Cryo-EM structure of CtBP2 confirms tetrameric architecture

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Modeling the acute- and late-phase responses to peripheral airway cooling and desiccation.

Acute bronchoconstriction after isocapnic hyperpnea can be produced in most asthmatic individuals. However, the existence of a late-phase response is less certain. We used a canine model of isocapnic hyperpnea to test the hypothesis that this discrepancy is due to differences in the challenge threshold for the responses. Acute-phase and late-phase bronchoconstriction was measured in nine dogs after peripheral airway exposure to unconditioned air. Additionally, bronchoalveolar lavage fluid (BALF) was obtained during the late-phase response. The acute-phase response was a polynomial function with a decreasing slope at higher challenges, whereas the late-phase response suggested that a minimum threshold of challenge severity was needed to produce late-phase bronchoconstriction. BALF leukocyte and eicosanoid concentrations had linear relationships with challenge severity. Our data support the hypothesis that acute- and late-phase posthyperpnea responses have different dose-response relationships, a fact that may explain the frequent lack of a late-phase response. However, our data suggest that mild inflammation can be induced with relatively lower challenge severity.     

Oligomerization of the EK18 mutant of the trp repressor of Escherichia coli as observed by NMR spectroscopy.

The regulation of the trp repressor system of Escherichia coli is frequently modeled by a single equilibrium, that between the aporepressor (TR) and the corepressor, l-tryptophan (Trp), at their intracellular concentrations. The actual mechanism, which is much more complex and more finely tuned, involves multiple equilibria: TR and Trp association, TR oligomerization, specific and nonspecific binding of various states of TR to DNA, and interactions between these various species and ions. TR in isolation exists primarily as a homodimer, but the state of oligomerization increases as the TR concentration goes up and/or the salt concentration goes down, leading to species with lower affinity for DNA. We have used multinuclear, multidimensional NMR spectroscopy to investigate structural changes that accompany the oligomerization of TR. For these investigations, the superrepressor mutant EK18 (TR with Glu 18 replaced by Lys) was chosen because it exhibits less severe oligomerization at higher protein concentration than other known variants; this made it possible to study the dimer to tetramer oligomerization step by NMR. The NMR results suggest that the interaction between TR dimers is structurally linked to folding of the DNA binding domain and that it likely involves direct contacts between the C-terminal residues of the C-helix of one dimer with the next dimer. This implies that oligomerization can compete with DNA binding and thus serves as a factor in the fine-tuning of gene expression.     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.