Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein

N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.

The agonism and synergistic potentiation of weak partial agonists by triethylamine in alpha(1)-adrenergic receptor activation: Evidence for a salt bridge as the initiating process

alpha(1)-adrenergic receptor (AR) activation is thought to be initiated by disruption of a constraining interhelical salt bridge (Porter et al., 1996). Disruption of this salt bridge is achieved through a competition for the aspartic acid residue in transmembrane domain three by the protonated amine of the endogenous ligand norepinephrine and a lysine residue in transmembrane domain seven. To further test this hypothesis, we investigated the possibility that a simple amine could mimic an important functional group of the endogenous ligand and break this alpha(1)-AR ionic constraint leading to agonism. Triethylamine (TEA) was able to generate concentration-dependent increases of soluble inositol phosphates in COS-1 cells transiently transfected with the hamster alpha(1b)-AR and in Rat-1 fibroblasts stably transfected with the human alpha(1a)-AR subtype. TEA was also able to synergistically potentiate the second messenger production by weak partial alpha(1)-AR agonists and this effect was fully inhibited by the alpha(1)-AR antagonist prazosin. However, this synergistic potentiation was not observed for full alpha(1)-AR agonists. Instead, TEA caused a parallel rightward shift of the dose-response curve, consistent with the properties of competitive antagonism. TEA specifically bound to a single population of alpha(1)-ARs with a K-i of 28.7 +/- 4.7 mM. In addition, the site of binding by TEA to the alpha(1)-AR is at the conserved aspartic acid residue in transmembrane domain three, which is part of the constraining salt bridge. These results indicate a direct interaction of TEA in the receptor agonist binding pocket that leads to a disruption of the constraining salt bridge, thereby initiating alpha(1)-AR activation.

Treatment of intestinal Behçet's syndrome with chimeric tumour necrosis factor alpha antibody

Few patients with Behçet's syndrome have gastrointestinal ulceration. Such patients are difficult to treat and have a higher mortality. Faced with refractory symptoms in two patients with intestinal Behçet's, we used the tumour necrosis factor alpha (TNF-alpha) monoclonal antibody infliximab to induce remission. Both women (one aged 27 years, the other 30 years) presented with orogenital ulceration, pustular rash, abdominal pain, bloody diarrhoea due to colonic ulceration, weight loss, and synovitis. One had thrombophlebitis, digital vasculitis, perianal fistula, and paracolic abscess; the other had conjunctivitis and an ulcer in the natal cleft. Treatment with prednisolone, methyl prednisolone, and thalidomide in one and prednisolone, colchicine, and cyclosporin in the other was ineffective. After full discussion, infliximab (3 mg/kg, dose reduced because of recent sepsis in one, and 5 mg/kg in the other) was administered. Within 10 days the ulcers healed, with resolution of bloody diarrhoea and all extraintestinal manifestations. A second infusion of infliximab was necessary eight weeks later in one case, followed by sustained (>15 months) remission on low dose thalidomide. Remission was initially sustained for 12 months in the other but thalidomide had to be stopped due to intolerance, and a good response to retreatment lasted only 12 weeks without immunosuppression, before a third infusion. The cause of Behçet's syndrome is unknown but peripheral blood CD45 gammadelta T cells in Behçet's produce >50-fold more TNF-alpha than controls when stimulated with phorbol myristate acetate and anti-CD3. Infliximab could have a role for inducing remission in Behçet's syndrome.

Autosomal dominant Alport syndrome linked to the type IV collage alpha 3 and alpha 4 genes (COL4A3 and COL4A4)

Alport syndrome is a hereditary nephritis that may lead to end-stage renal disease (ESRD) in young adult life and is often associated with sensorineural deafness and/or ocular abnormalities. The majority of families are X-linked due to mutations in the COL4A5 gene at Xq22. Autosomal forms of the disease are also recognized with recessive disease, having been shown to be due to mutations in the COL4A3 and COL4A4 genes on chromosome 2. Familial benign haematuria has also been mapped to this region in some families.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1) . The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

Selective Hydrogenation of alpha,beta-Unsaturated Aldehydes and Ketones using Novel Manganese Oxide and Platinum Supported on Manganese Oxide Octahedral Molecular Sieves as Catalysts

The selective hydrogenation of ,-unsaturated aldehydes and ketones has been studied using ketoisophorone and cinnamaldehyde as model substrates using manganese oxide octahedral molecular sieve (OMS-2) based catalysts. For the first time, OMS-2 has been shown to be an efficient and selective hydrogenation catalyst. High selectivities for either the CC or CO double bond at approximate to 100% conversion were achieved by using OMS-2 and platinum supported on OMS-2 catalysts. Density functional theory (DFT) calculations showed that the dissociation of H2 on OMS-2 was water assisted and occurred on the surface Mn of OMS-2(001) that had been modified by an adsorbed H2O molecule. The theoretically calculated activation barrier was in good agreement with the experimentally determined value for the hydrogenation reactions, indicating that H2 dissociation on OMS-2 is likely to be the rate-determining step. A significant increase in the rate of reaction was observed in the presence of Pt as a result of the enhancement of H2 dissociative adsorption and subsequent reaction on the Pt or spillover of the hydrogen to the OMS-2 support. The relative adsorption strengths of ketoisophorone and cinnamaldehyde on the OMS-2 support compared with the Pt were found to determine the product selectivity.

CD69, CD25, and HLA-DR activation antigen expression on CD3+ lymphocytes and relationship to serum TNF-alpha, IFN-gamma, and sIL-2R levels in aging

Aging is associated with changes in lymphocyte subsets and unexplained HLA-DR upregulation on T-lymphocytes. We further investigated this activation, by measuring early (CD69), middle (CD25), and late (HLA-DR) T-lymphocyte activation markers on CD3+ lymphocytes, across subjects (20-100 years) together with serum tumor necrosis factor (TNF-alpha), interferon-gamma (IFN-gamma), and soluble interleukin-2 receptor (sIL-2R). HLA-DR was present as a CD3+ HLA-DR+ subset that constituted 8% of total lymphocytes, increased twofold with age and included CD4+, CD8+, and CD45RA+ phenotypes. HLA-DR was also expressed on a CD8+ CD57+ subset. The CD3+ CD25+ subset constituted 13% of lymphocytes, fell with age but was weakly associated with the CD3+ HLA-DR+ subset especially in older subjects. A small 3-5% CD3+ CD69+ subsets showed no age effect. Serum sIL-2R, TNF-alpha, but not IFN-gamma, were associated with CD3+ HLA-DR+ lymphocytes, TNF-alpha with CD8+ CD57+ count and sIL-2R and IFN-gamma with the CD3+ CD25+/CD3+ CD4+ ratio. The study confirms age-related upregulation of HLA-DR on CD3+ lymphocytes, shows some evidence for associated upregulation of CD25 on CD3+ cells in older subjects, and links serum TNF-alpha, IFN-gamma, and sIL2-R to T-lymphocyte activation.

Local-field and excitonic effects in the optical response of alpha-alumina

This paper introduces key ingredients of the dielectric response of a-alumina that go beyond an independent-particle (IP) treatment of the valence-electron excitations. The optical-response functions were calculated from first-principles both at the Bethe-Salpeter and the random-phase approximation (RPA) levels. Excitonic effects obtained within the Bethe-Salpeter framework were found essential for reproducing the low-energy part of the experimental spectra (below 15 eV) and the bound exciton in particular. For higher energies, local-field effects introduced through the RPA modified considerably the IP results and provided a satisfactory account of the reflectivity spectra and of the position and shape of the dominant bulk plasmon resonance in the electron energy-loss spectra.