Relationships between renal cytoplasmic and nuclear aldosterone-receptors

Relationships between renal cytoplasmic and nuclear aldosteronereceptors.Three 3H-aldosterone receptor complexes have been recovered from rat kidneys: 1) cytosol (high speed supernatants), 2) Tris-soluble nuclear (obtained by an osmotic shock procedure), and 3) chromatin-bound (prepared by extracting post-shock nuclei with 0.4 M KCl).Glycerol density gradient analyses of cytosol labelled in vivo or in vitro with 3H-aldosterone yielded two specific peaks -4.5S and 8.5S.These peaks were sensitive to salt concentration; 0.4 M KCl shifted the 8.5S to 4.5S and the addition of Ca++ (6 mM) resulted in a further shift to 3.5S.The Tris-soluble nuclear species sedimented at 3S and the chromatin-bound species at 4S.The time-course of generation of the 3H-aldosterone-labelled cytosol and nuclear receptor species was studied in vivo and in vitro by tissue slice and reconstitution methods.The results obtained are consistent with a three-step mechanism: cytosol (8.5S or 4.5S)→ Tris-soluble nuclear (3S)→ chromatin-bound (4S).Alternatively, the 3S and 4S complexes may be attached to independent nuclear sites.The formation of the chromatin-bound species was temperature sensitive and failed to form at 0°C.Pre-treatment with DNase but not RNase impaired the generation of both the Tris-soluble nuclear and chromatin-bound species.These results imply a close association between nuclear aldosterone-receptor complexes and intact DNA

Concepción de un plan de negocios para la creación de una agencia de representación de nuevos músicos: modelo de aplicación basado en el género pop- rock

Al realizar un plan de negocio a partir de la idea de creación de una agencia de representación de artistas musicales, se analizaron los distintos factores que intervienen en el mismo, para de esta manera evaluar si la idea de negocio es factible o no y así mismo aplicar los conocimiento adquiridos en la maestría al emplearlos en un caso aplicativo que es el de IOSSA, un músico italiano con gran potencial. En el capítulo II, se realizó un análisis general del mercado en el cuál se determinó la oferta y la demanda de la música grabada y en streaming, para así poder determinar el tipo de estrategias a realizar. En el capítulo III, se realizó un estudio de la competencia y de acuerdo al análisis, realizó también un plan de marketing con las estrategias a realizar, seguido por una descripción de la estructura organizacional de la Agencia de Representación. Adicionalmente, se realizó un estudio de factibilidad económica y financiera para medir la factibilidad de la idea de negocio. Por último se realizó un caso aplicativo en el cuál se muestra cómo trabajará la Agencia de representación de artistas musicales. Líneas de investigación y desarrollo futuras: La información que se ha considerado necesaria presentar va desde la situación general del mercado, para luego realizar un estudio de la competencia y finalmente realizar un análisis de factibilidad del plan de negocios internacional. Así el proyecto pueda ser entendido, aceptado y también podría ser útil para solicitar créditos o buscar inversores o socios si el caso lo amerite.Facultad de Ciencias Económica

Absence of Kre increases xylose induced GFP-ComK expression.

<p>(A) GFP and phase contrast images of strains PG508 (<i>amyE</i>::<i>Pxyl-gfp-comK</i>, Δ<i>comK</i>, Δ<i>mecA</i>) and PG505 (<i>amyE</i>::<i>Pxyl-gfp-comK</i>, Δ<i>comK</i>, Δ<i>mecA</i>, <i>kre</i>:<i>Tn</i>) 60, 120 and 180 min after induction of GFP-ComK with 0.05% xylose. Fluorescence levels are indicated by a colour intensity scale using the same contrast settings. (B) Quantification of GFP-ComK levels after 60 min of xylose induction. (C) Induction of GFP-ComK causes a stronger reduction in cell growth when Kre is inactivated.</p

Kre affects <i>comK</i> mRNA stability.

