Banana (Musa spp.) as a model to study the meristem proteome: acclimation to osmotic stress

Banana (Musa spp.) multiple shoot meristems are an excellent model to study the meristem proteome. Using a 2-DE protocol developed for small amounts of tissue and MS-based cross species polypeptide identification, we have revealed the meristem proteome and investigated the influence of sucrose-mediated osmotic stress in a dehydration-tolerant variety. Proteins that were significantly up- or down-regulated due to the high-sucrose treatment were classified using non-parametric univariate statistics. Our results suggest that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energy-conserving glycolysis and the conservation of the cell wall integrity are essential to maintain homeostasis, to acclimate and to survive dehydration. By comparing the dehydration-tolerant variety with a dehydration-sensitive variety, we were able to distinguish several genotype-specific proteins (isoforms), and could associate the dehydration-tolerant variety with proteins involved in energy metabolism (e.g., phosphoglycerate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase) and proteins that are associated with stress adaptation (e.g., OSR40-like protein, abscisic stress ripening protein-like protein). This work shows that proteome analysis can be used successfully to perform quantitative difference analysis and to characterize genetic variations in a recalcitrant crop.     

Overview of cell shape: cytoskeletons shape bacterial cells

An evolving hypothesis is that bacterial cell shape is determined by cytoskeletal elements that localize peptidoglycan synthetic machineries. In most bacteria FtsZ assembles into the Z ring which recruits the machinery necessary for cytokinesis. Most rod shaped cells require MreB which assembles into cables that run between the poles of the cell and distribute various components of peptidoglycan metabolism along the cell length. Cells with other shapes have additional cytoskeletal elements that either localize synthetic machineries or possibly influence their activity.     

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K⁺ current, I_Kr is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I_Kr block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC₇₀ of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC₅₀ of dofetilide from 38.7 to 76.3 nm. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.     

ERGOSTEROL BIOSYNTHESIS: A FUNGAL PATHWAY FOR LIFE ON LAND?

Sterols, essential lipids of most eukaryotic cells, ensure important structural and signaling functions. The selection pressure that has led to different dominant sterols in the three eukaryotic kingdoms remains unknown. Here, we investigated the influence of the progression in the different steps of the ergosterol biosynthetic pathway (EBP) on the yeast resistance to transitions from aqueous to aerial media, typical perturbations of the higher fungi habitats. Five mutants of the EBP (ergΔ), accumulating different sterol intermediates in the EBP, and the wild‐type (WT) strain were exposed to drying under atmospheric air or nitrogen and wetting. Results show that the progression in the EBP parallels an increase in the yeast resistance to air‐drying with a maximal survival rate for the WT strain. When drying/wetting was performed under nitrogen, yeast survival was higher, particularly for the earlier mutants of the EBP. Thus, ergosterol, through its protective role against mechanical and oxidative stress, might have been selected by the pressure induced by drying/wetting cycles occurring in the fungi habitats. These results support the Bloch hypothesis, which postulates that the properties of sterols are gradually optimized for function along the biosynthetic pathway and provide a response to the enduring question “why ergosterol in fungi?”.     

The Exosome Secretory Pathway Transports Amyloid Precursor Protein Carboxyl-terminal Fragments from the Cell into the Brain Extracellular Space

In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-β precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-β (Aβ). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphology were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were observed in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the number of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs associated with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and Aβ, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted Aβ, is harmful to the brain.     

4-Phenyl-7-azaindoles as potent, selective and bioavailable IKK2 inhibitors demonstrating good in vivo efficacy

The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent and selective IKK2 inhibitor showing good efficacy in a rat model of neutrophil activation.     

Synthesis and biological characterisation of sirtuin inhibitors based on the tenovins

The tenovins are small molecule inhibitors of the NAD(+)-dependent family of protein deacetylases known as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible applications in both cancer and neurodegenerative disease therapy. Through the synthesis of novel tenovin analogues, further insights into the structural requirements for activity against the sirtuins in vitro are provided. In addition, the activity of one of the analogues in cells led to an improved understanding of the function of SirT1 in cells.