Antibiotic chloramphenicol (CLM) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino-acid analogues of CLM. The L-histidyl analogue binds to the ribosome with the affinity exceeding that of CLM by 10 fold. Several of the newly synthesized analogues were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CLM. However, the inhibitory properties of the semi-synthetic CLM-analogues did not correlate with their affinity and in general, the amino-acid analogues of CLM were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogues showed that CLM derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.
We have hypothesized that the adaptive response to low doses of ionizing radiation (IR) is mediated by oxidized cell-free DNA (cfDNA) fragments. Here, we summarize our experimental evidence for this model. Studies involving measurements of ROS, expression of the NOX (superoxide radical production), induction of apoptosis and DNA double-strand breaks, antiapoptotic gene expression and cell cycle inhibition confirm this hypothesis. We have demonstrated that treatment of mesenchymal stem cells (MSCs) with low doses of IR (10 cGy) leads to cell death of part of cell population and release of oxidized cfDNA. cfDNA has the ability to penetrate into the cytoplasm of other cells. Oxidized cfDNA, like low doses of IR, induces oxidative stress, ROS production, ROS-induced oxidative modifications of nuclear DNA, DNA breaks, arrest of the cell cycle, activation of DNA reparation and antioxidant response, and inhibition of apoptosis. The MSCs pretreated with low dose of irradiation or oxidized cfDNA were equally effective in induction of adaptive response to challenge further dose of radiation. Our studies suggest that oxidized cfDNA is a signaling molecule in the stress signaling that mediates radiation-induced bystander effects and that it is an important component of the development of radioadaptive responses to low doses of IR.
The influence of a water-soluble [60] fullerene derivative containing five residues of 3-phenylpropionic acid and a chlorine addend appended to the carbon cage (F-828) on serum-starving human embryo lung diploid fibroblasts (HELFs) was studied. Serum deprivation evokes oxidative stress in HELFs. Cultivation of serum-starving HELFs in the presence of 0.1–1 µM F-828 significantly decreases the level of free radicals, inhibits autophagy, and represses expression of NOX4 and NRF2 proteins. The activity of NF-κB substantially grows up in contrast to the suppressed NRF2 activity. In the presence of 0.2–0.25 µM F-828, the DSB rate and apoptosis level dramatically decrease. The maximum increase of proliferative activity of the HELFs and maximum activity of NF-κB are observed at these concentration values. Conclusion. Under the conditions of oxidative stress evoked by serum deprivation the water-soluble fullerene derivative F-828 used in concentrations of 0.1 to 1 µM strongly stimulates the NF-κB activity and represses the NRF2 activity in HELFs.
Cell-free DNA (cfDNA) is a circulating DNA of nuclear and mitochondrial origin mainly derived from dying cells. Recent studies have shown that cfDNA is a stress signaling DAMP (damage-associated molecular pattern) molecule. We report here that the expression profiles of cfDNA-induced factors NRF2 and NF-κB are distinct depending on the target cell's type and the GC-content and oxidation rate of the cfDNA. Stem cells (MSC) have shown higher expression of NRF2 without inflammation in response to cfDNA. In contrast, inflammatory response launched by NF-κB was dominant in differentiated cells HUVEC, MCF7, and fibroblasts, with a possibility of transition to massive apoptosis. In each cell type examined, the response for oxidized cfDNA was more acute with higher peak intensity and faster resolution than that for nonoxidized cfDNA. GC-rich nonoxidized cfDNA evoked a weaker and prolonged response with proinflammatory component (NF-κB) as predominant. The exploration of apoptosis rates after adding cfDNA showed that cfDNA with moderately increased GC-content and lightly oxidized DNA promoted cell survival in a hormetic manner. Novel potential therapeutic approaches are proposed, which depend on the current cfDNA content: either preconditioning with low doses of cfDNA before a planned adverse impact or eliminating (binding, etc.) cfDNA when its content has already become high.
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