10 research outputs found
Inhibition of M2 channel activity in Oocyte assay.
<p>The inhibition efficiency of each compound was examined at the concentration of 100 μM. The values in the table represent how much the M2 channel activity is inhibited. WT, wild type M2 channel. V27A, M2 channel with V27 A mutation. The Oocyte assay was conducted as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054070#pone.0054070-Balannik2" target="_blank">[12]</a>.</p
Screening for the inhibitors of M2 proton channel.
<p>(<b>A</b>) Expression of AcGFP-M2 protein in the presence of a group of compounds to be screened. Experiment procedure is the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054070#pone-0054070-g001" target="_blank">Figure 1A</a>. Lane 1, 1 mM IPTG is added to the culture at 0 hr, Lane 2, compound 10, Lane 3, compound 15, Lane 4, compound 34, Lane 5, compound 35, Lane 6, compound 180, Lane 7, compound 189, Lane 8, compound 206, Lane 9, compound 282, Lane 10, compound 293, Lane 11, compound 314, Lane 12, compound 332, Lane 13, compound 343, Lane 14, compound 352, Lane 15, compound 360, Lane 16, compound 360, Lane 17, amantadine, Lane 18, control without any additional compounds. (<b>C</b>) Expression of AcGFP-M2 (V27A) fusion protein in the presence of a group of compounds to be screened as described in (A). Positions of AcGFP-M2 fusion protein are indicated by arrowheads. (<b>B</b>) and (<b>D</b>), Cell density was measured as OD<sub>600</sub> of each overnight culture that expressing AcGFP-M2 fusion protein, and plotted as histogram corresponding to the compounds added. (<b>E</b>) After adding 1 mM IPTG for induction of AcGFP-M2 fusion protein, 100 μl of cell culture was loaded to a 96-well plate, in which a certain compound is added to each well (n = 3). OD<sub>600</sub> was collected after overnight incubation. All the compounds' concentration was 50 μM.</p
The expression profile of M2 protein and AcGFP-M2 fusion protein.
<p>(<b>A</b>) Expression of AcGFP-M2 fusion protein in the presence or absence of amantadine. Experiment procedures are the same as described in a. Positions of M2 protein and AcGFP-M2 fusion protein are indicated by arrowheads. (B) Expression of M2 protein in the presence or absence of amantadine. Cells harboring pColdII(sp-4) <i>m2</i> were cultured as described previously until O.D.<sub>600</sub> reached 0.5–0.6 and incubated at 15°C for 45 to 60 mins. 1 mM IPTG was added to at 5-hr and the culture was divided into two, one of which contains 50 μM amantadine. Cells from 1.5 ml culture were collected at 0 hr, 1 hr, 3 hr, 5 hr, 7 hr, and 19 hr after induction and subjected to SDS-PAGE assay.</p
Expression of M2 protein in the SPP system.
<p>(<b>A</b>) E. coli cells harboring pColdII(sp-4) <i>m2</i> (from residue 2 to 49 of M2 protein) and pACYC<i>mazF</i> were grown at 37°C to OD<sub>600</sub> = 0.5∼0.6, followed by cold-shock at 15°C for about 60 min. 1 mM of IPTG was added at 0 hr (Lane 1) for induction of M2 protein and MazF. Expression of M2 protein in the SPP system was examined in the presence of amantadine at different concentrations. Lane 2, 0 μM; Lane 3, 50 μM; Lane 4, 100 μM; Lane 5, 200 μM. After overnight incubation for 19 hours, cells from each culture were collected and subjected to SDS-PAGE. (<b>B</b>) Expression of M2 protein in the presence of other compounds besides amantadine. The final concentration of each compound in the culture is 50 μM. The experiments were carried out as described in (A). Lane 1, 1 mM IPTG is added to the culture at 0 hr, Lane 2: C, control without any additional compounds. Lane 3, compound 10, Lane 4, compound 15, Lane 5, compound 34, Lane 6, compound 35, Lane 7, compound 282, Lane 8, compound 293, Lane 9, compound 314, Lane 10, A, amantadine. (<b>C</b>) Expression of AcGFP-M2 fusion protein in the SPP system was carried out as described in (B). Positions of M2 protein and AcGFP-M2 fusion protein are indicated by arrowheads. (<b>D</b>) Cell density was measured as OD<sub>600</sub> of each overnight culture that expressing AcGFP-M2 fusion protein, and plotted as histogram corresponding to the compounds added. (<b>E</b>) Growth curve of cultures to express M2 or AcGFP-M2 fusion protein. Cultures were started at 0 hr and the following experiment procedures are similar to that described in (A). OD <sub>600</sub> of each culture is measured at every time point. M2 protein was induced at 5 hr with (▪) or without (•) amantadine. AcGFP-M2 fusion protein was induced at 5 hr with (♦) or without amantadine (▴).</p
A) Schematic representation of the <i>S. lividans</i> Antitoxin-Toxin bicistronic operon.
