Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the α-1,6 mannose to dolichollinked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulu

Beam-based alignment of TTF RF-gun using V-Code

The beam dynamics simulation code V [1,2], based on the Ensemble Model [3], is being developed for on-line simulations. One practical application of the V-Code is the beam-based alignment (BBA) of accelerator (TESLA Test Facility) elements. Before we started with BBA thefirst beam position monitor (BPM1), located after the RFgun cavity, showed non-zero readings. Moreover the readings depended on RF-power, RF-phase and primary and secondary solenoid currents. This effect could be explained by misalignments of the gun and the solenoids. Such beam offsets must be compensated by means of steering coils but such a procedure can be one of the sources of increased emittances. Based on the V-Code solver a dedicated utility was developed for alignment studies. The laser beam mismatch at the cathode, as well as the primary and secondary solenoid displacements were considered as probable reasons for the misalignment of the beam. A new method for the correction of these misalignments combines a sequence of measurements, simulations and the elimination of the largest imperfections. This semi-automatic method applied to the TTF RF-gun yields a centering of the beam within the accuracy of the BPM1

Investigation of TTF injector alignment with the simulation Code V

The exact alignment of accelerator components is of crucial importance for the production of low emittance beams. Once a beam-line section is set up, a supplementary correction of misalignments implies the knowledge of its magnitude which is difficult to determine using conventional adjusting instruments. An excellent alternative to measure existing misalignments of accelerator components is to vary machine parameters and compare the behaviour of the beam with results obtained from a simulation. It is obvious that time consuming particle tracking programmes are notappropriate to reach this aim. Regarding computing time, the on-line simulation code V is advantageous compared to other beam dynamics programmes. The theoretical basis of V-Code, the “Ensemble Model”, consists of selfconsistent equations for the ensemble parameters that arederived from the Vlasov equation. The requirement to simulate misalignments such as offsets and tilts led to the development of the ALIGNMENT UTILITY which utilizes the solver of V-Code. The new utility enabled us to investigate the beam-line alignment of the TESLA Test Facility injector.This contribution presents the theoretical background and an illustrating example of the optimization process

Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the α-1,6 mannose to dolichollinked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulu

Novel glucose-1-phosphatase with high phytase activity and unusual metal ion activation from soil bacterium Pantoea sp. strain 3.5.1

© 2015, American Society for Microbiology. Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values toward phytate, glucose-6- phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify DL-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features

Pathogenitätsfaktoren bei Pilzinfektionen

Für die starke Zunahme von Infektionen durch opportunistische Pilze sind vor allem prädisponierende Faktoren verantwortlich,welche die natürliche Abwehr des Wirtes abschwächen.Trotzdem müssen pathogene Pilze, wie die medizinisch bedeutende Hefe Candida albicans, über Virulenzfaktoren verfügen, die dem Mikroorganismus das Überleben auf und in einem Wirt sichern oder das Vordringen zu tieferen Geweben und Organen ermöglichen.Das Ziel der Forschungsprojekte der Nachwuchsgruppe „Pathogenitätsfaktoren bei Pilzinfektionen“ am Robert Koch-Institut (RKI) ist, solche Faktoren von Pilzen zu identifizieren und zu analysieren, von denen vermutet wird, dass sie bei einer Infektion eine wichtige Rolle spielen.Damit sollen nicht nur die Pathogenitätsmechanismen besser verstanden werden, sondern auch Ansatzpunkte für neue Medikamente gefunden werden.Schließlich gilt es,aufgrund der zunehmenden Resistenzen gegenüber den wenigen zur Verfügung stehenden Antimykotika, die Wirkungsweise existierender Medikamente zu verstehen

Ribonuclease Activity of Dis3 Is Required for Mitotic Progression and Provides a Possible Link between Heterochromatin and Kinetochore Function

BACKGROUND: Cellular RNA metabolism has a broad range of functional aspects in cell growth and division, but its role in chromosome segregation during mitosis is only poorly understood. The Dis3 ribonuclease is a key component of the RNA-processing exosome complex. Previous isolation of the dis3-54 cold-sensitive mutant of fission yeast Schizosaccharomyces pombe suggested that Dis3 is also required for correct chromosome segregation. METHODOLOGY/PRINCIPAL FINDINGS: We show here that the progression of mitosis is arrested in dis3-54, and that segregation of the chromosomes is blocked by activation of the mitotic checkpoint control. This block is dependent on the Mad2 checkpoint protein. Double mutant and inhibitor analyses revealed that Dis3 is required for correct kinetochore formation and function, and that this activity is monitored by the Mad2 checkpoint. Dis3 is a member of the highly conserved RNase II family and is known to be an essential subunit of the exosome complex. The dis3-54 mutation was found to alter the RNaseII domain of Dis3, which caused a reduction in ribonuclease activity in vitro. This was associated with loss of silencing of an ura4(+) reporter gene inserted into the outer repeats (otr) and central core (cnt and imr) regions of the centromere. On the other hand, centromeric siRNA maturation and formation of the RITS RNAi effector complex was normal in the dis3-54 mutant. Micrococcal nuclease assay also suggested the overall chromatin structure of the centromere was not affected in dis3-54 mutant. CONCLUSIONS/SIGNIFICANCE: RNase activity of Dis3, a core subunit of exosome, was found to be required for proper kinetochore formation and establishment of kinetochore-microtubule interactions. Moreover, Dis3 was suggested to contribute to kinetochore formation through an involvement in heterochromatic silencing at both outer centromeric repeats and within the central core region. This activity is likely monitored by the mitotic checkpoint, and distinct from that of RNAi-mediated heterochromatin formation directly targeting outer centromeric repeats

Proteasome Nuclear Import Mediated by Arc3 Can Influence Efficient DNA Damage Repair and Mitosis in Schizosaccharomyces Pombe

Proteasomes must efficiently remove their substrates throughout the cells in a timely manner as many of these proteins can be toxic. This study shows that proteasomes can do so efficiently because they are highly mobile. Furthermore this study uncovers that proteasome mobility requires functional Arc3, a subunit of the Arp2/3 complex