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The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.

Using equilibrium dialysis and sedimentation velocity analysis, we have characterized the binding of the antitumor drug daunomycin to chicken erythrocyte chromatin before and after depletion of linker histones and to its constitutive DNA under several ionic strengths (5, 25, and 75 mM NaCl). The equilibrium dialysis experiments reveal that the drug binds cooperatively to both the chromatin fractions and to the DNA counterpart within the range of ionic strength used in this study. A significant decrease in the binding affinity was observed at 75 mM NaCl. At any given salt concentration, daunomycin exhibits higher binding affinity for DNA than for linker histone-depleted chromatin or chromatin (in decreasing order). Binding of daunomycin to DNA does not significantly affect the sedimentation coefficient of the molecule. This is in contrast to binding to chromatin and to its linker histone-depleted counterpart. In these instances, preferential binding of the drug to the linker DNA regions induces an unfolding of the chromatin fiber that is followed by aggregation, presumably because of histone-DNA interfiber interactions.Daunomycin is an anthracycline antibiotic widely used as a potent chemotherapeutic agent in the treatment of various cancers (1, 2). Studies on its mechanism of action indicate that nuclear DNA is an important target for daunomycin. The structure of this pharmacologically active drug consists of two distinct domains ( Fig. 1): a planar aglycon chromophore that intercalates between adjacent base pairs of DNA and an amino sugar ring that lies in the minor groove of the DNA double helix (3, 4). Binding of daunomycin to DNA results in the inhibition of both DNA replication and RNA transcription (5-7).The binding of daunomycin to DNA has been studied in detail in the past (8 -11). However, in the cell, DNA does not exist as a naked structure but is associated with histones and other nuclear proteins in a complex that is known as chromatin (12-13). Thus, chromatin, not DNA, is the major target for daunomycin in vivo. How the presence of chromosomal proteins might modulate the binding of this drug to DNA and how in turn chromatin structure is affected by the binding of the drug are important questions that need to answered to understand the biological mechanism of action of daunomycin. Although several attempts in this direction have already been carried out using isolated nucleosome (14, 15) and crude chromatin fractions (16 -18), the mechanism of daunomycin binding to chromatin and the conformational transitions involved are still poorly understood. In this paper we report several experiments designed to address these questions in more detail.We have studied the ionic strength dependence of the binding of daunomycin to a well defined fraction of chicken erythrocyte chromatin consisting of an average number of 43 nucleosomes. The results are compared with those obtained with the same chromatin fraction upon depletion of linker histones and with the purified constitutive DNA counterpart. EXPERIMENTAL PROC...

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This work evaluated the effects of extra virgin olive oil (EVOO) phenols, hydroxytyrosyl acetate (2) and 3,4-dihydroxyphenylglycol (3), as well as two new acyl derivatives of 3, 4-(1,2-di(butanoyloxy)ethyl)benzene-1,2-diol (7) and 4-(1,2-di(lauroyloxy)ethyl)benzene-1,2-diol (8), on LPS-stimulated murine peritoneal macrophages in comparison with hydroxytyrosol (HTy, 1). Compounds 2, 3, 7, and 8 showed a strong reactive oxygen species (ROS)-scavenging activity, reducing significantly nitrite levels with a significant decrease on iNOS expression [2 (50 μM, 0.44 ± 0.03; 100 μM, 0.44 ± 0.01; p < 0.01); 3 (50 μM, 0.37 ± 0.03; 100 μM, 0.37 ± 0.01; p < 0.001); 7 (50 μM, 0.45 ± 0.06; p < 0.01)] . However, only 2 and 3 down-regulated COX-2 expression [2 (50 μM, 0.72 ± 0.04, p < 0.05; 100 μM, 0.54 ± 0.06, p < 0.01); 3 (50 μM, 0.56 ± 0.05, p < 0.05; 100 μM, 0.37 ± 0.04; p < 0.001)] and prevented IKBα degradation [2 (100 μM, 1.63 ± 0.14, p < 0.01); 3 (100 μM, 1.82 ± 0.09; p < 0.01)] ; the diacylated compounds 7 and 8 showed worse anti-inflammatory activity than the parent 3. In conclusion, 2 and 3 phenolic derivatives could play an important role in the anti-inflammatory effect of EVOO. The implication of this study for the nutrition and general health of the population rests in the possible use of natural HTy derivatives with better hydrophilic/lipophilic balance, thus improving its pharmacodynamic and pharmacokinetic profiles, as new dietary supplements in foods.