Luteolin attenuates doxorubicin-induced derangements of liver and kidney by reducing oxidative and inflammatory stress to suppress apoptosis.

Doxorubicin is an effective anti-neoplastic agent; the reported toxicities of DOX limit its use. Luteolin is a polyphenolic phytochemical that exhibits beneficial biological effects via several mechanisms. We investigate luteolin protective effects on hepatorenal toxicity associated with doxorubicin treatment in rats. For 2 weeks, randomly assigned rat cohorts were treated as follows: control, luteolin (100 mg/kg; per os), doxorubicin alone (2mg/kg; by intraperitoneal injection), co-treated cohorts received luteolin (50 and 100 mg/kg) in addition to doxorubicin. Treatment with doxorubicin alone significantly (p < 0.05) increased biomarkers of hepatorenal toxicities in the serum. Doxorubicin also reduced relative organ weights, antioxidant capacity, and anti-inflammatory cytokine interleukine-10. Doxorubicin also increased reactive oxygen and nitrogen species, lipid peroxidation, pro-inflammatory-interleukin-1ß and tumour necrosis factor-α-cytokine, and apoptotic caspases-3 and -9). Morphological damage accompanied these biochemical alterations in the rat's liver and kidney treated with doxorubicin alone. Luteolin co-treatment dose-dependently abated doxorubicin-mediated toxic responses, improved antioxidant capacity and interleukine-10 level. Luteolin reduced (p < 0.05) lipid peroxidation, caspases-3 and -9 activities and marginally improved rats' survivability. Similarly, luteolin co-treated rats exhibited improvement in hepatorenal pathological lesions observed in rats treated with doxorubicin alone. In summary, luteolin co-treatment blocked doxorubicin-mediated hepatorenal injuries linked with pro-oxidative, inflammatory, and apoptotic mechanisms. Therefore, luteolin can act as a chemoprotective agent in abating toxicities associated with doxorubicin usage and improve its therapeutic efficacy.

Heterogeneous diazo-transfer reaction: a facile unmasking of azide groups on amine-functionalized insoluble supports for solid-phase synthesis.

Solid-supported azides are commonly generated through direct nucleophilic displacement of appropriately activated supports by the azide ion. This reaction usually proceeds rather sluggishly under harsh conditions. Here, we report that triflyl azide rapidly reacts with a series of amine-functionalized solid supports to generate azide-coated supports under mild conditions. Further, we demonstrate that the "azide coat" allows facile loading of alkyne-functionalized leader nucleoside monomers by click chemistry. Finally, we show that the nucleoside-functionalized supports are suitable for solid-phase oligonucleotide synthetic applications. The approach herein described extends the scope of the amine-azide conversion reaction and may be adaptable for the introduction of azide to diverse amine-terminated solid supports that are not easily accessible by the conventional nucleophilic displacement method.

Site specific incorporation of 6-azauridine into the genomic HDV ribozyme active site.

The HDV ribozyme is proposed to catalyze its self cleavage reaction by a proton transfer mechanism wherein the N3 of its C75 acts as a general acid. The C75 to U mutation, which raises the N3 pKa from about 4 to almost 10. abolishes all enzymatic activity. To test if a U analogue with a neutral pKa can restore ribozyme function we incorporated 6-azauridine (n6U), a uridine analogue with histidine-like N3 pKa. into the genomic HDV ribozyme active site by 2'-O-ACE oligoribonucleotide protection chemistry. The resulting ribozymes were analyzed for their ability to undergo the HDV ribozyme cis-cleavage reaction. Incorporation of n6U at nucleotide position 75 did not restore ribozyme function compared to the U75 mutant. This suggests that the HDV ribozyme reaction mechanism involves more than positioning of a neutral nucleobase at the active site and implies that the exocyclic amino group of C75 participates in establishing the proper active site fold.

Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping.

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of a phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5'-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid-base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.

An important base triple anchors the substrate helix recognition surface within the Tetrahymena ribozyme active site.

Key to understanding the structural biology of catalytic RNA is determining the underlying networks of interactions that stabilize RNA folding, substrate binding, and catalysis. Here we demonstrate the existence and functional importance of a Hoogsteen base triple (U300.A97-U277), which anchors the substrate helix recognition surface within the Tetrahymena group I ribozyme active site. Nucleotide analog interference suppression analysis of the interacting functional groups shows that the U300.A97-U277 triple forms part of a network of hydrogen bonds that connect the P3 helix, the J8/7 strand, and the P1 substrate helix. Product binding and substrate cleavage kinetics experiments performed on mutant ribozymes that lack this base triple (C A-U, U G-C) or replace it with the isomorphous C(+).G-C triple show that the A97 Hoogsteen triple contributes to the stabilization of both substrate helix docking and the conformation of the ribozyme's active site. The U300. A97-U277 base triple is not formed in the recently reported crystallographic model of a portion of the group I intron, despite the presence of J8/7 and P3 in the RNA construct [Golden, B. L., Gooding, A. R., Podell, E. R. & Cech, T. R. (1998) Science 282, 259-264]. This, along with other biochemical evidence, suggests that the active site in the crystallized form of the ribozyme is not fully preorganized and that substantial rearrangement may be required for substrate helix docking and catalysis.