolismo , Medios de Cultivo , TemperaturaRESUMEN
Pulsed electric field (PEF) treatment, or electroporation, can be used to load molecules into cells. The permeabilizing effect of the PEF treatment on the cellular membrane can be either reversible or irreversible depending on the severity of the PEF treatment conditions. The influence of PEF on the reversibility of membrane permeabilization in Lactobacillus plantarum WCFS1 by two different fluorescent staining methods was investigated in this study. Whereas staining with propidium iodide (PI) before and after PEF treatment indicated small reversible permeabilized fractions of maximum 14%, the use of a double staining method with PI and SYTOX Green suggested larger reversible permeabilized fractions up to 40% of the population. This difference shows that the choice for a fluorescent staining method affects the conclusions drawn regarding reversibility of membrane permeabilization. Additionally, the effect of PEF treatment conditions on membrane integrity was compared, indicating a relation between critical electric field strength, cell size and membrane permeabilization. Overall this study showed the possibilities and limitations of fluorescent membrane integrity staining methods for PEF studies.
Asunto(s)
Fenómenos Biofísicos , Permeabilidad de la Membrana Celular , Electroporación , Lactobacillus plantarum/fisiología , Biomarcadores , Membrana Celular/metabolismo , Supervivencia Celular , Citometría de FlujoRESUMEN
To study the role of Rad50 in the DNA damage response, we cloned and deleted the Schizosaccharomyces pombe RAD50 homologue. The deletion is sensitive to a range of DNA-damaging agents and shows dynamic epistatic interactions with other recombination-repair genes. We show that Rad50 is necessary for recombinational repair of the DNA lesion at the mating-type locus and that rad50Delta shows slow DNA replication. We also find that Rad50 is not required for slowing down S phase in response to hydroxy urea or methyl methanesulfonate (MMS) treatment. Interestingly, in rad50Delta cells, the recombination frequency between two homologous chromosomes is increased at the expense of sister chromatid recombination. We propose that Rad50, an SMC-like protein, promotes the use of the sister chromatid as the template for homologous recombinational repair. In support of this, we found that Rad50 functions in the same pathway for the repair of MMS-induced damage as Rad21, the homologue of the Saccharomyces cerevisiae Scc1 cohesin protein. We speculate that Rad50 interacts with the cohesin complex during S phase to assist repair and possibly re-initiation of replication after replication fork collapse.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Ciclo Celular , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Intercambio de Cromátides Hermanas , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Separación Celular , Proteínas Cromosómicas no Histona , Deleción Cromosómica , Clonación Molecular , Cruzamientos Genéticos , Daño del ADN , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Epistasis Genética , Citometría de Flujo , Proteínas Fúngicas/fisiología , Rayos gamma , Eliminación de Gen , Genotipo , Humanos , Hidroxiurea/farmacología , Metilmetanosulfonato/farmacología , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Recombinación Genética , Fase S , Schizosaccharomyces/metabolismo , Homología de Secuencia de Aminoácido , Telómero/metabolismo , Factores de TiempoRESUMEN
When, after prolonged viewing of a moving stimulus, a stationary (test) pattern is presented to an observer, this results in an illusory movement in the direction opposite to the adapting motion. Typically, this motion aftereffect (MAE) does not occur after adaptation to a second-order motion stimulus (i.e. an equiluminous stimulus where the movement is defined by a contrast or texture border, not by a luminance border). However, a MAE of second-order motion is perceived when, instead of a static test pattern, a dynamic test pattern is used. Here, we investigate whether a second-order motion stimulus does affect the MAE on a static test pattern (sMAE), when second-order motion is presented in combination with first-order motion during adaptation. The results show that this is indeed the case. Although the second-order motion stimulus is too weak to produce a convincing sMAE on its own, its influence on the sMAE is of equal strength to that of the first-order motion component, when they are adapted to simultaneously. The results suggest that the perceptual appearance of the sMAE originates from the site where first-order and second-order motion are integrated.