Chemotaxonomic characterisation of the thaumarchaeal lipidome

Thaumarchaeota are globally distributed and abundant microorganisms occurring in diverse habitats and thus represent a major source of archaeal lipids. The scope of lipids as taxonomic markers in microbial ecological studies is limited by the scarcity of comparative data on the membrane lipid composition of cultivated representatives, including the phylum Thaumarchaeota. Here, we comprehensively describe the core and intact polar lipid (IPL) inventory of ten ammonia‐oxidising thaumarchaeal cultures representing all four characterized phylogenetic clades. IPLs of these thaumarchaeal strains are generally similar and consist of membrane‐spanning, glycerol dibiphytanyl glycerol tetraethers with monoglycosyl, diglycosyl, phosphohexose and hexose‐phosphohexose headgroups. However, the relative abundances of these IPLs and their core lipid compositions differ systematically between the phylogenetic subgroups, indicating high potential for chemotaxonomic distinction of thaumarchaeal clades. Comparative lipidomic analyses of 19 euryarchaeal and crenarchaeal strains suggested that the lipid methoxy archaeol is synthesized exclusively by Thaumarchaeota and may thus represent a diagnostic lipid biomarker for this phylum. The unprecedented diversity of the thaumarchaeal lipidome with 118 different lipids suggests that membrane lipid composition and adaptation mechanisms in Thaumarchaeota are more complex than previously thought and include unique lipids with as yet unresolved properties.     

Design and synthesis of 2-amino-isoxazolopyridines as Polo-like kinase inhibitors

A series of 2-amino-isoxazolopyridines was designed and synthesized as Polo-like kinase (Plk) inhibitors. Key SAR and crystallographic data are discussed. More advanced analogues inhibit Plk1 with good enzymatic activity and modest cell-based activity. Differential selectivity among the three Plk isoforms is observed.     

UDP-N-Acetyl-α-D-glucosamine as acceptor substrate of β-1,4-galactosyltransferase. Enzymatic synthesis of UDP-N-acetyllactosamine

The capacity of UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for beta-1,4-galactosyltransferase (beta4GalT1, EC 2.4.1.38) from human and bovine milk and for recombinant human beta4GalT1, expressed in Saccharomyces cerevisiae, was evaluated. It turned out that each of the enzymes is capable to transfer Gal from UDP-alpha-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(beta1-4)GlcNAc(alpha1-UDP (UDP-LacNAc). Using beta4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Studies with all three beta4GalTs in the presence of alpha-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-alpha-D-glucose is not an acceptor substrate. This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate. Interestingly, in these studies beta4GalT1 accepts an alpha-glycosidated GlcNAc derivative. The results imply that beta4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk.     

Mouse Aurora A: expression in Escherichia coli and purification

Aurora kinases have recently become some of the most intensely pursued oncology targets for the design of small-molecule inhibitors. Most of the active Aurora-A protein variants are currently being expressed from baculoviruses in insect cells, while catalytically impaired proteins can also be generated in and purified from Escherichia coli. In this study we present a method of expressing large quantities of active mouse Aurora-A kinase domain as an N-terminal glutathione-S-transferase fusion protein in bacteria and outline a simple purification method that produces greater than 99% pure protein samples suitable for enzymatic assays and X-ray crystallography. The methods described in this report simplify mouse Aurora-A expression and purification, and may aid in the production of other difficult kinases in prokaryotes.     

Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2

Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium ([Ca(2+)](c)) is a potent inducer of macroautophagy. Various Ca(2+) mobilizing agents (vitamin D(3) compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca(2+)/calmodulin-dependent kinase kinase-beta and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the [Ca(2+)](ER) and attenuates agonist-induced Ca(2+) fluxes. Thus, an increase in the [Ca(2+)](c) serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2.     

Satellog: A database for the identification and prioritization of satellite repeats in disease association studies

Background  

To date, 35 human diseases, some of which also exhibit anticipation, have been associated with unstable repeats. Anticipation has been reported in a number of diseases in which repeat expansion may have a role in etiology. Despite the growing importance of unstable repeats in disease, currently no resource exists for the prioritization of repeats. Here we present Satellog, a database that catalogs all pure 1–16 repeat unit satellite repeats in the human genome along with supplementary data. Satellog analyzes each pure repeat in UniGene clusters for evidence of repeat polymorphism.