Phospholipid chain immobilization and steriod rotational immobilization in acetylcholine receptor-rich membranes from torpedo marmorata

1. The ESR spectra of both phosphatidylcholine and phosphatidylethanolamine spin labels reveal an immobilized lipid component (τ_R ? 50 ns), in addition to a fluid component (τ_R ～ 1 ns), in acetylcholine receptorrich membranes prepared from Torpedo marmorata electroplax according to the method of Cohen et al. (Cohen, J.B., Weber, M., Huchet, M. and Changeux, J.P. (1972) FEBS Lett. 26, 43–47). 2. The ESR spectra of the androstanol spin label display a component corresponding to molecules which are immobilized with respect to rotation about the long molecular axis (

), in addition to the fluid lipid bilayer component in which the molecules are rotating rapidly about their long axes (

). This immobilized component is observed throughout the temperature range 2–22°C, at an approximately constant relative intensity of approx. 45% of the total, which is quantitatively the same as previously observed with fatty acid spin labels.     

Characterization of hydrogen peroxide production by Duox in bronchial epithelial cells exposed to Pseudomonas aeruginosa

Hydrogen peroxide production by the NADPH oxidase Duox1 occurs during activation of respiratory epithelial cells stimulated by purified bacterial ligands, such as lipopolysaccharide. Here, we characterize Duox activation using intact bacterial cells of several airway pathogens. We found that only Pseudomonas aeruginosa, not Burkholderia cepacia or Staphylococcus aureus, triggers H₂O₂ production in bronchial epithelial cells in a calcium-dependent but predominantly ATP-independent manner. Moreover, by comparing mutant Pseudomonas strains, we identify several virulence factors that participate in Duox activation, including the type-three secretion system. These data provide insight on Duox activation by mechanisms unique to P. aeruginosa.     

The psychoactive substance of cannabis Δ9-tetrahydrocannabinol (THC) negatively regulates CFTR in airway cells

Background

Marijuana consumption is on the rise in the US but the health benefits of cannabis smoking are controversial and the impact of cannabis components on lung homeostasis is not well-understood. Lung function requires a fine regulation of the ion channel CFTR, which is responsible for fluid homeostasis and mucocilliary clearance. The goal of this study was to assess the effect that exposure to Δ9-tetrahydrocannabinol (THC), the psychoactive substance present in marijuana, has on CFTR expression and function.

The Role of L1 Stalk-tRNA Interaction in the Ribosome Elongation Cycle

The ribosomal L1 stalk is a mobile structure implicated in directing tRNA movement during translocation through the ribosome. This article investigates three aspects of L1 stalk-tRNA interaction. First, by combining data from cryo electron microscopy, X-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different tRNAs occupying the hybrid P/E state interacting with the L1 stalk. These models confirm the assignment of fluorescence resonance energy transfer states from previous single-molecule investigations of L1 stalk dynamics. Second, the models reconcile how initiator tRNA^fMet interacts less strongly with the L1 stalk compared to elongator tRNA^Phe, as seen in previous single-molecule experiments. Third, results from a simulation of the entire ribosome in which the L1 stalk is moved from a half-closed conformation to its open conformation are found to support the hypothesis that L1 stalk opening is involved in tRNA release from the ribosome.     

Preferences of AAA/AAG codon recognition by modified nucleosides, τm5s2U34 and t6A37 present in tRNALys

Deficiency of 5-taurinomethyl-2-thiouridine, τm⁵s²U at the 34th ‘wobble’ position in tRNA^Lys causes MERRF (Myoclonic Epilepsy with Ragged Red Fibers), a neuromuscular disease. This modified nucleoside of mt tRNA^Lys, recognizes AAA/AAG codons during protein biosynthesis process. Its preference to identify cognate codons has not been studied at the atomic level. Hence, multiple MD simulations of various molecular models of anticodon stem loop (ASL) of mt tRNA^Lys in presence and absence of τm⁵s²U₃₄ and N⁶-threonylcarbamoyl adenosine (t⁶A₃₇) along with AAA and AAG codons have been accomplished. Additional four MD simulations of multiple ASL mt tRNA^Lys models in the context of ribosomal A-site residues have also been performed to investigate the role of A-site in recognition of AAA/AAG codons. MD simulation results show that, ASL models in presence of τm⁵s²U₃₄ and t⁶A₃₇ with codons AAA/AAG are more stable than the ASL lacking these modified bases. MD trajectories suggest that τm⁵s²U recognizes the codons initially by ‘wobble’ hydrogen bonding interactions, and then tRNA^Lys might leave the explicit codon by a novel ‘single’ hydrogen bonding interaction in order to run the protein biosynthesis process smoothly. We propose this model as the ‘Foot-Step Model’ for codon recognition, in which the single hydrogen bond plays a crucial role. MD simulation results suggest that, tRNA^Lys with τm⁵s²U and t⁶A recognizes AAA codon more preferably than AAG. Thus, these results reveal the consequences of τm⁵s²U and t⁶A in recognition of AAA/AAG codons in mitochondrial disease, MERRF.     

Efficiency of pretreatments for optimal enzymatic saccharification of soybean fiber

The effectiveness of several pretreatments [high-power ultrasound, sulfuric acid (H₂SO₄), sodium hydroxide (NaOH), and ammonium hydroxide (NH₃OH)] to enhance glucose production from insoluble fractions recovered from enzyme-assisted aqueous extraction processing of extruded full-fat soybean flakes (FFSF) was investigated. Sonication of the insoluble fraction at 144 μm_{pp (peak-to-peak)} for 30 and 60 s did not improve the saccharification yield. The solid fractions recovered after pretreatment with H₂SO₄ [1% (w/w), 90 °C, 1.5 h], NaOH [15% (w/w), 65 °C, 17 h], and NH₃OH [15% (w/w), 65 °C, 17 h] showed significant lignin degradation, i.e., 81.9%, 71.2%, and 75.4%, respectively, when compared to the control (7.4%). NH₃OH pretreatment resulted in the highest saccharification yield (63%) after 48 h of enzymatic saccharification. A treatment combining the extraction and saccharification steps and applied directly to the extruded FFSF, where oil extraction yield and saccharification yield reached 98% and 43%, respectively, was identified.     

The lactoperoxidase system links anion transport to host defense in cystic fibrosis

Chronic respiratory infections in cystic fibrosis result from CFTR channel mutations but how these impair antibacterial defense is less clear. Airway host defense depends on lactoperoxidase (LPO) that requires thiocyanate (SCN-) to function and epithelia use CFTR to concentrate SCN- at the apical surface. To test whether CFTR mutations result in impaired LPO-mediated host defense, CF epithelial SCN- transport was measured. CF epithelia had significantly lower transport rates and did not accumulate SCN- in the apical compartment. The lower CF [SCN-] did not support LPO antibacterial activity. Modeling of airway LPO activity suggested that reduced transport impairs LPO-mediated defense and cannot be compensated by LPO or H2O2 upregulation.