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

Transfer of pESBL-283 from ESBL242 (donor, amp^R) to <i>E</i>. <i>coli</i> MG1655 (acceptor, chlor^R).

<p>After donor and acceptor cells reached steady states in separate chemostats, cultures were mixed in a ratio of 1:1 in an empty reactor vessel and immediately supplied with fresh medium at the same dilution rate. Experimental conditions in each of the panels: (A) 0 μg/ml at D = 0.2 h^-1, (B) 512 μg/ml ampicillin at D = 0.2 h^-1, (C) 0 μg/ml at D = 0.4 h^-1 and (D) 512 μg/ml ampicillin at D = 0.4 h^-1.</p

Plasmids found in the acceptor, donor and selected transconjugants.

<p>^a Accession number CP006784</p><p>^b Accession number CP008736</p><p>Plasmids of ESBL242 were isolated, separated and identified by sequencing. The sequences can be accessed at NCBI SRA database: SRX878242. Transconjugants that evolved in different experiments were selected and sequenced.</p><p>Plasmids found in the acceptor, donor and selected transconjugants.</p

Number of transconjugants during continuous cultivation of ESBL242 (donor) and E. coli MG1655 (acceptor) cells at t = 6h or 24h.

<p><i>E</i>. <i>coli</i> MG1655 and ESBL242 cells were cultivated separately and continuously at a dilution rate of D = 0.2 or 0.4 h^-1 and subsequently mixed with a ratio of 1:1 (t = 0h).</p

Minimum inhibitory concentration (MIC), maximum growth rate (μ_max) and β-lactamase activity of acceptor, donor and transconjugant cells.

<p>^a Average maximum growth rate of three transconjugants randomly chosen from three different transfer experiments. All individual transconjugants were grown in two replicates.</p><p>^b Specific activity is reported in nanomoles of nitrocefin hydrolyzed per minute per milligram of protein. The results are presented as the means and standard deviations of two independent measurements.</p><p>^c β-lactamase activity was averaged from transconjugants obtained in two individual experiments</p><p>Minimum inhibitory concentration (MIC), maximum growth rate (μ_max) and β-lactamase activity of acceptor, donor and transconjugant cells.</p

Number of evolved transconjugants/ml after co-culturing ESBL242 (donor, amp^R) and <i>E</i>. <i>coli</i> MG1655 (acceptor, chlor^R) for 24 hours in batch culture.

<p>Cells were mixed in a ratio of 1:1 with varying antibiotic concentrations.</p

Genetic interference following ingestion of anti-GFP dsRNA-expressing <i>B</i>. <i>subtilis</i> by <i>C</i>. <i>elegans</i>.

<p>(A) Physical map of the pBSR vector. The DNA sequence corresponding to dsRNA of interest was cloned between flanking copies of the P<i>spac</i> promoter to replace the spacer. <i>B</i>. <i>subtilis</i> strain BG322 was used as a host. GFP-expressing <i>C</i>. <i>elegans</i> strains TJ356 (B) and SD1084 (C) were fed on BG322 strains transformed with original pBSR vector and on bacteria expressing dsRNA corresponding to the <i>gfp</i> coding region. Under these conditions, 95% of the animals showed dramatic decrease in GFP expression after 24 hours of feeding. DAF-16::GFP and <i>SUR-5</i>::GFP expression is significantly decreased in the GFP (RNAi) treated animals. RNAi was induced starting at L4 larvae stage by feeding worms <i>B</i>. <i>subtilis</i> bacteria expressing dsRNA against GFP. GFP expression was measured at day 2 of adulthood. The y-axis denotes GFP expression (arbitrary units). Average expression and Standard Error from 20 animals are shown. *-p-value < 0.001 (t-test p-values). Scale bar = 100 μm.</p

Effects of various <i>E</i>. <i>coli</i> and <i>B</i>. <i>subtilis</i> strains on longevity.

<p>(A) Worms fed on wild type <i>B</i>. <i>subtilis</i> strain (PY79) live 65% longer compared to wild type, standard laboratory food <i>E</i>. <i>coli</i> strain (OP50). (B) Worms fed on RNase III-null <i>B</i>. <i>subtilis</i> (BG322) strain live 55% longer compared to DE3, <i>E</i>. <i>coli</i> strain used for expressing stable dsRNA for RNA interference. Median survival on: OP50 = 21 days, PY79 = 35 days DE3 = 18 days, BG322 = 28 days. Age refers to days of adulthood. Three biological replicates were observed for each experiment (n = 70–80 worms per experiment); error bars indicate Standard Error. In both graphs p < 0.0001.</p

DAF-16/FOXO activated in <i>daf-2</i> (RNAi) and <i>glp-1</i>(RNAi) mutants when grown on <i>E</i>. <i>coli</i> or <i>B</i>. <i>subtilis</i>.

<p>Green fluorescent protein images of adult TJ356 (Is<i>daf-16</i>::GFP) transgenic animals shown at 40X. (A) Images of an animal raised on <i>E</i>. <i>coli</i> expressing empty vector for RNA interference. (B) Images of an animal grown on <i>E</i>. <i>coli</i> and exposed to <i>E</i>. <i>coli</i> expressing <i>daf-2</i> dsRNA for 48 hours post development. Note strong nuclear localization in most tissues, including the intestine. (C) Image of an animal grown on <i>B</i>. <i>subtilis</i> expressing the empty vector for RNA interference. Images of animals fed <i>B</i>. <i>subtilis</i> expressing either (D) <i>daf-2</i> dsRNA, (E) <i>glp-1</i> dsRNA, or (F) <i>unc-62</i> dsRNA for 48 hours post development. Note weak activation of DAF-16/FOXO in the head area upon treatment with daf-2 and glp-1 dsRNA. Scale bar = 100μm</p

Number of evolved transconjugants/ml after co-culturing ESBL242 (donor, amp^R) and <i>E</i>. <i>coli</i> MG1655 (acceptor, chlor^R).

<p>Cells were mixed in a ratio of 1:1 at different total cell densities and incubated for 1 hour in batch culture.</p