Over-expression of a functionally active human G M2 -activator protein in Escherichia coli

The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17. The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus. The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded. The hexahistidine tail could be removed when desired by digestion with Factor Xa. In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator

Quantification of Spent Coffee Ground Extracts by Roast and Brew Method, and Their Utility in a Green Synthesis of Gold and Silver Nanoparticles

Nanotechnology has become increasingly important in modern society, and nanoparticles are routinely used in many areas of technology, industry, and commercial products. Many species of nanoparticle (NP) are typically synthesized using toxic or hazardous chemicals, making these methods less environmentally friendly. Consequently, there has been growing interest in green synthesis methods, which avoid unnecessary exposure to toxic chemicals and reduce harmful waste. Synthesis methods which utilize food waste products are particularly attractive because they add value and a secondary use for material which would otherwise be disposed of. Here, we show that spent coffee grounds (SCGs) that have already been used once in coffee brewing can be easily used to synthesize gold and silver NPs. SCGs derived from medium and dark roasts of the same bean source were acquired after brewing coffee by hot brew, cold brew, and espresso techniques. The total antioxidant activity (TAC) and total caffeoylquinic acid (CQA) of the aqueous SCG extracts were investigated, showing that hot brew SCGs had the highest CQA and TAC levels, while espresso SCGs had the lowest. SCG extract proved effective as a reducing agent in synthesizing gold and silver NPs regardless of roast or initial brew method

Discovery of Dual-Action Membrane-Anchored Modulators of Incretin Receptors

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptors are considered complementary therapeutic targets for type 2 diabetes. Using recombinant membrane-tethered ligand (MTL) technology, the present study focused on defining optimized modulators of these receptors, as well as exploring how local anchoring influences soluble peptide function.Serial substitution of residue 7 in membrane-tethered GIP (tGIP) led to a wide range of activities at the GIP receptor, with [G(7)]tGIP showing enhanced efficacy compared to the wild type construct. In contrast, introduction of G(7) into the related ligands, tGLP-1 and tethered exendin-4 (tEXE4), did not affect signaling at the cognate GLP-1 receptor. Both soluble and tethered GIP and GLP-1 were selective activators of their respective receptors. Although soluble EXE4 is highly selective for the GLP-1 receptor, unexpectedly, tethered EXE4 was found to be a potent activator of both the GLP-1 and GIP receptors. Diverging from the pharmacological properties of soluble and tethered GIP, the newly identified GIP-R agonists, (i.e. [G(7)]tGIP and tEXE4) failed to trigger cognate receptor endocytosis. In an attempt to recapitulate the dual agonism observed with tEXE4, we conjugated soluble EXE4 to a lipid moiety. Not only did this soluble peptide activate both the GLP-1 and GIP receptors but, when added to receptor expressing cells, the activity persists despite serial washes.These findings suggest that conversion of a recombinant MTL to a soluble membrane anchored equivalent offers a means to prolong ligand function, as well as to design agonists that can simultaneously act on more than one therapeutic target

Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings. We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance. We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter

A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection

The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py) binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER) where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We show that Py is transported initially to the endolysosome where the low pH imparts a conformational change that enhances its subsequent ER-to-cytosol membrane penetration. GD1a stimulates not viral binding or entry, but rather sorting of Py from late endosomes and/or lysosomes to the ER, suggesting that GD1a binding is responsible for ER targeting. Consistent with this, an artificial particle coated with a GD1a antibody is transported to the ER. Our results provide a rationale for transport of Py through the endolysosome, demonstrate a novel endolysosome-to-ER transport pathway that is regulated by a lipid, and implicate ganglioside binding as a general ER targeting mechanism

Comparison of the virulence of exopolysaccharide-producing Prevotella intermedia to exopolysaccharide non-producing periodontopathic organisms

<p>Abstract</p> <p>Background</p> <p>Evidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built.</p> <p>Methods</p> <p>This study evaluates the role of EPS in <it>Prevotella intermedia </it>for the expression of virulence. This evaluation was accomplished by comparing EPS-producing <it>P. intermedia </it>strains 17 and OD1-16 with non-producing <it>P. intermedia </it>ATCC 25611 and <it>Porphyromonas gingivalis </it>strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis.</p> <p>Results</p> <p>EPS-producing <it>P. intermedia </it>strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 10⁷colony-forming units (CFU). In comparison, <it>P. intermedia </it>ATCC 25611 and <it>P. gingivalis </it>ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (10⁹CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, <it>P. intermedia </it>strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes.</p> <p>Conclusions</p> <p>These results demonstrate that the production of EPS by <it>P. intermedia </it>strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the host's innate defence response.</p

Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments

The Anti-interferon Activity of Conserved Viral dUTPase ORF54 is Essential for an Effective MHV-68 Infection

Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR−/− mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68