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Now showing items 33 - 48 of 2542

  • [Subcellular Biochemistry] Macromolecular Protein Complexes Volume 83 || α2-Macroglobulins: Structure and Function

    Harris, J. Robin   Marles-Wright, Jon  

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  • Keyhole limpet hemocyanin: Molecular structure of a potent marine immunoactivator. A review

    Harris, J. Robin   Markl, Juergen  

    Objectives: In this short review we present a survey of the available biochemical and electron microscopic data on keyhole limpet hemocyanin (KLH). Results: The biosynthesis of KLH and its biological role are discussed and the purification of the two isoforms of KLH (KLH1 and KLH2) presented in some detail. The determination of the molecular mass of KLH, its functional unit structure, carbohydrate content, immunological analysis and aspects of the molecular biology of KLH are all dealt with. Transmission electron microscopy (TEM) and crossed immunoelectrophoresis have played a significant part in the understanding of KLH structure. We present a summary of TEM studies on the native oligomers of KLH, the experimental manipulation of the different oligomeric states, immunological analysis and subunit reassociation. Conclusion: This fundamental structural information provides the scientific background upon which the understanding of the in vivo immunostimulatory function of KLH can be based.
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  • Cell Biology Protocols (Harris/Cell Biology Protocols) || Isolation and Functional Analysis of Organelles

    Harris, J. Robin   Graham, John   Rickwood, David  

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  • [Subcellular Biochemistry] Macromolecular Protein Complexes Volume 83 || The Peroxiredoxin Family: An Unfolding Story

    Harris, J. Robin   Marles-Wright, Jon  

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  • Negative Staining: Historical Background and Technical Development

    Harris, J. Robin  

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  • Negative Staining and Cryoelectron Microscopy

    Harris, J. Robin  

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  • [Subcellular Biochemistry] Macromolecular Protein Complexes Volume 83 || “Pyruvate Carboxylase, Structure and Function”

    Harris, J. Robin   Marles-Wright, Jon  

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  • Immunonegative staining: Epitope localization on macromolecules

    Harris, J. Robin  

    Relevant literature relating to immunonegative staining is reviewed and integrated with current research of the author and others. The immunonegative staining procedure has been utilized for the study of epitope localization on immune complexes formed from keyhole limpet hemocyanin type 2 (KLH2) di- and multidecamers, and the 20S and 26S proteasome from Xenopus laevis. The IgG linkage pattern of molecules in small immune complexes is considered to provide the most reliable indication of epitope location. For both KLH2 and the 20S proteasome, using domain-specific monoclonal antibodies and a 32-kDa (p32) subunit-specific polyclonal antibody, respectively, it is shown that epitopes (KLH2, subunit fg domain pair and the proteasome p32) are located on the ends of the two cylindrical molecules. Also, for KLH2 with a monoclonal antibody directed against subunit domain a, it is shown that this domain is located at the center of the KLH2 didecamer. Interpretation of the data for KLH2 indicates that the 10 subunits in each decamer are likely to be oriented in parallel. The epitopes of the 10 fg domain pairs are located on the ends, near the edge of the didecamer cylinder (i.e., at the "collar" region of the didecamer) and the epitope of the 10 a domains is located on the side wall of the didecamer cylinder, where the two decamers meet. In view of the varying shape of the Xenopus 20S proteasome Immune complexes, it is tentatively proposed that there may be two p32 subunits located in each of the heptameric a rings rather than one, but the possibility of macromolecular heterogeneity with respect to p32 subunit composition cannot be discounted.
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  • Comparative 11 angstrom structure of two molluscan hemocyanins from D cryo-electron microscopy RID G-4433-2011

    Meissner, Ulrich   Gatsogiannis, Christos   Moeller, Arne   Depoix, Frank   Harris, J. Robin   Markl, Juergen  

