ABSTRACT
Simple surface modification of nano-hydroxyapatite, through acid-basic reactions, allows expanding the properties of this material. Introduction of organic groups such as hydrophobic alkyl chains, carboxylic acid, and amide or amine basic groups on the hydroxyapatite surface systematically change the polarity, surface area, and reactivity of hydroxyapatite without modifying its phase. Physical and chemical properties of the new derivative particles were analyzed. The biocompatibility of modified Nano-Hap on Raw 264.7 cells was also assessed.
Subject(s)
Bone Substitutes/chemical synthesis , Bone Substitutes/toxicity , Durapatite/chemical synthesis , Durapatite/toxicity , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Animals , Cell Survival/drug effects , Materials Testing , Mice , Nanoparticles/toxicity , Particle Size , Phase Transition , RAW 264.7 Cells , Surface PropertiesABSTRACT
Sesquiterpene lactones (SLs) are potent anti-inflammatory substances. It was previously shown that the anti-inflammatory effect could be partly explained by the inhibition of the transcription factor NF-kappaB. Whether they inhibit the DNA binding of NF-kappaB, the activation of the IkappaB-kinase, or both is still a matter of debate. The data supporting these hypotheses were obtained using different cell systems. In this contribution we analyzed the mechanism of the sesquiterpene lactone-mediated inhibition using different cell systems, and showed that in all the cell lines analyzed, SLs inhibited both NF-kappaB binding and the IkappaB-kinase, but that the former played a more preponderant role in the inhibition. These results again confirm the importance of cysteine 38 in the inhibition and regulation of NF-kappaB's function. Moreover, we compared the selectivity of the SL parthenolide with that of N-ethyl maleimide (NEM). We showed that NEM directly alkylated p65 as well as p50 of NF-kappaB, whereas SLs possess a selectivity towards p65. Finally, we studied the transactivating properties of various p65 mutants, to analyze the effect of exchanged cysteine residues in the DNA binding domain of NF-kappaB/p65 on its function and demonstrated that the transactivating potential of the mutants did not correlate with their DNA binding strenght.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Calcium-Binding Proteins , Cysteine , DNA-Binding Proteins/antagonists & inhibitors , Ethylmaleimide/pharmacology , NF-kappa B , Sesquiterpenes/pharmacology , Transcriptional Activation/drug effects , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , I-kappa B Kinase , Jurkat Cells , Macrophages , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mutation , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , NF-kappa B/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Synaptotagmin I , Synaptotagmins , Transcription Factor RelA , TransfectionABSTRACT
Sesquiterpene lactones (SLs) have potent anti-inflammatory properties. We have shown previously that they exert this effect in part by inhibiting activation of the transcription factor NF-kappaB, a central regulator of the immune response. We have proposed a molecular mechanism for this inhibition based on computer molecular modeling data. In this model, SLs directly alkylate the p65 subunit of NF-kappaB, thereby inhibiting DNA binding. Nevertheless, an experimental evidence for the proposed mechanism was lacking. Moreover, based on experiments using the SL parthenolide, an alternative mode of action has been proposed by other authors in which SLs inhibit IkappaB-alpha degradation. Here we report the construction of p65/NF-kappaB point mutants that lack the cysteine residues alkylated by SLs in our model. In contrast to wild type p65, DNA-binding of the Cys(38) --> Ser and Cys(38,120) --> Ser mutants is no longer inhibited by SLs. In addition, we provide evidence that parthenolide uses a similar mechanism to other SLs in inhibiting NF-kappaB. Contrary to previous reports, we show that parthenolide, like other SLs, inhibits NF-kappaB most probably by alkylating p65 at Cys(38). Although a slight inhibition of IkappaB degradation was detected for all SLs, the amount of remaining IkappaB was too low to explain the observed NF-kappaB inhibition.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cysteine , DNA-Binding Proteins/antagonists & inhibitors , I-kappa B Proteins , Lactones/pharmacology , NF-kappa B/antagonists & inhibitors , Sesquiterpenes/pharmacology , DNA-Binding Proteins/metabolism , Drug Design , NF-KappaB Inhibitor alpha , Protein Binding , Protein Subunits , Quercetin/pharmacology , Transcription Factor RelAABSTRACT
The new 3-desoxyanthocyanidins 6,7,3'-trihydroxy-5,4'-dimethoxy-flavylium and 6,7,3',4'-tetrahydroxy-5-methoxy-flavylium and the known 6,7-dihydroxy-5,4'-dimethoxy-flavylium (Carajurin) were isolated by bioguided fractionation from the leaves of Arrabidaea chica, with transcription factor NF-kappaB as target. The structure of Carajurone was revised to be 6,7,4'-trihydroxy-5-methoxy-flavylium. Additionally, the flavone acacetin was found. All structures were mainly established on the basis of MS- and NMR data (1H, 1H-1H COSY and partly 13C, GHMQCR and GHSQCR). Carajurin, which failed to give a positive result in the DPPH TLC assay completely inhibited NF-kappaB, but not NF-AT at a 500 microM concentration.
