Assembly and activation of a kinase ribozyme.

RNA activities can be regulated by modulating the relative energies of all conformations in a folding landscape; however, it is often unknown precisely how peripheral elements perturb the overall landscape in the absence of discrete alternative folds (inactive ensemble). This work explores the effects of sequence and secondary structure in governing kinase ribozyme activity. Kin.46 catalyzes thiophosphoryl transfer from ATPγS onto the 5' hydroxyl of polynucleotide substrates, and is regulated 10,000-fold by annealing an effector oligonucleotide to form activator helix P4. Transfer kinetics for an extensive series of ribozyme variants identified several dispensable internal single-stranded segments, in addition to a potential pseudoknot at the active site between segments J1/4 and J3/2 that is partially supported by compensatory rescue. Standard allosteric mechanisms were ruled out, such as formation of discrete repressive structures or docking P4 into the rest of the ribozyme via backbone 2' hydroxyls. Instead, P4 serves both to complete an important structural element (100-fold contribution to the reaction relative to a P4-deleted variant) and to mitigate nonspecific, inhibitory effects of the single-stranded tail (an additional 100-fold contribution to the apparent rate constant, k(obs)). Thermodynamic activation parameters ΔH(‡) and ΔS(‡), calculated from the temperature dependence of k(obs), varied with tail length and sequence. Inhibitory effects of the unpaired tail are largely enthalpic for short tails and are both enthalpic and entropic for longer tails. These results refine the structural view of this kinase ribozyme and highlight the importance of nonspecific ensemble effects in conformational regulation by peripheral elements.

Comparison of tobramycin 0.3%/dexamethasone 0.1% and tobramycin 0.3%/loteprednol 0.5% in the management of blepharo-keratoconjunctivitis.

In this clinical trial, investigators compared the effectiveness of 2 commercially formulated antibiotic/steroid combinations - tobramycin 0.3%/dexamethasone 0.1% (Tobradex; Alcon, Fort Worth, Tex) and tobramycin 0.3%/loteprednol 0.5% (Zylet; Bausch & Lomb Inc., Rochester, NY) - for rapidly controlling inflammation in patients with blepharo-keratoconjunctivitis. Investigators in this randomized, parallel-group, double-masked study examined 40 eyes of 40 patients with blepharo-keratoconjunctivitis. Patients received tobramycin 0.3%/dexamethasone 0.1% or tobramycin 0.3%/loteprednol 0.5% twice daily in the test eye, according to the randomization schedule. At baseline, the ocular surface was graded on a scale of 3 (extensive) to 0 (minimum) for 4 components: blepharitis, conjunctivitis, ocular discharge, and corneal punctate epithelial keratopathy (PEK). Only those patients with moderate to extensive inflammation (cumulative score >6) were included in the study. At follow-up 3 to 5 d later, the ocular surface was regraded so that treatment response could be evaluated. No statistically significant difference was noted between groups in pretreatment scores for blepharitis (P=.31), discharge (P=.62), conjunctivitis (P=1.0), and PEK (P=.57), or for total ocular inflammation (P=.87). Mean posttreatment scores were as follows: total ocular surface scores, 1.8 and 3.4 (P=.002); blepharitis scores, 0.9 and 1.35 (P=.017); discharge scores, 0.2 and 0.6 (P=.025); and conjunctivitis scores, 0.15 and 0.6 (P=.013) for tobramycin/dexamethasone and tobramycin/loteprednol, respectively. Corneal PEK scores were not significantly different between treatments. Tobramycin 0.3%/dexamethasone 0.1% significantly decreased clinical signs of ocular inflammation (ie, blepharitis, discharge, conjunctivitis) and total ocular inflammation scores when compared with tobramycin 0.3%/loteprednol 0.5% in patients with moderate to severe blepharo-keratoconjunctivitis. The 2 regimens also provided comparably rapid decreases in corneal PEK.

DNA cross-linking, double-strand breaks, and apoptosis in corneal endothelial cells after a single exposure to mitomycin C.

PURPOSE: To investigate the cellular effects of mitomycin C (MMC) treatment on corneal endothelial (CE) cells at clinically relevant applications and dosages. METHODS: Radial and posterior diffusion of MMC was determined by an Escherichia coli growth inhibition bioassay. A modified version of the comet assay (single cell gel electrophoresis) was used to detect DNA cross-linking. Immunostaining detected the nuclear phosphorylated histone variant H2AX (gamma-H2AX) indicating DNA double-strand breaks. Apoptosis in MMC-treated cells was detected with annexin V staining. RESULTS: Topical application of 0.02% MMC to intact goat globes resulted in MMC in the CE at 0.37 microg/mL and produced a significant increase in CE DNA cross-linking with as little as 6 seconds of topical MMC treatment. DNA cross-linking was also demonstrated in cultured CE cells by using MMC exposures similar to those detected in CE of intact eyes. Such MMC treatment of CE produced elevated and persistent gamma-H2AX-positive cells indicative of DNA double-strand breaks. Similarly, there was an increase in the proportion of apoptotic CE cells, evidenced by positive annexin V staining. CONCLUSIONS: The results demonstrate that exposure to MMC at times and concentrations commonly used in refractive surgery produces cross-linking of corneal endothelial DNA, persistent DNA damage, and endothelial death via apoptosis. Current practices of MMC application during refractive surgeries may increase the potential for long-term and permanent deleterious effects on the health of the corneal endothelium.

Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry.

We assessed the utility of the sulfhydryl reductant Tris(2-carboxyethyl)phosphine (TCEP) for both nucleic acid and thiophosphate chemistry, including its effects on organomercurial gel electrophoresis, RNA catalysis, RNA backbone stability, and the intrinsic stability of TCEP. The sulfhydryls of dithiothreitol (DTT) compete with thiophosphates for binding to the mercury within [(N-acryloylamino)phenyl] mercuric chloride (APM) polyacrylamide gels, whereas millimolar concentrations of TCEP gave no difference in the fraction of thiophosphorylated RNA retained on the APM interface relative to samples containing no reductant. Ribozyme activity in TCEP, assessed by the self-thiophosphorylating Kin.46 ribozyme, was unaffected by the presence of DTT or TCEP or by the absence of reductant, as measured on APM gels and evaluated by Michaelis-Menten kinetics. Unexpectedly, TCEP more than doubled the half-life of full-length RNA at 50 and 70 degrees C, whether in 5 or 50mM MgCl(2), relative to DTT and the absence of reductant. Under these same conditions, the 5(')-thiophosphate showed negligible decay, and TCEP was more stable than DTT. TCEP thermostability was equivalent in the presence of 5 or 50mM MgCl(2) and 10mM adenosine or ATP.