Three serendipitous pathways in E. coli can bypass a block in pyridoxal-5′-phosphate synthesis

Overexpression of seven different genes restores growth of a ΔpdxB strain of E. coli, which cannot make pyridoxal phosphate (PLP), on M9/glucose.None of the enzymes encoded by these genes has a promiscuous 4-phosphoerythronate dehydrogenase activity that can replace the activity of PdxB.Overexpression of these genes restores PLP synthesis by three different serendipitous pathways that feed into the normal PLP synthesis pathway downstream of the blocked step.Reactions in one of these pathways are catalyzed by low-level activities of enzymes of unknown function and a promiscuous activity of an enzyme that normally has a role in another pathway; one reaction appears to be non-enzymatic

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

Antimicrobial Peptides and Skin: A Paradigm of Translational Medicine

Antimicrobial peptides (AMPs) are small, cationic, amphiphilic peptides with broad-spectrum microbicidal activity against both bacteria and fungi. In mammals, AMPs form the first line of host defense against infections and generally play an important role as effector agents of the innate immune system. The AMP era was born more than 6 decades ago when the first cationic cyclic peptide antibiotics, namely polymyxins and tyrothricin, found their way into clinical use. Due to the good clinical experience in the treatment of, for example, infections of mucus membranes as well as the subsequent understanding of mode of action, AMPs are now considered for treatment of inflammatory skin diseases and for improving healing of infected wounds. Based on the preclinical findings, including pathobiochemistry and molecular medicine, targeted therapy strategies are developed and first results indicate that AMPs influence processes of diseased skin. Importantly, in contrast to other antibiotics, AMPs do not seem to propagate the development of antibiotic-resistant micro-organisms. Therefore, AMPs should be tested in clinical trials for their efficacy and tolerability in inflammatory skin diseases and chronic wounds. Apart from possible fields of application, these peptides appear suited as an example of the paradigm of translational medicine for skin diseases which is today seen as a `two-way road' - from bench to bedside and backwards from bedside to bench. Copyright (c) 2012 S. Karger AG, Base

The relationship between workers' self-reported changes in health and their attitudes towards a workplace intervention: lessons from smoke-free legislation across the UK hospitality industry

Background: The evaluation of smoke-free legislation (SFL) in the UK examined the impacts on exposure to second-hand smoke, workers’ attitudes and changes in respiratory health. Studies that investigate changes in the health of groups of people often use self-reported symptoms. Due to the subjective nature it is of interest to determine whether workers’ attitudes towards the change in their working conditions may be linked to the change in health they report. Methods: Bar workers were recruited before the introduction of the SFL in Scotland and England with the aim of investigating their changes to health, attitudes and exposure as a result of the SFL. They were asked about their attitudes towards SFL and the presence of respiratory and sensory symptoms both before SFL and one year later. Here we examine the possibility of a relationship between initial attitudes and changes in reported symptoms, through the use of regression analyses. Results: There was no difference in the initial attitudes towards SFL between those working in Scotland and England. Bar workers who were educated to a higher level tended to be more positive towards SFL. Attitude towards SFL was not found to be related to change in reported symptoms for bar workers in England (Respiratory, p = 0.755; Sensory, p = 0.910). In Scotland there was suggestion of a relationship with reporting of respiratory symptoms (p = 0.042), where those who were initially more negative to SFL experienced a greater improvement in self-reported health. Conclusions: There was no evidence that workers who were more positive towards SFL reported greater improvements in respiratory and sensory symptoms. This may not be the case in all interventions and we recommend examining subjects’ attitudes towards the proposed intervention when evaluating possible health benefits using self-reported methods. Keywords: ‘Self-Reported Health’, Attitudes, ‘Workplace Intervention’, ‘Public Health Intervention