<p>(A) Relative increase in <i>veg</i>, <i>pksA</i> and <i>comK</i> mRNA levels in a <i>kre</i> mutant determined by quantitative real-time PCR (qPCR). RNA was isolated from PG500 (<i>amyE</i>::<i>Pveg-lacZ-gfp</i>) and PG512 (<i>amyE</i>::<i>Pveg-lacZ-gfp</i>, Δ<i>kre</i>), and results shown are the average of 3 biological replicates. (B & C) Strains PG500 (<i>amyE</i>::<i>Pveg-lacZ-gfp</i>) and PG512 (<i>amyE</i>::<i>Pveg-lacZ-gfp</i>, Δ<i>kre</i>) were grown in LB at 37°C. At OD₆₀₀ ~0.2 T0-samples were collected immediately before rifampicin (150 μg/ml) was added. Subsequent samples were taken 2, 4, 6, 8 and 16 min after rifampicin addition. Relative abundance of <i>comK</i> (B) and <i>ftsZ</i> (C) transcripts were quantified over 3 independent experiments using qPCR. (D & E) Strain PG474 (<i>amyE</i>::<i>Physp</i>-<i>kre</i>) was grown in LB at 37°C in the presence or absence of 1 mM IPTG. At OD₆₀₀ ~0.25, T0-samples were collected immediately before rifampicin was added. Subsequent samples were taken 1, 2, 3, 4, 6, and 8 minutes after rifampicin addition. Relative abundance of <i>comK</i> (D) and <i>ftsZ</i> (E) transcripts were quantified over 3 independent experiments using qPCR.</p

Altered levels of Kre affect competence development in wild-type strains.

<p>(A) Fraction of <i>PcomG</i> expressing cells in the presence (wt) and absence of <i>kre</i> (<i>kre</i>:<i>Tn</i> and Δ<i>kre</i>). Strains PG389 (<i>amyE</i>::<i>PcomG-lacZ-gfp</i>), PG433 (<i>amyE</i>::<i>PcomG-lacZ-gfp</i>, <i>kre</i>:<i>Tn</i>) and PG488 (<i>amyE</i>::<i>PcomG-lacZ-gfp</i>, Δ<i>kre</i>) were grown overnight at 37°C on competence medium plates and GFP levels were measured using fluorescence light microscopy. Cells were counted as <i>PcomG</i> ‘ON’ when the GFP intensity exceeded 200 A.U. At least 300 cells were measured for each strain, and the results of 4 independent experiments are shown. (B) Luciferase expression from <i>PcomG</i> in wild-type (●) and Δ<i>kre</i> mutant (▲) strains. Strains PG710 (<i>PcomG-luc</i>) and PG724 (<i>PcomG-luc</i>, Δ<i>kre</i>) were grown in competence medium at 37°C in a plate reader in the presence of luciferin. Relative luminescence readings and O.D.₆₀₀ are plotted. (C) Western blot analysis of ComK levels in wild type (BSB1) and Δ<i>kre</i> mutant strain (PG479). Cultures were grown in competence medium at 37°C. Time is given in hours relative to the point of transition to the stationary growth phase (T₀). T_on indicate samples that were taken after prolonged stationary phase growth (overnight incubation). Arrow indicates ComK band and star indicates an aspecific protein band. (D) Transformation frequencies of wild type (wt) strain BSB1 and Δ<i>kre</i> mutant strain (PG479) grown in competence medium at 37°C. DNA was added 0, 1 and 2 hours (T0, T1, T2) relative to the point of transition to stationary phase. Transformation frequencies were determined by plating on selective and unselective plates and results of 3 independent experiments are shown. (E) Fraction of <i>PcomG</i> expressing cells when <i>kre</i> is overexpressed. Strains PG342 (<i>comG</i>:<i>comG-gfp</i>), PG490 (<i>comG</i>:<i>comG-gfp</i>, <i>amyE</i>::<i>Physp-kre</i>) and PG491 (<i>comG</i>:<i>comG-gfp</i>, <i>amyE</i>::<i>Physp-kre</i>, Δ<i>kre</i>) were grown overnight at 37°C on competence medium plates supplemented with 0, 0.1 or 1 mM IPTG, and the fractions of ‘<i>PcomG</i> ON’ cells were determined as in (A). Results of 2 independent experiments are shown. (F) Transformation frequencies when <i>kre</i> is overexpressed. Wild type (wt) strain BSB1 and strain PG474 (<i>amyE</i>::<i>Physp-kre</i>) were grown in competence medium in the presence or absence of 1 mM IPTG and transformed using a two-step starvation protocol used for routine transformations. Results of 3 independent experiments are shown.</p

Strains and plasmids used in this study.

<p>Unless stated otherwise, all strains were made in the BSB1 wild type background [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005047#pgen.1005047.ref038" target="_blank">38</a>]. Genes responsible for resistance to antibiotics are abbreviated as follows: <i>bla</i> (ampicillin), <i>cat</i> (chloramphenicol), <i>erm</i> (erythromycin), <i>kan</i> (kanamycin), <i>phleo</i> (phleomycin), <i>spc</i> (spectinomycin), <i>tet</i> (tetracycline).</p

General effect of Kre on gene expression.

