5 research outputs found
Table1.DOC
<p>The MOB<sub>V1</sub> family of relaxases is broadly distributed in plasmids and other mobile genetic elements isolated from staphylococci, enterococci, and streptococci. The prototype of this family is protein MobM encoded by the streptococcal promiscuous plasmid pMV158. MobM cleaves the phosphodiester bond of a specific dinucleotide within the origin of transfer (oriT) to initiate conjugative transfer. Differently from other relaxases, MobM and probably other members of the family, cleaves its target single-stranded DNA through a histidine residue rather than the commonly used tyrosine. The oriT of the MOB<sub>V1</sub> family differs from other well-known conjugative systems since it has sequences with three inverted repeats, which were predicted to generate three mutually-exclusive hairpins on supercoiled DNA. In this work, such hypothesis was evaluated through footprinting experiments on supercoiled plasmid DNA. We have found a change in hairpin extrusion mediated by protein MobM. This conformational change involves a shift from the main hairpin generated on “naked” DNA to a different hairpin in which the nick site is positioned in a single-stranded configuration. Our results indicate that the oriT<sub>pMV158</sub> acts as a molecular switch in which, depending on the inverted repeat recognized by MobM, pMV158 mobilization could be turned “on” or “off.”</p
Effect of etomoxir, etoposide and cisplatin on cell proliferation, cycle phase distribution and apoptosis generation in different cell models.
<p>Cells were incubated for 24 h with the indicated concentrations of etomoxir (Eto), ATO, etoposide (ETP) or cisplatin (CDDP), alone or in combination. When nothing is indicated, etomoxir was used at 100 µM. (A) Apoptosis generation, measured by flow cytometry, in NB4 cells. (B) The same, in mitogen-stimulated human PBLs. (C, D) Changes in proliferation activity, measured by cell counting (C), and apoptosis generation, measured by flow cytometry (D), in HL60 cells. Changes in proliferation are expressed in relation to untreated (Cont) cultures. (E) Flow cytometry histograms showing changes in cycle distribution in HL60 cell cultures treated with etoposide and cisplatin. The bar charts in (A–D) represent the mean ± S.D. of at least three determinations. Symbols and n.s. indicate significant and non-significant differences, respectively, between the indicated pairs of values (C), or between the combined treatment and the sum of values in the corresponding individual treatments (A, B, D). For other conditions see legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115250#pone-0115250-g001" target="_blank">Fig. 1</a>.</p
Scheme summarizing the main results in this work.
<p>Etomoxir decreases respiration while increasing glycolytic activity. In addition, it causes oxidative stress (ROS over-production, GSH depletion) which, together with AMPK activation, may explain the potentiation of ATO-provoked apoptosis. Etomoxir may also cooperate in some cell models with glycolytic inhibitors (2-DG, lonidamine) to cause apoptosis. This response is further enhanced by co-incubation with ATO, due to the capacity of this agent to attenuate the 2-DG/etomoxir-provoked Akt and ERK activation.</p
Effect of etomoxir and ATO on cell viability, cycle phase distribution and apoptosis generation in HL60 cells.
<p>Cells were incubated for 24 h with the indicated concentrations of etomoxir (Eto) and ATO, alone and in combination. When nothing is indicated, ATO was used at 2 µM. For convenience, the drug concentrations are sometimes indicated as subheadings. When indicated, the cells were co-incubated with the pan-caspase inhibitor z-VAD-fmk (50 µM). (A) Changes in cell viability, as evidenced by the MTT assay. Absorption values are indicated in relation to untreated (Cont) cultures. (B) Frequency of cells at the different phases of the growth cycle, namely G<sub>1</sub>, S and G<sub>2</sub>/M, and with sub-G<sub>1</sub> DNA content (apoptotic). Examples of flow cytometry histograms are presented in (E). (C) Frequency of apoptotic cells, as determined by flow cytometry. (D) Caspase-3 cleavage/activation, determined by immunoblot. β-actin is included as a loading control. The bar charts in (A–C) represent the mean ± S.D. of at least three determinations. Symbols mean: (*) significant differences between the indicated pair values; (<sup>#</sup>) significant differences between the combined treatment and the sum of values in the corresponding individual treatments (e.g., co-incubation with 100 µM Eto and 2 µM ATO, in relation to the sum of 100 µM Eto alone plus 2 µM ATO alone) (n.s., non-significant). To better discern differences, in this case the sum of values in individual treatments is indicated by a horizontal white line within the bar corresponding to the combined treatment. Single symbol, <i>p</i><0.05; double symbol, <i>p</i><0.01; triple symbol, <i>p</i><0.001.</p
Energy metabolism and oxidative stress upon incubation with etomoxir, 2-DG and ATO in HL60 cells.
<p>(A, B) Relative fraction of ATP in relation to total nucleotide (ATP+ADP+AMP) content (A), and AMP/ATP ratio (B), in untreated cells (Cont) and cells incubated for 6 h with the indicated concentrations of 2-DG and etomoxir, alone and in combination, and in the presence or absence of ATO. For more complete information see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115250#pone.0115250.s005" target="_blank">S1 Table</a>. (C, D) Changes in intracellular ROS accumulation (C) and GSH content (D), in cells incubated for 4 h with etomoxir and 2-DG, alone or in combination, and in the presence or absence of ATO. 3-BrP (60 µM) was used as a positive control in D (see Ref. 23). The values represent the mean ± S.D. of at least five determinations. Symbols in A–C indicate significant differences in relation to the control or between the indicated pairs of treatments. Symbols in D indicate significant differences in relation to the control (n.s., non-significant). ATO was always used at 2 µM. For other conditions, see legends of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115250#pone-0115250-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115250#pone-0115250-g004" target="_blank">4</a>.</p