Effect of acute hypoxia on respiratory muscle fatigue in healthy humans

<p>Abstract</p> <p>Background</p> <p>Greater diaphragm fatigue has been reported after hypoxic versus normoxic exercise, but whether this is due to increased ventilation and therefore work of breathing or reduced blood oxygenation per se remains unclear. Hence, we assessed the effect of different blood oxygenation level on isolated hyperpnoea-induced inspiratory and expiratory muscle fatigue.</p> <p>Methods</p> <p>Twelve healthy males performed three 15-min isocapnic hyperpnoea tests (85% of maximum voluntary ventilation with controlled breathing pattern) in normoxic, hypoxic (SpO₂= 80%) and hyperoxic (FiO₂= 0.60) conditions, in a random order. Before, immediately after and 30 min after hyperpnoea, transdiaphragmatic pressure (P_di,tw) was measured during cervical magnetic stimulation to assess diaphragm contractility, and gastric pressure (P_ga,tw) was measured during thoracic magnetic stimulation to assess abdominal muscle contractility. Two-way analysis of variance (time x condition) was used to compare hyperpnoea-induced respiratory muscle fatigue between conditions.</p> <p>Results</p> <p>Hypoxia enhanced hyperpnoea-induced P_di,twand P_ga,twreductions both immediately after hyperpnoea (P_di,tw: normoxia -22 ± 7% vs hypoxia -34 ± 8% vs hyperoxia -21 ± 8%; P_ga,tw: normoxia -17 ± 7% vs hypoxia -26 ± 10% vs hyperoxia -16 ± 11%; all <it>P </it>< 0.05) and after 30 min of recovery (P_di,tw: normoxia -10 ± 7% vs hypoxia -16 ± 8% vs hyperoxia -8 ± 7%; P_ga,tw: normoxia -13 ± 6% vs hypoxia -21 ± 9% vs hyperoxia -12 ± 12%; all <it>P </it>< 0.05). No significant difference in P_di,twor P_ga,twreductions was observed between normoxic and hyperoxic conditions. Also, heart rate and blood lactate concentration during hyperpnoea were higher in hypoxia compared to normoxia and hyperoxia.</p> <p>Conclusions</p> <p>These results demonstrate that hypoxia exacerbates both diaphragm and abdominal muscle fatigability. These results emphasize the potential role of respiratory muscle fatigue in exercise performance limitation under conditions coupling increased work of breathing and reduced O₂transport as during exercise in altitude or in hypoxemic patients.</p

Reduced Stability and Increased Dynamics in the Human Proliferating Cell Nuclear Antigen (PCNA) Relative to the Yeast Homolog

Proliferating Cell Nuclear Antigen (PCNA) is an essential factor for DNA replication and repair. PCNA forms a toroidal, ring shaped structure of 90 kDa by the symmetric association of three identical monomers. The ring encircles the DNA and acts as a platform where polymerases and other proteins dock to carry out different DNA metabolic processes. The amino acid sequence of human PCNA is 35% identical to the yeast homolog, and the two proteins have the same 3D crystal structure. In this report, we give evidence that the budding yeast (sc) and human (h) PCNAs have highly similar structures in solution but differ substantially in their stability and dynamics. hPCNA is less resistant to chemical and thermal denaturation and displays lower cooperativity of unfolding as compared to scPCNA. Solvent exchange rates measurements show that the slowest exchanging backbone amides are at the β-sheet, in the structure core, and not at the helices, which line the central channel. However, all the backbone amides of hPCNA exchange fast, becoming undetectable within hours, while the signals from the core amides of scPCNA persist for longer times. The high dynamics of the α-helices, which face the DNA in the PCNA-loaded form, is likely to have functional implications for the sliding of the PCNA ring on the DNA since a large hole with a flexible wall facilitates the establishment of protein-DNA interactions that are transient and easily broken. The increased dynamics of hPCNA relative to scPCNA may allow it to acquire multiple induced conformations upon binding to its substrates enlarging its binding diversity

A strong 13C chemical shift signature provides the coordination mode of histidines in zinc-binding proteins

International audienceZinc is the second most abundant metal ion incorporated in proteins, and is in many cases a crucial component of protein three-dimensional structures. Zinc ions are frequently coordinated by cysteine and histidine residues. Whereas cysteines bind to zinc via their unique Sγ atom, histidines can coordinate zinc with two different coordination modes, either Nδ1 or Nε2 is coordinating the zinc ion. The determination of this coordination mode is crucial for the accurate structure determination of a histidine-containing zinc-binding site by NMR. NMR chemical shifts contain a vast amount of information on local electronic and structural environments and surprisingly their utilization for the determination of the coordination mode of zinc-ligated histidines has been limited so far to 15N nuclei. In the present report, we observed that the 13C chemical shifts of aromatic carbons in zinc-ligated histidines represent a reliable signature of their coordination mode. Using a statistical analysis of 13C chemical shifts, we show that 13Cδ2 chemical shift is sensitive to the histidine coordination mode and that the chemical shift difference δ{13Cε1} - δ{13Cδ2} provides a reference-independent marker of this coordination mode. The present approach allows the direct determination of the coordination mode of zinc-ligated histidines even with non-isotopically enriched protein samples and without any prior structural information