No abstract

Chondrocytes have been shown to produce superoxide and hydrogen peroxide, suggesting possible formation of hydroxyl radical in these cells. In this study, we used electron spin resonance/spin trapping technique to detect hydroxyl radicals in chondrocytes. We found that hydroxyl radicals could be detected as alpha-hydroxyethyl spin trapped adduct of 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN) in chondrocytes stimulated with phorbol 12-myristate 13-acetate in the presence of ferrous ion. The formation of hydroxyl radical appears to be mediated by the transition metal-catalyzed Haber-Weiss reaction since no hydroxyl radical was detected in the absence of exogenous iron. The hydroxyl radical formation was inhibited by catalase but not by superoxide dismutase, suggesting that the hydrogen peroxide is the precursor. Cytokines, IL-1 and TNF enhanced the hydroxyl radical formation in phorbol 12-myristate 13-acetate treated chondrocytes. Interestingly, hydroxyl radical could be detected in unstimulated fresh human and rabbit cartilage tissue pieces in the presence of iron. These results suggest that the formation of hydroxyl radical in cartilage could play a role in cartilage matrix degradation.

Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor represents a unique posttranslational modification that is ubiquitously present in eukaryotic cells and archaebacteria. Specific inhibition of deoxyhypusine synthase leads to growth arrest and cell death. The precise cellular function of eIF-5A and the physiological significance of hypusine modification are not clear. Although the methionyl-puromycin synthesis has been suggested to be the functional assay for eIF-5A activity in vitro, the role of eIF-5A in protein synthesis has not been established. Recent studies have suggested that eIF-5A may be the cellular target of the human immunodeficiency virus type 1 Rev and human T cell leukemia virus type 1 Rex proteins. Motif analysis suggested that eIF-5A resembles a bimodular RNA-binding protein in that it contains a stretch of basic amino acids clustered at the N-terminal region and a leucine-rich stretch at the C-terminal region. Using Rev target RNA, RRE, as a model, we tested the hypothesis that eIF-5A may be an RNA-binding protein. We found that both deoxyhypusine and hypusine-containing eIF-5A can bind to the 252-nt RRE RNA, as determined by a gel mobility shift assay. In contrast, the unmodified eIF-5A precursor cannot. Deoxyhypusine-containing eIF-5A, but not its precursor, could also cause supershift of the Rev stem-loop IIB RRE complex. Preliminary studies also indicated that eIF-5A can bind to RNA such as U6 snRNA and that deoxyhypusine modification appears to be required for the binding. The ability of eIF-5A to directly interact with RNA suggests that deoxyhypusine formation of eIF-5A may be related to its role in RNA processing and protein synthesis. Our study also suggests the possibility of using a gel mobility shift assay for eIF-5A-RNA binding as a functional assay for deoxyhypusine and hypusine formation.

Kinetic analysis of ribosomal peptidyltransferase activity in a methanolic puromycin reaction with wild type and drug-resistant 23 S RNA mutants was used to probe the structural basis of catalysis and mechanism of resistance to antibiotics. 23 S RNA mutants G2032A and G2447A are resistant to oxazolidinones both in vitro and in vivo with the latter displaying a 5-fold increase in the value of K m for initiator tRNA and a 100-fold decrease in V max in puromycin reaction. Comparison of the K i values for oxazolidinones, chloramphenicol, and sparsomycin revealed partial cross-resistance between oxazolidinones and chloramphenicol; no cross-resistance was observed with sparsomycin, a known inhibitor of the peptidyltransferase A-site. Inhibition of the mutants using a truncated CCA-Phe-X-Biotin fragment as a P-site substrate is similar to that observed with the intact initiator tRNA, indicating that the inhibition is substrateindependent and that the peptidyltransferase itself is the oxazolidinone target. Mapping of all known mutations that confer resistance to these drugs onto the spatial structure of the 50 S ribosomal subunit allows for docking of an oxazolidinone into a proposed binding pocket. The model suggests that oxazolidinones bind between the P-and A-loops, partially overlapping with the peptidyltransferase P-site. Thus, kinetic, mutagenesis, and structural data suggest that oxazolidinones interfere with initiator fMet-tRNA binding to the P-site of the ribosomal peptidyltransferase center.Oxazolidinones, the only novel class of antibiotics identified in the last two decades, are the focus of intensive discovery efforts (1-9). Linezolid, an oxazolidinone, is approved for treatment of infections caused by Gram-positive bacteria that are resistant to other antibiotics. Emerging resistance to all known drugs, including the "last resort" vancomycin family, poses a serious threat to the public health worldwide. Understanding the mechanism of action of oxazolidinones at the molecular level, therefore, has a great importance for the development of the next generation of these novel antibiotics and, ultimately, for the outcome of the ongoing battle against drug-resistant pathogens.Oxazolidinones impose their action at the initiation stage of translation (4, 5), apparently via inhibition of preinitiation complex formation (9). Our recent finding that oxazolidinones interfere with binding of initiator tRNA to the ribosomal P-site (1), thus inhibiting formation of the first peptide bond, prompted a search for similarities between oxazolidinones and known inhibitors of peptidyltransferase. To address this and other mechanistic questions, we have studied catalytic properties of oxazolidinone-resistant ribosomes and compared the mechanism of oxazolidinone inhibition with the action of known peptidyltransferase inhibitors, such as chloramphenicol and sparsomycin. To further define the oxazolidinone binding site, we have mapped resistant mutations onto the three-dimensional structure of the ribosomal 50 S subunit to reveal a...

Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA templates but have no significant effect on poly(A)-dependent translation. Here we show that various oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70 S ribosomes using initiator-tRNA or N-protected CCA-Phe as a P-site substrate and puromycin as an A-site substrate. Steadystate kinetic analysis shows that oxazolidinones display a competitive inhibition pattern with respect to both the P-site and A-site substrates. This is consistent with a rapid equilibrium, ordered mechanism of the peptidyltransferase reaction, wherein binding of the A-site substrate can occur only after complex formation between peptidyltransferase and the P-site substrate. We propose that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiator fMet-tRNA i Met to the ribosomal peptidyltransferase Psite, which is vacant only prior to the formation of the first peptide bond.