@article{802, abstract = {Glycoinositolphosphoceramides (GIPCs) are complex sphingolipids present at the plasma membrane of various eukaryotes with the important exception of mammals. In fungi, these glycosphingolipids commonly contain an alpha-mannose residue (Man) linked at position 2 of the inositol. However, several pathogenic fungi additionally synthesize zwitterionic GIPCs carrying an alpha-glucosamine residue (GlcN) at this position. In the human pathogen Aspergillus fumigatus, the GlcNalpha1,2IPC core (where IPC is inositolphosphoceramide) is elongated to Manalpha1,3Manalpha1,6GlcNalpha1,2IPC, which is the most abundant GIPC synthesized by this fungus. In this study, we identified an A. fumigatus N-acetylglucosaminyltransferase, named GntA, and demonstrate its involvement in the initiation of zwitterionic GIPC biosynthesis. Targeted deletion of the gene encoding GntA in A. fumigatus resulted in complete absence of zwitterionic GIPC; a phenotype that could be reverted by episomal expression of GntA in the mutant. The N-acetylhexosaminyltransferase activity of GntA was substantiated by production of N-acetylhexosamine-IPC in the yeast Saccharomyces cerevisiae upon GntA expression. Using an in vitro assay, GntA was furthermore shown to use UDP-N-acetylglucosamine as donor substrate to generate a glycolipid product resistant to saponification and to digestion by phosphatidylinositol-phospholipase C as expected for GlcNAcalpha1,2IPC. Finally, as the enzymes involved in mannosylation of IPC, GntA was localized to the Golgi apparatus, the site of IPC synthesis.}, author = {Engel, Jakob and Schmalhorst, Philipp S and Kruger, Anke and Muller, Christina and Buettner, Falk and Routier, Françoise}, journal = {Glycobiology}, number = {12}, pages = {1423 -- 1430}, publisher = {Oxford University Press}, title = {{Characterization of an N-acetylglucosaminyltransferase involved in Aspergillus fumigatus zwitterionic glycoinositolphosphoceramide biosynthesis}}, doi = {10.1093/glycob/cwv059}, volume = {25}, year = {2015}, } @article{815, abstract = {The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason- Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ~8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. }, author = {Schur, Florian and Dick, Robert and Hagen, Wim and Vogt, Volker and Briggs, John}, journal = {Journal of Virology}, number = {20}, pages = {10294 -- 10302}, publisher = {ASM}, title = {{The structure of immature virus like Rous sarcoma virus gag particles reveals a structural role for the p10 domain in assembly}}, doi = {10.1128/JVI.01502-15}, volume = {89}, year = {2015}, } @article{814, abstract = {Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.}, author = {Florian Schur and Hagen, Wim J and Rumlová, Michaela and Ruml, Tomáš and Müller B and Kraüsslich, Hans Georg and Briggs, John A}, journal = {Nature}, number = {7535}, pages = {505 -- 508}, publisher = {Nature Publishing Group}, title = {{Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution}}, doi = {10.1038/nature13838}, volume = {517}, year = {2015}, } @article{8242, author = {Einhorn, Lukas and Fazekas, Judit and Muhr, Martina and Schoos, Alexandra and Oida, Kumiko and Singer, Josef and Panakova, Lucia and Manzano-Szalai, Krisztina and Jensen-Jarolim, Erika}, issn = {0091-6749}, journal = {Journal of Allergy and Clinical Immunology}, number = {2}, publisher = {Elsevier}, title = {{Generation of recombinant FcεRIα of dog, cat and horse for component-resolved allergy diagnosis in veterinary patients}}, doi = {10.1016/j.jaci.2014.12.1263}, volume = {135}, year = {2015}, } @article{832, abstract = {Plants maintain capacity to form new organs such as leaves, flowers, lateral shoots and roots throughout their postembryonic lifetime. Lateral roots (LRs) originate from a few pericycle cells that acquire attributes of founder cells (FCs), undergo series of anticlinal divisions, and give rise to a few short initial cells. After initiation, coordinated cell division and differentiation occur, giving rise to lateral root primordia (LRP). Primordia continue to grow, emerge through the cortex and epidermal layers of the primary root, and finally a new apical meristem is established taking over the responsibility for growth of mature lateral roots [for detailed description of the individual stages of lateral root organogenesis see Malamy and Benfey (1997)]. To examine this highly dynamic developmental process and to investigate a role of various hormonal, genetic and environmental factors in the regulation of lateral root organogenesis, the real time imaging based analyses represent extremely powerful tools (Laskowski et al., 2008; De Smet et al., 2012; Marhavy et al., 2013 and 2014). Herein, we describe a protocol for real time lateral root primordia (LRP) analysis, which enables the monitoring of an onset of the specific gene expression and subcellular protein localization during primordia organogenesis, as well as the evaluation of the impact of genetic and environmental perturbations on LRP organogenesis.}, author = {Peter Marhavy and Eva Benková}, journal = {Bio-protocol}, number = {8}, publisher = {Bio-protocol LLC}, title = {{Real time analysis of lateral root organogenesis in arabidopsis}}, doi = {10.21769/BioProtoc.1446}, volume = {5}, year = {2015}, }