ALG9 mannosyltransferase is involved in two different steps of lipid-linked oligosaccharide biosynthesis

N-linked protein glycosylation follows a conserved pathway in eukaryotic cells. The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for the oligosaccharyltransferase (OST), is catalyzed by different glycosyltransferases located at the membrane of the endoplasmic reticulum (ER). The substrate specificity of the different glycosyltransferase guarantees the ordered assembly of the branched oligosaccharide and ensures that only completely assembled oligosaccharide is transferred to protein. The glycosyltransferases involved in this pathway are highly specific, catalyzing the addition of one single hexose unit to the lipid-linked oligosaccharide (LLO). Here, we show that the dolichylphosphomannose-dependent ALG9 mannosyltransferase is the exception from this rule and is required for the addition of two different α-1,2-linked mannose residues to the LLO. This report completes the list of lumen-oriented glycosyltransferases required for the assembly of the LL

Customer Relationship Management : strategische Ausrichtung statt IT-getriebenem Aktivismus

Der Begriff Customer Relationship Management (CRM) ist heute in aller Munde. Wie immer, wenn ein solches „buzzword“ auftaucht, besteht viel Unklarheit darüber, was es eigentlich bedeutet und inwieweit es etwas wirklich Neues darstellt. Insbesondere IT-Unternehmen suggerieren häufig, daß ein erfolgreiches Kundenbeziehungsmanagement im wesentlichen eine Frage der richtigen Hard- und Software sei. Das Resultat ist häufig ein IT-getriebener Aktivismus, der die hochgesteckten Erwartungen natürlich nicht erfüllen kann. Für eine erfolgreiches CRM sind insbesondere drei Aspekte von besonderer Bedeutung: 1. Eine systematische Aufnahme und Auswertung kundenbezogener Informationen 2. Die Entwicklung eines klaren CRM-Konzeptes sowie 3. Die Schaffung entsprechender Implementierungsvoraussetzungen im Unternehmen. Auf diese Aspekte geht das vorliegende Papier ein. Es soll Managern einen systematischen Zugang zur strategisch konzeptionellen Facette des Customer Relationship Management geben. Ein saubere Ausarbeitung dieser strategisch-konzeptionellen Aspekte ist unerläßliche Voraussetzung dafür, daß in einem zweiten Schritt entsprechende IT-Konzepte wirkungsvoll eingesetzt werden können

Use Your Strategic Entrepreneurs to Build Your Strategic Partnerships

Internationalisation through strategic partnerships is a goal for many higher education institutions and their upper-level management teams. Yet for institutional objectives to truly flourish, they should get the most out of the various skills that different actors bring to be table. This piece explores the interesting role that can be played by resourceful academic staff in materialising institutional, and individual, aims

Distinct Flippases Translocate Glycerophospholipids and Oligosaccharide Diphosphate Dolichols across the Endoplasmic Reticulum†

Transbilayer movement, or flip-flop, of lipids across the endoplasmic reticulum (ER) is required for membrane biogenesis, protein glycosylation, and GPI anchoring. Specific ER membrane proteins, flippases, are proposed to facilitate lipid flip-flop, but no ER flippase has been biochemically identified. The glycolipid Glc 3Man 9GlcNAc 2-PP-dolichol is the oligosaccharide donor for protein N-glycosylation reactions in the ER lumen. Synthesis of Glc 3Man 9GlcNAc 2-PP-dolichol is initiated on the cytoplasmic side of the ER and completed on the lumenal side, requiring flipping of the intermediate Man 5GlcNAc 2-PP-dolichol (M5-DLO) across the ER. Here we report the reconstitution of M5-DLO flipping in proteoliposomes generated from Triton X-100-extracted Saccharomyces cerevisiae microsomal proteins. Flipping was assayed by using the lectin Concanavalin A to capture M5-DLOs that had been translocated from the inner to the outer leaflet of the vesicles. M5-DLO flipping in the reconstituted system was ATP-independent and trypsin-sensitive and required a membrane protein(s) that sedimented at ∼4 S. Man 7GlcNAc 2-PP-dolichol, a higher-order lipid intermediate, was flipped >10-fold more slowly than M5-DLO at 25°C. Chromatography on Cibacron Blue dye resin enriched M5-DLO flippase activity ∼5-fold and resolved it from both the ER glycerophospholipid flippase activity and the genetically identified flippase candidate Rft1 [Helenius, J., et al. (2002) Nature 415, 447-450]. The latter result indicates that Rft1 is not the M5-DLO flippase. Our data (i) demonstrate that the ER has at least two distinct flippase proteins, each specifically capable of translocating a class of phospholipid, and (ii) provide, for the first time, a biochemical means of identifying the M5-DLO flippase. © 2008 American Chemical Society.link_to_subscribed_fulltex

