Conformational Selection and Substrate Binding Regulate the Monomer/Dimer Equilibrium of the C-terminal domain of Escherichia coli Enzyme I

The bacterial phosphotransferase system (PTS) is a signal transduction pathway that couples phosphoryl transfer to active sugar transport across the cell membrane. The PTS is initiated by the binding of phosphoenolpyruvate (PEP) to the C-terminal domain (EIC) of enzyme I (EI), a highly conserved protein that is common to all sugar branches of the PTS. EIC exists in a dynamic monomer/dimer equilibrium that is modulated by ligand binding and is thought to regulate the overall PTS. Isolation of EIC has proven challenging, and conformational dynamics within the EIC domain during the catalytic cycle are still largely unknown. Here, we present a robust protocol for expression and purification of recombinant EIC from Escherichia coli and show that isolated EIC is capable of hydrolyzing PEP. NMR analysis and residual dipolar coupling measurements indicate that the isolated EIC domain in solution adopts a stable tertiary fold and quaternary structure that is consistent with previously reported crystallographic data. NMR relaxation dispersion measurements indicate that residues around the PEP binding site and in the β3α3 turn (residues 333-366), which is located at the dimer interface, undergo a rapid transition on the sub-millisecond time scale (with an exchange rate constant of ～1500 s(-1)) between major open (～97%) and minor closed (～3%) conformations. Upon PEP binding, the β3α3 turn is effectively locked in the closed state by the formation of salt bridges between the phosphate group of PEP and the side chains of Lys(340) and Arg(358), thereby stabilizing the dimer.     

Relation of left ventricular mass to QTc in normotensive severely obese patients

Prolongation of the corrected QT interval (QTc) has been described in obese subjects. This study assesses the relation of left ventricular (LV) mass to QTc in normotensive severely obese subjects. Fifty normotensive patients whose BMI was ≥40 kg/m(2) (mean age: 38 ± 7 years) were studied. QTc was derived using Bazett's formula. LV mass was calculated using the formula of Devereux et al. and was indexed to height(2.7). Mean QTc was 428.8 ± 19.0 ms and was significantly longer in those with than in those without LV hypertrophy (P < 0.01) QTc correlated positively and significantly with BMI (r = 0.392, P < 0.025), LV mass/height(2.7) (r = 0.793, P < 0.0005), systolic blood pressure (r = 0.742, P < 0.001), LV end - systolic wall stress (r = 0.746, P < 0.001) and LV internal dimension in diastole (r = 0.788, P < 0.0005). Among five variables tested, LV mass/height(2.7) was identified as the sole predictor of QTc by multivariate analysis. In conclusion, LV mass and loading conditions that may affect LV mass are important determinants of QTc in normotensive severely obese subjects.     

Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy

We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-'visible' free species and an NMR-'invisible' species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. (15)N-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (ΔR(2)) in (15)N transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The (15)N-DEST and ΔR(2) data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-β (Aβ) at concentrations of ~100 μM.     

Small‐plot studies comparing pheromone and juice baits for mass‐trapping invasive Synanthedon myopaeformis in Canada

Recent introduction of Synanthedon myopaeformis (Borkhausen) (Lepidoptera: Sesiidae) into organic apple‐growing areas of Canada has stimulated research on semiochemical‐based management of this European pest. Replicated, small‐plot (0.16 ha) experiments were conducted to compare sex pheromone, 3Z,13Z‐octadecadienyl acetate (10 mg), Concord grape juice (300 ml), or their combination, as mass‐trapping lures at trap densities equivalent to 12.5, 25, 50, and 100 traps ha^?1. Total numbers of male and female moths removed from test plots increased significantly with trap density in all juice‐based mass‐trapping experiments. In pheromone mass‐trapping experiments, however, total catches of males did not increase significantly as trap densities were increased and catches appeared to plateau with 25–50 traps ha^?1. With pheromone‐based mass‐trapping, significantly fewer males were caught in pheromone‐baited assessment traps at the centre of each mass‐trapping plot than in identical traps in untreated plots. This reduction is indicative of significant trap interference or trap ‘shut‐down’. Increasing the density of juice‐based mass‐trapping had no effect on catches of male or female moths in juice‐baited assessment traps, indicating a short range of attraction and lack of interference between juice traps. Pheromone‐ and juice‐based mass trapping removed similar numbers of males at each trap density tested, respectively, but summed catches of males and females were greatest with juice baits. Combining pheromone and juice into a single mass‐trapping treatment (50 traps ha^?1) did not significantly increase catches of males or females relative to either treatment alone. If a practical bisexual mass‐trapping system is going to be developed for S. myopaeformis, then identification of volatile kairomones in Concord grape juice may be useful.     

Identification of a Common Non-Apoptotic Cell Death Mechanism in Hereditary Retinal Degeneration

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.     

Development and laboratory-scale testing of a fully automated online flow cytometer for drinking water analysis

Accurate and sensitive online detection tools would benefit both fundamental research and practical applications in aquatic microbiology. Here, we describe the development and testing of an online flow cytometer (FCM), with a specific use foreseen in the field of drinking water microbiology. The system incorporated fully automated sampling and fluorescent labeling of bacterial nucleic acids with analysis at 5-min intervals for periods in excess of 24 h. The laboratory scale testing showed sensitive detection (< 5% error) of bacteria over a broad concentration range (1 × 10(3) -1 × 10(6) cells mL(-1) ) and particularly the ability to track both gradual changes and dramatic events in water samples. The system was tested with bacterial pure cultures as well as indigenous microbial communities from natural water samples. Moreover, we demonstrated the possibility of using either a single fluorescent dye (e.g., SYBR Green I) or a combination of two dyes (SYBR Green I and Propidium Iodide), thus broadening the application possibilities of the system. The online FCM approach described herein has considerable potential for routine and continuous monitoring of drinking water, optimization of specific drinking water processes such as biofiltration or disinfection, as well as aquatic microbiology research in general.     

High-resolution crystal structures of transient intermediates in the phytochrome photocycle

1. Download : Download high-res image (234KB)
2. Download : Download full-size image

     

High-precision coding in visual cortex

1. Download : Download high-res image (195KB)
2. Download : Download full-size image