<p>(A) Absence of <i>kre</i> leads to increased GFP expression from a <i>Pxyl</i> promoter. Strains PG537 (<i>amyE</i>::<i>Pxyl-gfp</i>, Δ<i>comK</i>, Δ<i>mecA</i>) and PG538 (<i>amyE</i>::<i>Pxyl-gfp</i>, Δ<i>comK</i>, Δ<i>mecA kre</i>:<i>Tn</i>) were grown to logarithmic phase in LB at 37°C in the presence of 0.1% xylose. Graph shows the results of one representative experiment (3 biological replicates). (B) Absence of <i>kre</i> leads to increased GFP expression from the <i>Physp</i> promoter in a wild type background. Strains PG820 (<i>amyE</i>::<i>Physp-gfp</i>) and PG821 (<i>Physp-gfp</i>, <i>kre</i>:<i>Tn</i>) were grown to logarithmic phase in LB at 37°C in the presence of 50 μM IPTG. Graph shows the results of one representative experiment (3 biological replicates). (C) Increase in β-galactosidase expression when <i>kre</i> is deleted. PG500 (<i>amyE</i>::<i>Pveg-lacZ-gfp</i>), PG512 (<i>amyE</i>::<i>Pveg-lacZ-gfp</i>, Δ<i>kre</i>), PG811 (<i>amyE</i>::<i>PpksA-lacZ-gfp</i>), PG815 (<i>amyE</i>::<i>PpksA-lacZ-gfp</i>, Δ<i>kre</i>) were grown in LB at 37°C and samples were collected at O.D.₆₀₀ ~0.2–0.3 for β-galactosidase activity measurements. Graphs show the ratio between <i>kre</i> mutant and wild-type strain averaged over 3 independent experiments.</p

Transposon insertion in <i>ykyB</i> increases the activation of an artificial ComK feedback loop.

<p>Strains PG401 (<i>amyE</i>::<i>PcomG-lacZ-gfp</i>, <i>PcomG-comK</i>, Δ<i>mecA</i>) and PG401-Tn4 (<i>amyE</i>::<i>PcomG-lacZ-gfp</i>, <i>PcomG-comK</i>, Δ<i>mecA</i>, <i>ykyB</i>:<i>Tn</i>) were grown on nutrient agar plates (A) or competence medium plates (B) supplemented with X-gal. Cells from plates were imaged by fluorescent light microscopy. Insets show related phase contrast images and arbitrary GFP colour intensity scales. Pictures and microscopy images were taken after overnight incubation at 37°C.</p

Negative feedback regulation of <i>kre</i>.

<p>(A) Schematic representation of the <i>kre</i> promoter region. Three potential AT-boxes and the putative -35 promoter region [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005047#pgen.1005047.ref038" target="_blank">38</a>] are highlighted in red and grey, respectively. (B) Overproduction of ComK in a <i>mecA</i> mutant causes repression of <i>kre</i> expression. Strains PG501 (<i>amyE</i>::<i>Pkre-lacZ-gfp</i>), PG763 (<i>amyE</i>::<i>Pkre-lacZ-gfp</i>, Δ<i>mecA</i>), PG764 (<i>amyE</i>::<i>Pkre-lacZ-gfp</i>, Δ<i>comK</i>) and PG765 (<i>amyE</i>::<i>Pkre-lacZ-gfp</i>, Δ<i>mecA</i>, Δ<i>comK</i>) were grown in competence medium at 37°C, and samples were collected at OD₆₀₀ ~0.1 for β-galactosidase measurements. (C) Schematic representation of the double negative feedback regulation exerted by Kre and ComK. (D) Reciprocal correlation between <i>Pkre</i> and <i>PcomG</i> expression. Strain PG688 (<i>kre</i>:<i>Pkre-gfp</i>, <i>amyE</i>::<i>PcomG-mcherry</i>) was grown in competence medium at 37°C, and phase contracts and fluorescent images were taken after overnight incubation. (E) Average GFP and mCherry levels in single cells from the same culture in D (n = 211).</p

Phylogenetic relation between <i>comK</i> and its key regulators.

<p>Phylogenetic display of <i>kre</i> and other ComK regulators in bacterial species. Data and presentation is based on information from the STRING interaction database [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005047#pgen.1005047.ref075" target="_blank">75</a>]. Colour intensity indicates measure of homology with the corresponding genes in <i>B</i>. <i>subtilis</i>.</p