The clinically approved antioxidant cardioprotective agent dexrazoxane (ICRF-187) was examined for its ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle, both through reductive activation to its semiquinone form, and by the production of hydroxyl radicals mediated by its complex with iron. Hydrolyzed dexrazoxane metabolites prevent site-specific iron-based oxygen radical damage by displacing iron from doxorubicin and chelating free and loosely bound iron. The mitochondrial stain MitoTracker Green FM and doxorubicin were shown by epifluorescence microscopy to accumulate in the myocyte mitochondria. An epifluorescence microscopic image analysis method to measure mitochondrial damage was developed using the mitochondrial membrane potential sensing ratiometric dye JC-1. This method was used to show that dexrazoxane protected against doxorubicin-induced depolarization of the myocyte mitochondrial membrane. Dexrazoxane also attenuated doxorubicin-induced oxidation of intracellular dichlorofluorescin. Annexin V-FITC/propidium iodide staining of myocytes was used to demonstrate that, depending on the concentration, doxorubicin caused both apoptotic and necrotic damage. These results suggest that doxorubicin may be cardiotoxic by damaging the mitochondria and dexrazoxane may be protective by preventing iron-based oxidative damage.

Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit. Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA. [Fe-Cys31] S4 was then complexed with 16S rRNA or incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins. Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain. One region spans positions [419][420][421][422][423][424][425][426][427][428][429][430][431][432] and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies. A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome. These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.Understanding the molecular mechanism of translation depends on detailed knowledge of the three-dimensional structure of the ribosome. In the absence of an x-ray crystal structure, a wide variety of alternative biochemical and physical approaches have been devised to obtain information concerning the relative locations of ribosomal proteins and rRNA and other macromolecular components of translation in the ribosome (1). Indeed, even with a well-resolved electron density map in hand, information of this kind will likely be essential for its interpretation.In the studies presented here, we describe a biochemical method for obtaining information about the three-dimensional structure of RNA-protein complexes such as the ribosome. It consists of generating hydroxyl radicals locally from Fe(II) tethered to a single position in the ribosome, which results in cleavage of the rRNA backbone at positions that are in close proximity to the Fe(II) ion. Because of the short lifetime of hydroxyl radicals in aqueous solution, cleavage is usually restricted to positions in the RNA that are within about 10 A of the Fe(II) ion (2, 3). We use ribosomal protein S4 as a model system for these studies. The binding of S4 to 16S rRNA has been extensively characterized (4-8); it is one of six small-subunit ribosomal proteins that bind specifically to the RNA in the absence of the other proteins (9). Fe(II) is tethered to the unique cysteine residue at position 31 of protein S4 via 1-(p-bromoacetamidobenzyl)-EDTA (BABE), a reagent that has successfully been used to map intramolecular proximities in proteins (10, 11). Iron-derivatized protein S4 S4) is then bound to 16S rRNA, either alone, or in a fully assembled 30S ribosomal subunit. Hydroxyl radicals are generated after assembly of the ribonucleopro...

No abstract

No abstract