Activation of nicotinic cholinergic receptors prevents ventilator-induced lung injury in rats

Respiratory distress syndrome is responsible for 40 to 60 percent mortality. An over mortality of about 10 percent could result from additional lung injury and inflammation due to the life-support mechanical ventilation, which stretches the lung. It has been recently demonstrated, in vitro, that pharmacological activation of the alpha 7 nicotinic receptors (α7-nAChR) could down regulate intracellular mediators involved in lung cell inflammatory response to stretch. Our aim was to test in vivo the protective effect of the pharmacological activation of the α7-nAChR against ventilator-induced lung injury (VILI). Anesthetized rats were ventilated for two hours with a high stretch ventilation mode delivering a stroke volume large enough to generate 25-cmH(2)O airway pressure, and randomly assigned to four groups: pretreated with parenteral injection of saline or specific agonist of the α7-nAChR (PNU-282987), or submitted to bilateral vagus nerve electrostimulation while pre-treated or not with the α7-nAChR antagonist methyllycaconitine (MLA). Controls ventilated with a conventional stroke volume of 10 mL/kg gave reference data. Physiological indices (compliance of the respiratory system, lung weight, blood oxygenation, arterial blood pressure) and lung contents of inflammatory mediators (IL-6 measured by ELISA, substance P assessed using HPLC) were severely impaired after two hours of high stretch ventilation (sham group). Vagal stimulation was able to maintain the respiratory parameters close to those obtained in Controls and reduced lung inflammation except when associated to nicotinic receptor blockade (MLA), suggesting the involvement of α7-nAChR in vagally-mediated protection against VILI. Pharmacological pre-treatment with PNU-282987 strongly decreased lung injury and lung IL-6 and substance P contents, and nearly abolished the increase in plasmatic IL-6 levels. Pathological examination of the lungs confirmed the physiological differences observed between the groups. In conclusion, these data suggest that the stimulation of α7-nAChR is able to attenuate VILI in rats.     

Intraspecific variability in leaf traits strongly affects alder leaf decomposition in a stream

This study assessed the intraspecific variability of senescent leaves of alder (Alnus glutinosa Gaertn.) and the effects of this variability on leaf decomposition in streams. Leaves were collected at five geographically distant locations in Europe. We analyzed 10 batches of leaf samples for seven quantitative leaf traits as well as leaf decomposition rate in coarse and fine mesh bags exposed in a single stream. The geographic origin of leaf samples largely explained the observed variation in litter quality and decomposition rate. Phosphorus (0.034–0.187%) and lignin (3.9–18.7%) concentrations in leaves varied widely. Together, these two traits accurately predicted leaf decomposition rate (r²=84.1%). Intraspecific variation in leaf decomposition rate was within a range similar to that reported for interspecific variation among co-occurring riparian plant species in Europe. Our study demonstrates extensive intraspecific variability in leaf traits on a continental scale, which can have enormous effects on major ecosystem processes such as leaf decomposition.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Open interface and large quaternary structure movements in 3D domain swapped proteins: insights from molecular dynamics simulations of the C-terminal swapped dimer of ribonuclease A

Bovine pancreatic ribonuclease (RNase A) forms two three-dimensional (3D) domain swapped dimers. Crystallographic investigations have revealed that these dimers display completely different quaternary structures: one dimer (N-dimer), which presents the swapping of the N-terminal helix, is characterized by a compact structure, whereas the other (C-dimer), which is stabilized by the exchange of the C-terminal end, shows a rather loose assembly of the two subunits. The dynamic properties of monomeric RNase A and of the N-dimer have been extensively characterized. Here, we report a molecular dynamics investigation carried out on the C-dimer. This computational experiment indicates that the quaternary structure of the C-dimer undergoes large fluctuations. These motions do not perturb the proper folding of the two subunits, which retain the dynamic properties of RNase A and the N-dimer. Indeed, the individual subunits of the C-dimer display the breathing motion of the beta-sheet structure, which is important for the enzymatic activity of pancreatic-like ribonucleases. In contrast to what has been observed for the N-dimer, the breathing motion of the two subunits of the C-dimer is not coupled. This finding suggests that the intersubunit communications in a 3D domain swapped dimer strongly rely on the extent of the interchain interface. Furthermore, the observation that the C-dimer is endowed with a high intrinsic flexibility holds interesting implications for the specific properties of 3D domain swapped dimers. Indeed, a survey of the quaternary structures of the other 3D domain swapped dimers shows that large variations are often observed when the structural determinations are conducted in different experimental conditions. The 3D domain swapping phenomenon coupled with the high flexibility of the quaternary structure may be relevant for protein-protein recognition, and in particular for the pathological aggregations.     

Cloning and over-expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermalis. Characterization and comparison of the native and the recombinant enzymes.

A NADP-dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 degrees C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over-expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH-dependent, being a 197-kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5-kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C2 to C8 but is also active towards methanol and glycerol and stereospecific for monoterpenes. T. hydrothermalis ADH is the first Thermococcale ADH to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics.     

The secE gene of Helicobacter pylori

Despite extensive annotation by two independent teams, the Helicobacter pylori genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing secE component of the major secretion machinery of Helicobacter pylori.     

CD16 promotes Escherichia coli sepsis through an FcR gamma inhibitory pathway that prevents phagocytosis and facilitates inflammation

Sepsis, a leading cause of death worldwide, involves proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here we show that the FcRgamma adaptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing signal transduction subunit of the Fc receptor family, has a deleterious effect on sepsis. FcRgamma(-/-) mice show increased survival during peritonitis, owing to markedly increased E. coli phagocytosis and killing and to lower production of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The FcRgamma-associated receptor that inhibits E. coli phagocytosis is FcgammaRIII (also called CD16), and its absence protects mice from sepsis. FcgammaRIII binds E. coli, and this interaction induces FcRgamma phosphorylation, recruitment of the tyrosine phosphatase SHP-1 and phosphatidylinositide-3 kinase (PI3K) dephosphorylation. Decreased PI3K activity inhibits E. coli phagocytosis and increases TNF-alpha production through Toll-like receptor 4. We identified the phagocytic receptor negatively regulated by FcRgamma on macrophages as the class A scavenger receptor MARCO. E. coli-FcgammaRIII interaction induces the recruitment of SHP-1 to MARCO, thereby inhibiting E. coli phagocytosis. Thus, by binding FcgammaRIII, E. coli triggers an inhibitory FcRgamma pathway that both impairs MARCO-mediated bacterial clearance and activates TNF-alpha secretion.     

Reversible inactivation of the transcriptional function of P53 protein by farnesylation

Background  

The use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function.