The aim of the present investigation was to evaluate the protective effect of a 70% methanolic leaf extract of Cyclea peltata Lam on cisplatin-induced renal toxicity. The concentration of creatinine, urea, sodium, and potassium in serum and levels of malonyldyaldehyde (MDA), glutathione (GSH), as well as gluathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) activities were determined in kidney tissue. The marked cisplatin-induced renal damage, characterized by a significant increase in creatinine and urea levels, decreased in extract-treated group, whereas sodium and potassium levels did not change significantly. C. peltata Lam extract significantly changed the increased MDA level and decreased GSH levels found in rats treated with cisplatin alone. The reduced activities of GSH-Px, SOD, and CAT in groups treated with cisplatin alone were significantly increased by the extract. The protective effect was greater in the post-treated than in the pre-treated group of animals. The results indicate that the post-treatment of C. peltata Lam extract might effectively ameliorate the oxidative stress parameters observed in cisplatin induced renal toxicity and could be used as a natural antioxidant against cisplatin-induced oxidative stress.
The formation of a stable G-quadruplex (GQ) can inhibit the increased telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most biochemical processes involving GQs might require local conformational changes at the guanine tetrad (G4) level. These local conformational changes of individual G4 layers during protein and drug interactions have not yet been explored in detail. In this study, we monitored the local conformations of individual G4 layers in GQs using 6methylisoxanthopterine (6MI) chromophores, which are circular dichroism (CD)-active fluorescent base analogues of guanine, as local conformational probes. A synthetic, tetramolecular, parallel GQ with site-specifically positioned 6MI monomers or dimers was used as the experimental construct. Analytical ultracentrifugation studies and gel electrophoretic studies showed that properly positioned 6MI monomers and dimers could form stable GQs with CDactive fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then tracked by monitoring the absorbance, fluorescence intensity, thermal melting, fluorescence quenching, and CD changes of the incorporated probes. Overall, these studies showed that site-specifically incorporated fluorescent base analogues could be used as probes to monitor the local conformational changes of individual G4 layers of a GQ structure. This method can be applied to explore the details of small molecule−GQ interaction at the level of the individual G4 layers, which may prove to be useful in designing drugs to treat GQ-related genetic disorders, cancer, and aging.
The formation of a stable G-quadruplex (GQ), a noncanonical DNA secondary structure, can inhibit the elevated telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most of the biochemical processes involving GQs might require local conformational changes at the guanine tetrad (G4) level. These local conformational changes of individual G4 layers during protein and drug interactions have not yet been explored in detail. This is because the spectroscopic signals of individual G4 layers are concealed in the total signal of GQ. Here we report a method to study the local conformations of individual G4 layers in GQs that uses 6-methylisoxanthopterine (6MI), a Circular Dichroism (CD)-active fluorescent base analogue of guanine. A synthetic, tetra molecular, parallel GQ with site-specifically positioned 6MI monomers or dimers was used as the substrate. Analytical ultracentrifugation studies and gel electrophoretic studies showed that properly positioned 6MI monomers and dimers could form stable GQs with CD-active fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then tracked by following the incorporated probes absorbance, fluorescence intensity, and circular dichroism changes. Further, thermal melting and acrylamide fluorescence quenching experiments were performed to confirm the stability and solvent accessibility of individual G4 layers. Overall, the study showed that site-specifically incorporated CD active fluorescent base analogues could be used as a probe to monitor the local conformational changes of individual G4 layers of a GQ structure. This method can be applied to explore the details of small molecule-GQ interaction at the G4 layer, which will be useful in designing drugs for GQ-related genetic disorders, cancer, and aging.
How fast can a cell locate a specific chromosomal DNA sequence specified by a single stranded oligonucleotide? To address this question we study the CRISPR-associated protein Cas9 which can be programmed by guide RNAs to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive dCas9 in living Escherichia coli by combining single molecule fluorescence microscopy and bulk restriction protection assays. We find that it takes a single dCas9~100 h to find and bind a specific target, in stark contrast to transcription factors such as LacI, which takes 5 minutes to locate its target. Thus, the price dCas9 pays for flexibility in targeting is time. We further identify a likely role for short-range (20-40) 1D sliding along DNA in dCas9 target search. The physical limitations for Cas9 likely generalize to all systems that are programmed by single stranded oligonucleotides to locate sequences in dsDNA, such as the homologous repair machinery.
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