mRNA degradation in bacteria

Messenger RNAs in prokaryotes exhibit short half-lives when compared with eukaryotic mRNAs. Considerable progress has been made during recent years in our understanding of mRNA degradation in bacteria. Two major aspects determine the life span of a messenger in the bacterial cell. On the side of the substrate, the structural features of mRNA have a profound influence on the stability of the molecule. On the other hand, there is the degradative machinery. Progress in the biochemical characterization of proteins involved in mRNA degradation has made clear that RNA degradation is a highly organized cellular process in which several protein components, and not only nucleases, are involved. In Escherichia coli, these proteins are organized in a high molecular mass complex, the degradosome. The key enzyme for initial events in mRNA degradation and for the assembly of the degradosome is endoribonuclease E. We discuss the identified components of the degradosome and its mode of action. Since research in mRNA degradation suffers from dominance of E. coli-related observations we also look to other organisms to ask whether they could possibly follow the E. coli standard model.     

Simultaneous on-line extraction and analysis of sirolimus (rapamycin) and ciclosporin in blood by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific semi-automated liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous quantification of sirolimus and ciclosporin in blood. Following a simple protein precipitation step, the supernatants were injected into the HPLC system and extracted on-line. After column switching, the analytes were backflushed from the extraction column onto the analytical narrow-bore column and eluted into the ESI-MS system. The assay was linear from 0.4 to 100 μg/l sirolimus and from 2 to 1500 μg/l ciclosporin. The mean recoveries of sirolimus and ciclosporin were 98 and 96%, respectively. The mean interday precision/accuracy was 8.6%/−4.8% for sirolimus and 9.3%/−2.9% for ciclosporin.     

High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a K_i of approximately 4 μM. Uptake studies with [1,2-dimethyl-¹⁴C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a K_m value of 4 ± 1 μM and a maximum rate of transport (V_max) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance ¹³C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline-O-sulfate but also of carnitine, crotonobetaine, and γ-butyrobetaine (R. Kappes and E. Bremer, Microbiology 144:83–90, 1998). Thus, our data underscore its crucial role in the acquisition of a variety of osmoprotectants from the environment by B. subtilis.     

Assembly of binding loops on aromatic templates as VCAM‐1 mimetics

The design and synthesis of cyclic mimetics of VCAM‐1 protein that reproduce the integrin‐binding domain are presented. The unprotected peptide precursor 37 – 43 , Thr‐Gln‐Ile‐Asp‐Ser‐Pro‐Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C‐ and N‐terminal oximes resulting in a mixture of four isomeric forms due to syn–anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene‐derived template was found to inhibit the VCAM‐1/VLA‐4 interaction more efficiently than previously reported for sulfur‐bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37 – 43 in the design of synthetic inhibitors of the VCAM‐1/integrin interaction. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.