    Hemocyanins are giant extracellular proteins that transport oxygen in the hemolymph of many molluscs. Molluscan hemocyanins are cylindrical decamers or didecamers of a 350-400 kDa subunit that contains seven or eight different covalently linked globular functional units (FUs), arranged in a linear manner. Each FU carries a single copper active site and reversibly binds one dioxygen molecule. As a consequence, the decamer can carry up to 70 or 80 02 molecules. Although complete sequence information is now available from several molluscan hemocyanins, many details of the quaternary structure are still unclear, including the topology of the 10 subunits within the decamer. Here we show 3D reconstructions from cryo-electron micrographs of the hemocyanin decamer of Nautilus pompilius (Cephalopoda) and Haliotis tuberculata (Gastropoda) at a resolution of 11 angstrom (FSC1/2-bit criterion). The wall structure of both hemocyanins is very similar and shows, as in previous reconstructions, three tiers with 20 functional units each that encircle the cylinder wall, and the 10 oblique minor and major wall grooves. However, the six types of wall FUs of the polypeptide subunit, termed a-b-c-d-e-f, are now for the first time individually discernable by their specific orientation, shape, and connections. Also, the internal collar complex of the decamers shows superior resolution which, in this case, reveals striking differences between the two hemocyanins. The five arcs (FU-g pairs) of the central collar (in both hemocyanins) and the five slabs (FU-h pairs) of the peripheral collar (only present in Haliotis hemocyanin), as well as their connections to the wall and to each other are now more clearly defined. The arc is attached to the wall through a feature termed the anchor, a previously undescribed structural element of the hemocyanin wall. (C) 2006 Elsevier Ltd. All rights reserved.
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  • [Subcellular Biochemistry] Alzheimer’s Disease Volume 38 || Amyloid-β Metal Interaction and Metal Chelation

    Harris, J. Robin   Fahrenholz, Falk  

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  • [Subcellular Biochemistry] Subcellular Biochemistry Volume 25 || Introduction

    Harris, J. Robin  

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  • Recombinant anthrax protective antigen: Observation of aggregation phenomena by TEM reveals specific effects of sterols

    Harris, J. Robin   Soliakov, Andrei   Watkinson, Allan   Lakey, Jeremy H.  

    rPA83 forms fibrous aggregates when heated at low temperatures (50 °C). rPA83 aggregates binds to the edges of cholesterol bilayer microcrystals. PA63 prepore oligomers bind to the edges of cholesterol bilayer microcrystals. PA63 prepores convert to PA63 pores when treated with sodium deoxycholate. PA63 pores produced by sodium deoxycholate aggregate as dimers and angular chains
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  • Negative Stains Containing Trehalose: Application to Tubular and Filamentous Structures

    Harris, J. Robin   Gerber, Max   Gebauer, Wolfgang   Wernicke, Wolfgang   Markl, Jürgen  

    Several examples are presented that show the successful application of uranyl acetate and ammonium molybdate negative staining in the presence of trehalose for TEM studies of filamentous and tubular structures. The principal benefit to be gained from the inclusion of trehalose stems from the considerably reduced flattening of the large tubular structures and the greater orientational freedom of single molecules due to an increased depth of the negative stain in the presence of trehalose. Trehalose is likely to provide considerable protection to protein molecules and their assemblies during the drying of negatively stained specimens. Some reduction in the excessive density imparted by uranyl acetate around large assemblies is also achieved. Nevertheless, in the presence of 1% (w/v) trehalose, it is desirable to increase the concentration of negative stain to 5% (w/v) for ammonium molybdate and to 4% for uranyl acetate to produce satisfactory image contrast. In general, the ammonium molybdate-trehalose negative stain is more satisfactory than the uranyl acetate-trehalose combination, because of the greater electron beam sensitivity of the uranyl negative stain. Reassembled taxol-stabilized pig brain microtubules, together with collagen fibrils, sperm tails, helical filaments, and reassociated hemocyanin (KLH2), all from the giant keyhole limpet Megathuracrenulata, have been studied by negative staining in the presence of trehalose. In all cases satisfactory TEM imaging conditions were readily obtained on the specimens, as long as regions of excessively deep stain were avoided.
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  • Human Islet Amyloid Polypeptide Fibril Binding to Catalase: A Transmission Electron Microscopy and Microplate Study

    Milton, Nathaniel G. N.   Harris, J. Robin  

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  • [Subcellular Biochemistry] Alzheimer’s Disease Volume 38 || Membrane Disordering Effects of β-Amyloid Peptides

    Harris, J. Robin   Fahrenholz, Falk  

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  • The collagen type I segment long spacing (SLS) and fibrillar forms: Formation by ATP and sulphonated diazo dyes

    Harris, J. Robin   Lewis, Richard J.  

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