Prediction of chronic toxicity of pharmaceuticals in Daphnia magna by combining ortholog prediction, pharmacological effects, and quantitative structure-activity relationship.

To develop a method for predicting chronic toxicity of pharmaceuticals in Daphnia, we investigated the feasibility of combining the presence of drug-target orthologs in Daphnia magna, classification based on pharmacological effects, and ecotoxicity quantitative structure-activity relationship (QSAR) prediction. We established datasets on the chronic toxicity of pharmaceuticals in Daphnia, including information on therapeutic categories, target proteins, and the presence or absence of drug-target orthologs in D. magna, using literature and databases. Chronic toxicity was predicted using ecotoxicity prediction QSAR (Ecological Structure Activity Relationship and Kashinhou Tool for Ecotoxicity), and the differences between the predicted and measured values and the presence or absence of drug-target orthologs were examined. For pharmaceuticals without drug-target orthologs in D. magna or without expected specific actions, the ecotoxicity prediction QSAR analysis yielded acceptable predictions of the chronic toxicity of pharmaceuticals. In addition, a workflow model to assess the chronic toxicity of pharmaceuticals in Daphnia was proposed based on these evaluations and verified using an additional dataset. The addition of biological aspects such as drug-target orthologs and pharmacological effects would support the use of QSARs for predicting the chronic toxicity of pharmaceuticals in Daphnia.

The A-site finger in 23 S rRNA acts as a functional attenuator for translocation.

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

Antibiotic susceptibility of mammalian mitochondrial translation.

All medically useful antibiotics should have the potential to distinguish between target microbes (bacteria) and host cells. Although many antibiotics that target bacterial protein synthesis show little effect on the translation machinery of the eukaryotic cytoplasm, it is unclear whether these antibiotics target or not the mitochondrial translation machinery. We employed an in vitro translation system from bovine mitochondria, which consists of mitochondrial ribosomes and mitochondrial elongation factors, to estimate the effect of antibiotics on mitichondrial protein synthesis. Tetracycline and thiostrepton showed similar inhibitory effects on both Escherichia coli and mitochondrial protein synthesis. The mitochondrial system was more resistant to tiamulin, macrolides, virginiamycin, fusidic acid and kirromycin than the E. coli system. The present results, taken together with atomic structure of the ribosome, may provide useful information for the rational design of new antibiotics having less adverse effects in humans and animals.