Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology.

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology.

In vitro selection of allosteric ribozymes: theory and experimental validation.

In vitro selection techniques offer powerful and versatile methods to isolate nucleic acid sequences with specific activities from huge libraries. We describe an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to pefloxacin and other quinolone derivatives. Within 16 selection cycles, highly sensitive clones responding to drug levels in the sub-micromolar range were obtained. The morpholine moiety of the quinolone derivatives was required for inhibition of the self-cleavage of the selected ribozymes: modifications of the aromatic system were tolerated better than modifications of the morpholine ring. We also present a theoretical model that analyzes the predicted fraction of ribozymes with a given binding constant and cleavage rate recovered after each selection cycle. This model precisely predicts the actual experimental values obtained with the selection procedure. It can thus be used to determine the optimal conditions for an in vitro selection of an allosteric ribozyme with a desired dissociation constant and cleavage rate for a given application.

[Protein-poor pleurisy and mesothelioma. Apropos of 2 cases]. / Pleurésie pauvre en protides et mésothéliome. A propos de 2 cas.

We report two cases of protein poor pleural effusions secondary to malignant mesothelioma which were proven histologically. In the absence of any extra pulmonary cause, in particular cardiac, the associated investigations carried out led to the exclusion of either a chylothorax or a mechanical effusion due to atelectasis. Pulmonary venous obstruction by the mesothelial masses which had developed on the mediastinal pleura were shown in the two cases. The high pressure in the pulmonary capillaries which resulted could be the origin of the low level of protein observed in the pleural fluid.