The cAMP effector EPAC activates Elk1 transcription factor in prostate smooth muscle, and is a minor regulator of alpha 1-adrenergic contraction

Background: Prostate smooth muscle tone is regulated by alpha 1-adrenoceptor-induced contraction and cAMP-mediated relaxation. EPAC is an effector of cAMP, being involved in smooth muscle relaxation and cell cycle control outside the lower urinary tract. Here, we investigated the expression and function of EPAC in human prostate tissues from patients undergoing radical prostatectomy. Results: mRNA and protein expression of EPAC was detected in all prostate tissues by RT-PCR and Western blot analysis. Immunoreactivity was observed in stromal cells, and colocalized with immunofluorescence for a-smooth muscle actin and calponin. Under normal conditions, noradrenaline-or phenylephrine-induced contraction of prostate strips in the organ bath was not affected by the EPAC activator pCPT (SP-8-pCPT-2'-O-Me-cAMPS.NA) (30 mu M). However, when the cyclooxygenase inhibitor indomethacin (50 mu M) was added, EPAC activators pCPT and OME (8-CPT-2'-O-Me-cAMP.Na) (30 mu M) significantly reduced contractions by low concentrations of phenylephrine. These effects were not observed on noradrenaline-induced contraction. OME and pCPT caused phosphorylation of the transcription factor Elk1 in prostate tissues. Elk1 activation was confirmed by EMSA (electrophoretic mobility shift assay), where OME and pCPT incresed Elk1 binding to a specific DNA probe. Conclusions: EPAC activation may reduce alpha 1-adrenergic prostate contraction in the human prostate, although this effect is masked by cyclooxygenases and beta-adrenoceptors. A main EPAC function in the human prostate may be the regulation of the transcription factor Elk1

Geochemical modelling of water-rock interaction

CO2 geological storage is one of the most promising technologies for reducing atmospheric emissions of greenhouse gas. In this work we present and discuss a new approach geochemical modelling for evaluating the effects of short-medium term CO2 disposal in deep geologic formations that has been tested in the Weyburn test site (Saskatchewan, Canada), where since September 2000 5000 t/day of supercritical CO2 are injected. The geochemical modeling has been performed by using the code PRHEEQC (V2.11) software package, via thermodynamic corrections to the code default database. First, we reconstructed the in-situ reservoir (62°C and 0.1 MPa) chemical composition, including pH, by the chemical equilibrium among the various phases, and we evaluated the boundary conditions (e.g. PCO2 , PH2S), which are necessary for the implementation of reaction path modeling. This is the starting point to assess the geochemical impact of CO2 into the oil reservoir and, as main target, to quantify water-gas-rock reactions. Furthermore, we identified possible compositions of the initially reservoir liquid phases by assuming the equilibrium conditions for the mineral assemblage with respect to a Na-Cl water (Cl/Na=1.2). Then we computed the kinetic evolution of the CO2-rich Weyburn brines interacting with the host-rock minerals, performed over 100 years after injection. Results of reaction path modeling suggest that, in this period, CO2 can be neutralized by solubility (as CO2 (aq)) and mineral trapping through Dawsonite precipitation. In order to validate our geochemical model we have simulated the geochemical impact of three years of CO2 injection (September 2000-2003) by kinetically controlled reactions and we have compared the computed and measured data. The calculated chemical composition after the CO2 injection is consistent with the analytical data of samples collected in 2003 with an error within 5 % for most analytical species, with the exception of the Ca and Mg contents (error > 90%), likely due to the complexation effect of carboxilic acid

