Clinical factors such as age, gender, alcohol use, and age-at-infection influence the progression to cirrhosis but cannot accurately predict the risk of developing cirrhosis in patients with chronic hepatitis C (CHC). The aim of this study was to develop a predictive signature for cirrhosis in Caucasian patients. All patients had well-characterized liver histology and clinical factors; DNA was extracted from whole blood for genotyping. We validated all significant markers from a genome scan in the training cohort, and selected 361 markers for the signature building. Using a "machine learning" approach, a signature consisting of markers most predictive for cirrhosis risk in Caucasian patients was developed in the training set (N ‫؍‬ 420). The Cirrhosis Risk Score (CRS) was calculated to estimate the risk of developing cirrhosis for each patient. The CRS performance was then tested in an independently enrolled validation cohort of 154 Caucasian patients. A CRS signature consisting of 7 markers was developed for Caucasian patients.

No abstract

Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem across the outer membrane. In this study, we examined the effects of deletions in six of the proposed eight surface loops of OprD on the in vivo and in vitro functions of this protein. Native OprD formed very small channels in planar lipid bilayers, with an average single-channel conductance in 1.0 M KCl of 20 pS. When large numbers of OprD channels were incorporated into lipid bilayer membranes, addition of increasing concentrations of imipenem to the bathing solutions resulted in a progressive blocking of the membrane conductance of KCl, indicating the presence of a specific binding site(s) for imipenem in the OprD channel. From these experiments, the concentration of imipenem value of resulting in 50% inhibition of the initial conductance was calculated as approximately 0.6 M. In contrast, no decrease in channel conductance was observed for the OprD⌬L2 channel upon addition of up to 2.4 M imipenem, confirming that external loop 2 was involved in imipenem binding. Deletion of four to eight amino acids from loops 1 and 6 had no effect on antibiotic susceptibility, whereas deletion of eight amino acids from loops 5, 7, and 8 resulted in supersusceptibility to ␤-lactams, quinolones, chloramphenicol, and tetracycline. Planar lipid bilayer analysis indicated that the OprD⌬L5 channel had a 33-fold increase in single-channel conductance in 1 M KCl but had retained its imipenem binding site. The disposition of these loop regions in the interior of the OprD channel is discussed.The porins of gram-negative bacteria form water-filled channels that permit the diffusion of hydrophilic solutes across the outer membrane (7, 24). They are generally divided into two classes: nonspecific porins which permit the general diffusion of hydrophilic molecules below a certain size, and specific porins which facilitate the diffusion of specific substrates by virtue of containing stereospecific binding sites (7). Pseudomonas aeruginosa OprD is a specific porin for imipenem (22), a carbapenem which shows excellent activity against P. aeruginosa. However, the natural substrate for OprD is not imipenem but its structural analogs, basic amino acids and small peptides containing these amino acids (23). In vivo functional studies indicated that OprD could also selectively facilitate the diffusion of gluconate under growth-rate-limiting conditions (11). Although the protein has been characterized in vitro in liposome swelling assays and the presence of the binding site has been confirmed (22), very limited work has been done to study its physical properties in planar lipid bilayers. Ishii and Nakae (13) measured the single-channel conductance of OprD, which was 20 to 30 pS. They also observed larger channels (400 pS) that they suggested were capable of being induced under certain conditions. In their studies, the ion selectivity of the OprD channel was not measured, and no direct evidence for the presence of a specific binding site(s)...

No abstract

Ribonuclease (RNase) E is an extensively studied enzyme from Escherichia coli whose site-specific endoribonuclease activity on single-stranded RNA has a central role in the processing of ribosomal RNA, the degradation of messenger RNA and the control of replication of ColE1-type plasmids. Here we report a previously undetected activity of RNase E: the ability to shorten 3' poly(A)- and poly(U)-homopolymer tails on RNA molecules. This activity, which leaves a 6-nucleotide adenylate or a 1-nucleotide uridylate remnant on primary transcripts, resides in the amino-terminal region of RNase E and does not require other protein cofactors. Addition of a 3'-terminal phosphate group prevents both removal of the poly(A) tail and endonucleolytic cleavage within primary transcripts, but has no effect on the cleavage of transcripts with tails that have already been truncated. The ability of RNase E to shorten poly(A) tails, together with the effect of tail length on endonucleolytic cleavage within primary transcripts, suggests a mechanism by which RNase E may exercise overall control over RNA decay.