RNA-binding activity and regulatory functions of the emerging sRNA-binding protein ProQ.

Regulatory small RNAs (sRNAs) ubiquitously impact bacterial physiology through antisense-mediated control of mRNA translation and stability. In Gram negative bacteria, sRNAs often associate with RNA-binding proteins (RBPs), both to gain cellular stability and to enable regulatory efficiency. The Hfq and CsrA proteins were for long the only known global RBPs implicated in sRNA biology. During the last five years, the FinO domain-containing protein ProQ has emerged as another global RBP with a broad spectrum of sRNA and mRNA ligands. This review provides a summary of the current knowledge of enterobacterial ProQ, with a special focus on RNA binding activity, RNA ligand preferences, influence on RNA stability and gene expression, and impact on bacterial physiology. Considering that characterization of ProQ is still in its infancy, we highlight aspects that, when addressed, will provide important clues to the physiological functions and regulatory mechanisms of this globally acting RBP.

Characterization of jejunal absorption and apical efflux of ropivacaine, lidocaine and bupivacaine in the rat using in situ and in vitro absorption models.

The purpose of this study was to characterize the rat jejunal passive transport and the possible active efflux of three local anaesthetics, ropivacaine, lidocaine and bupivacaine using two different absorption models, the in situ single-pass intestinal perfusion and the in vitro Ussing chamber model, as well as P-glycoprotein (Pgp)-mediated calcein transport inhibition in Caco-2 cells. Concentration and pH dependence, efflux inhibition by verapamil and digoxin and bi-directional permeability studies were performed to investigate the potential involvement of efflux carriers in the intestinal absorption of the local anaesthetics. In the jejunal perfusion the permeability of these agents appeared to be high, predicting complete intestinal absorption (>90%). There was no effect of the Pgp inhibitors on net absorption for any of the local anaesthetics in the two absorption models. However, in the Ussing chamber at an equal pH of 7.4 at mucosal and serosal sides, the observed jejunal permeability ratios (S-M)/(M-S), of 2.3, 1.8 and 3.0 for ropivacaine, lidocaine and bupivacaine, respectively, indicated at least some involvement of carrier-mediated intestinal secretion. This idea was supported in the calcein AM Pgp transport assay in which two of the tested local anaesthetic agents affected cellular calcein retention. As anticipated for these agents, the mucosal pH conditions were shown to largely affect the gut permeability. The jejunal permeabilities of the local anaesthetics as measured in the two absorption models fitted well in a model comparison that incorporated the permeabilities of six other structurally unrelated drugs. In conclusion, the jejunal permeability of ropivacaine, lidocaine and bupivacaine was high and although evidence was obtained for carrier-mediated intestinal efflux this process appeared not to have a significant influence on the rate and extent of in vivo intestinal absorption. Rather, passive diffusion of these agents seems to be the major mechanism for the intestinal absorption.

Regional transport and metabolism of ropivacaine and its CYP3A4 metabolite PPX in human intestine.

The major aim of this study was to investigate the CYP3A4 metabolism and polarized transport of ropivacaine and its metabolite 2',6'-pipecoloxylidide (PPX) in tissue specimens from the human small and large intestine. Ropivacaine has been shown to be effective in the treatment of ulcerative colitis in human colon. This study was conducted using a modified Ussing-chamber technique with specimens from jejunum, ileum and colon collected from 11 patients. The local kinetics of ropivacaine and PPX were assessed from their concentration-time profiles in mucosal and serosal compartments. The permeability (P(app)) in the absorptive direction for both ropivacaine and PPX increased regionally in the order jejunum < ileum < colon. Ropivacaine was not found to be subjected to any carrier-mediated intestinal efflux. However, the CYP3A4 metabolite left the human enterocyte in a polarized manner and both the extent of CYP3A4 metabolism of ropivacaine and the extrusion of its metabolite to the mucosal chamber were more efficient in jejunum than in ileum. P-glycoprotein was probably not involved in the metabolite extrusion. No other metabolite than PPX was found. This in-vitro study with human intestinal tissues provides new mechanistic insights into regional transport and metabolism of drugs.

Gene and protein expression of P-glycoprotein, MRP1, MRP2, and CYP3A4 in the small and large human intestine.

The cytochrome P450 3A4 enzyme and the ABC-transporters may affect the first-pass extraction and bioavailability of drugs and metabolites. Conflicting reports can be found in the literature on the expression levels of efflux transporters in human intestine and how they vary along the intestine. The relative levels of mRNA and protein of CYP3A4 and the ABC tranporters Pgp (ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2) were determined using RT-PCR and Western blot for human intestinal tissues (n = 14) from jejunum, ileum and colon. The expression of mRNA for CYP3A4, Pgp, and MRP2 was highest in jejunum and decreased toward more distal regions, whereas MRP1 was equally distributed in all intestinal regions. For CYP3A4, a more significant correlation could be established between mRNA and protein expression than for the ABC transporters. The samples showed considerable interindividual variability, especially at the protein level. The apically located Pgp and MRP2 showed a similar expression pattern along the human intestine as for CYP3A4. The gene expression of MRP1 exhibited a more uniform distribution.