Exploiting neutron-rich radioactive ion beams to constrain the symmetry energy

The Modular Neutron Array (MoNA) and 4 Tm Sweeper magnet were used to measure the free neutrons and heavy charged particles from the radioactive ion beam induced 32Mg + 9Be reaction. The fragmentation reaction was simulated with the Constrained Molecular Dynamics model(CoMD), which demonstrated that the of the heavy fragments and free neutron multiplicities were observables sensitive to the density dependence of the symmetry energy at sub-saturation densities. Through comparison of these simulations with the experimental data constraints on the density dependence of the symmetry energy were extracted. The advantage of radioactive ion beams as a probe of the symmetry energy is demonstrated through examination of CoMD calculations for stable and radioactive beam induced reactions

ALG12 mannosyltransferase defect in congenital disorder of glycosylation type lg

In the endoplasmic reticulum (ER) of eukaryotes, N-linked glycans are first assembled on the lipid carrier dolichyl pyrophosphate. The GlcNAc2Man9Glc3 oligosaccharide is transferred to selected asparagine residues of nascent polypeptides. Defects along the biosynthetic pathway of N-glycans are associated with severe multisystemic syndromes called congenital disorders of glycosylation. Here, we describe a deficiency in the ALG12 ER α1,6-mannosyltransferase resulting in a novel type of glycosylation disorder. The severe disease was identified in a child presenting with psychomotor retardation, hypotonia, growth retardation, dysmorphic features and anorexia. In the patient's fibroblasts, the biosynthetic intermediate GlcNAc2Man7 oligosaccharide was detected both on the lipid carrier dolichyl pyrophosphate and on newly synthesized glycoproteins, thus pointing to a defect in the dolichyl pyrophosphate-GlcNAc2Man7-dependent ALG12 α1,6 mannosyltransferase. Analysis of the ALG12 cDNA in the CDG patient revealed compound heterozygosity for two point mutations that resulted in the amino acid substitutions T67M and R146Q, respectively. The impact of these mutations on ALG12 protein function was investigated in the Saccharomyces cerevisiae alg12 glycosylation mutant by showing that the yeast ALG12 gene bearing the homologous mutations T61M and R161Q and the human mutant ALG12 cDNA alleles failed to normalize the growth defect phenotype of the alg12 yeast model, whereas expression of the normal ALG12 cDNA complemented the yeast mutation. The ALG12 mannosyltransferase defect defines a new type of congenital disorder of glycosylation, designated CDG-I

Space and Ground Based Pulsation Data of Eta Bootis Explained with Stellar Models Including Turbulence

The space telescope MOST is now providing us with extremely accurate low frequency p-mode oscillation data for the star Eta Boo. We demonstrate in this paper that these data, when combined with ground based measurements of the high frequency p-mode spectrum, can be reproduced with stellar models that include the effects of turbulence in their outer layers. Without turbulence, the l=0 modes of our models deviate from either the ground based or the space data by about 1.5-4.0 micro Hz. This discrepancy can be completely removed by including turbulence in the models and we can exactly match 12 out of 13 MOST frequencies that we identified as l=0 modes in addition to 13 out of 21 ground based frequencies within their observational 2 sigma tolerances. The better agreement between model frequencies and observed ones depends for the most part on the turbulent kinetic energy which was taken from a 3D convection simulation for the Sun.Comment: 13 pages, 7 figures, ApJ in pres