<p>The overlap between the two genes is shown. The stop codon of the antitoxin and the start codon of the toxin are shown in bold. B) Clustal alignment of the predicted amino acid sequence of the putative <i>S. lividans</i> antitoxin (YefMsl) with <i>E. coli</i> YefM (YefMec) and the putative <i>S. lividans</i> toxin (YoeBsl) with <i>E. coli</i> YoeB (YoeBec). Identical amino acids are boxed in black and conservative amino acid substitutions are boxed in grey. Semi-conservative substitutions are shown with one dot. C) DNA sequence of the putative promoter of the <i>S. lividans</i> TA system. The putative -35 and -10 regions as well as the ribosome-binding site (SD) are underlined.</p
Effect of YoeBsl and YefM/YoeBsl complexes on the viability of the wild-type <i>S. lividans</i> (A, B, C), and on the <i>S. lividans </i>
<p>Δ<b>TA (</b>Δ<b><i>yefM/yoeBsl</i></b><b>) mutant (D, E, F).</b> A) R2YE agar plates showing the colonies obtained in transformations with the same DNA amount of empty multicopy vector (pN702Gem3), the plasmid carrying the <i>yoeBsl</i> gene (pN702Gem3-Tox) or the plasmid containing <i>yefM/yoeBsl</i> genes (pN702Gem3-TA). B) Viability of the colonies obtained in the transformation after streaking them onto R2YE media containing 1% xylose (inducing conditions of the <i>xysAp</i> promoter). C) R2YE agar plates showing the colonies resulted from the transformation of the <i>S. lividans</i> wild type strain with the same DNA amount of empty integrative vector (pKC796) or with this plasmid carrying the <i>yoeBsl</i> gene (pKC796-Tox) or the plasmid carrying the <i>yefM/yoeBsl</i> genes (pKC796-TA). D) As in C, but using the <i>S. lividans</i> ΔTA <i>(ΔyefM/yoeBsl)</i> mutant as host. E and F) Effect of coexpression of YefMsl and YoeBsl, from different compatible plasmids, in <i>S. lividans ΔyefM/yoeBsl</i> mutant. Protoplasts of <i>S. lividans ΔyefM/yoeBsl</i> mutant carrying the empty multicopy vector pGM160 (E) or the plasmid pGM160-yefMsl (F) were transformed with the same amount of integrative plasmid pKC796 or its derivatives pKC796-Tox or pKC796-TA and inoculated in R2YE plates. The presence of pGM160-yefMsl in this strain eliminates the lethality originated by pKC796-Tox.</p
Effect of YoeBsl on protein synthesis in a cell-free system and Primer extension analysis of YoeBsl cleavage sites in the <i>ompA</i> mRNA <i>in vivo</i>.
<p>A) MazG protein synthesis was carried out using <i>E. coli</i> T7 S30 extract system for circular DNA (Promega) with pET11a-<i>mazG</i>. Lane 1, without YoeB-His<sub>6</sub>sl; lanes 2 to 5, 0.1, 0.2, 0.5 and 1 µM YoeB-His<sub>6</sub>sl were added, respectively; lane 6, 1 µM YoeB-His<sub>6</sub>sl plus 1 µM YefM-His<sub>6</sub>sl; and lane 7, 1 µM YefM-His<sub>6</sub>sl was added. B) Total RNA was prepared from <i>E. coli</i> BL21 cells harboring pFUS2-Tox at indicated time points before and after the induction of <i>yoeBsl</i> expression. The major cleavage site is indicated by an arrowhead on the right side of the gel. The sequence of the major cleavage site in the <i>ompA</i> mRNA is also shown on the right side of the gel. The sequence ladder for <i>ompA</i> was obtained using pCR®2.1-TOPO-<i>ompA</i> as template. The full-length RNA bands (FL) are indicated with an arrow.</p
Effect of the overproduction of YoeBsl and YefM/YoeBsl complexes in <i>E. coli</i> SC36 (MG1655 Δ<i>yefM</i>-<i>yoeB</i>).
<p>A) Cells transformed with control plasmid (pFUS2), with the plasmid carrying <i>yoeBsl</i> (pFUS2-Tox), or with the plasmid carrying the <i>yefM/yoeBsl</i> (pFUS2-TA) were grown in liquid LB medium to mid-logarithmic phase. At this time (time zero), 0.2% glucose (diamond) was added to one half of each culture and 0.2% arabinose (square) to the other half. Cell growth was monitored measuring the OD<sub>600</sub> of the cultures at different times. The means and standard deviation of three different experiments is presented. B) 5 µl of serial dilutions of the different cultures, taken one hour after protein induction, were inoculated in LB plates and incubated overnight at 37°C. The upper plates were inoculated with cells from media with glucose (repression), and the lower plates were inoculated with cells from media with arabinose (induction).</p