A Complete Pathway Model for Lipid A Biosynthesis in Escherichia coli.

Lipid A is a highly conserved component of lipopolysaccharide (LPS), itself a major component of the outer membrane of Gram-negative bacteria. Lipid A is essential to cells and elicits a strong immune response from humans and other animals. We developed a quantitative model of the nine enzyme-catalyzed steps of Escherichia coli lipid A biosynthesis, drawing parameters from the experimental literature. This model accounts for biosynthesis regulation, which occurs through regulated degradation of the LpxC and WaaA (also called KdtA) enzymes. The LpxC degradation signal appears to arise from the lipid A disaccharide concentration, which we deduced from prior results, model results, and new LpxK overexpression results. The model agrees reasonably well with many experimental findings, including the lipid A production rate, the behaviors of mutants with defective LpxA enzymes, correlations between LpxC half-lives and cell generation times, and the effects of LpxK overexpression on LpxC concentrations. Its predictions also differ from some experimental results, which suggest modifications to the current understanding of the lipid A pathway, such as the possibility that LpxD can replace LpxA and that there may be metabolic channeling between LpxH and LpxB. The model shows that WaaA regulation may serve to regulate the lipid A production rate when the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) concentration is low and/or to control the number of KDO residues that get attached to lipid A. Computation of flux control coefficients showed that LpxC is the rate-limiting enzyme if pathway regulation is ignored, but that LpxK is the rate-limiting enzyme if pathway regulation is present, as it is in real cells. Control also shifts to other enzymes if the pathway substrate concentrations are not in excess. Based on these results, we suggest that LpxK may be a much better drug target than LpxC, which has been pursued most often

Diffusion, Crowding & Protein Stability in a Dynamic Molecular Model of the Bacterial Cytoplasm

A longstanding question in molecular biology is the extent to which the behavior of macromolecules observed in vitro accurately reflects their behavior in vivo. A number of sophisticated experimental techniques now allow the behavior of individual types of macromolecule to be studied directly in vivo; none, however, allow a wide range of molecule types to be observed simultaneously. In order to tackle this issue we have adopted a computational perspective, and, having selected the model prokaryote Escherichia coli as a test system, have assembled an atomically detailed model of its cytoplasmic environment that includes 50 of the most abundant types of macromolecules at experimentally measured concentrations. Brownian dynamics (BD) simulations of the cytoplasm model have been calibrated to reproduce the translational diffusion coefficients of Green Fluorescent Protein (GFP) observed in vivo, and “snapshots” of the simulation trajectories have been used to compute the cytoplasm's effects on the thermodynamics of protein folding, association and aggregation events. The simulation model successfully describes the relative thermodynamic stabilities of proteins measured in E. coli, and shows that effects additional to the commonly cited “crowding” effect must be included in attempts to understand macromolecular behavior in vivo

Novel roles for GAPDH in cell death and carcinogenesis

Publicado en línea el 25 de septiembre de 2009Growing evidence points to the fact that glucose metabolism has a central role in carcinogenesis. Among the enzymes controlling this energy production pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is of particular interest. Initially identified as a glycolytic enzyme and considered as a housekeeping gene, this enzyme is actually tightly regulated and is involved in numerous cellular functions. Particularly intriguing are recent reports describing GAPDH as a regulator of cell death. However, its role in cell death is unclear; whereas some studies point toward a proapoptotic function, others describe a protective role and suggest its participation in tumor progression. In this study, we highlight recent findings and discuss potential mechanisms through which cells regulate GAPDH to fulfill its diverse functions to influence cell fate.This work was supported in part by l’Association pour la Recherche sur le Cancer, by l’Agence Nationnal de la Recherche, la Fondation de France, Plan Nacional I+D SAF2008-04974 and by grants from The U.S. National Institutes of Health. J-E.R. is a recipient of a contrat d’interface INSERM-CHU de Nice.Peer reviewe

Outcomes and Treatment of Lumbosacral Spinal Tuberculosis: A Retrospective Study of 53 Patients

A retrospective clinic study.To evaluate the efficacy of conservative and surgical treatment for lumbosacral tuberculosis.This study retrospectively reviewed 53 patients with lumbosacral tuberculosis who were treated in our institution between January 2005 and January 2011. There were 29 males and 24 females with average ages of 37.53 ± 17.28 years (range 6-72 years). 11 patients were given only anti-TB drugs; the remainder underwent anterior debridement, interbody fusion with and without instrumentation, or one-stage anterior debridement combined with posterior instrumentation. Outcome data for these patients included neurologic status, lumbosacral angle, erythrocyte sedimentation rate value(ESR) and C-reactive protein value(CRP) were assessed before and after treatment.The mean lumbosacral angles were 23.00°± 2.90° in the conservatively treated patients and 22.36°± 3.92o in the surgically treated patients. At the final follow-up, this had improved to 24.10o ± 2.96° in the conservatively treated patients and 28.13° ± 1.93° in the surgically treated patients (all P < 0.05). There were statistically significant differences before and after treatment in terms of ESR and CRP (all P < 0.05). All patients achieved bone fusion. The mean follow-up period was 32.34 ± 8.13 months (range 18 to 55 months). The neurological deficit did not worsen in any of the patients.It has been proven that conservative and surgical treatments are safe and effective and produce good clinical outcomes for patients with lumbosacral tuberculosis. The advantages of operation include thoroughness of debridement, decompression of the spinal cord, and adequate spinal stabilization