ABSTRACT
We present a unique pipe flow rig capable of simultaneous particle tracking and flow velocity measurements in a dilute, neutrally buoyant particulate pipe flow in regimes of transition to turbulence. The flow consists of solid glass spheres for the disperse phase and a density-matching fluid for the carrier phase. The measurements are conducted using a bespoke, combined two-dimensional particle image velocimetry and particle tracking velocimetry technique. The technique takes advantage of a phase discrimination approach that involves separating the disperse and carrier phases based on their respective image characteristics. Our results show that the rig and the technique it implements can effectively be employed to study transitional particulate pipe flows at dilute concentrations.
ABSTRACT
The recombinant 18.5-kDa charge isoform of murine myelin basic protein (rmMBP) is unmodified posttranslationally and was used to study the effects of deimination, i.e., the conversion of arginyl to citrullinyl residues, on the protein's interactions with itself and with lipids. The unmodified species rmMBP-Cit(0) (i.e., containing no citrullinyl residues) interacted with binary monolayers containing acidic (phosphatidylinositol) and nickel-chelating lipids to form paracrystalline arrays with 4.8-nm spacing. A sample of protein was deiminated to an average of 9 citrullinyl residues per molecule of protein, yielding rmMBP-Cit(9). Under both low- and high-salt conditions, this species formed better-ordered domains than rmMBP-Cit(0), viz., planar crystalline assemblies. Thus, deimination of MBP resulted in a significant alteration of its lipid-organizing and self-interaction properties that might be operative in myelin in vivo, especially in progression of the autoimmune disease multiple sclerosis. Comparisons of amino acid sequences indicated significant similarities of MBP with filaggrin, a protein that is deiminated in another autoimmune disease, rheumatoid arthritis, suggesting that comparable epitopes could be targeted in both pathologies. In contrast, binary lipid monolayers consisting of phosphatidylinositol-4-phosphate (or phosphatidylinositol-4,5-bisphosphate) and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by rmMBP, especially in its deiminated form. Sequence comparisons with other actin- and phosphoinositide-binding proteins (vinculin, ActA, MARCKS) suggested that the carboxyl-terminal segment of MBP could form an amphipathic alpha helix and was the phosphoinositide binding site.
Subject(s)
Lipids/chemistry , Myelin Basic Protein/chemistry , Myelin Basic Protein/metabolism , Phosphatidylinositols/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cattle , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Hydrolases/chemistry , Mice , Microscopy, Electron , Molecular Sequence Data , Myelin Sheath/chemistry , Phosphatidylinositol Phosphates/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
A recombinant hexahistidine-tagged 18.5-kDa isoform of murine myelin basic protein has been characterized biochemically and immunogenically, by mass spectrometry, by circular dichroism under various conditions (in aqueous solution, with monosialoganglioside G(M1), and in 89% 2-propanol), and by transmission electron microscopy. The preparations of this protein indicated a high degree of purity and homogeneity, with no significant posttranslational modifications. Circular dichroic spectra showed that this preparation had the same degree of secondary structure as the natural bovine 18.5-kDa isoform of myelin basic protein. Incubation of the recombinant protein with lipid monolayers containing a nickel-chelating lipid resulted in the formation of fibrous assemblies that formed paracrystals of spacings 4.8 nm between fibers and 3-4 nm along them.
Subject(s)
Myelin Basic Protein/chemistry , Myelin Basic Protein/immunology , Animals , Cattle , Chromatography, Ion Exchange , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Epitopes/immunology , Escherichia coli , Gangliosides/metabolism , Lipid Metabolism , Lipids , Mass Spectrometry , Mice , Microscopy, Electron , Myelin Basic Protein/genetics , Myelin Basic Protein/ultrastructure , Nickel/metabolism , Peptide Fragments/immunology , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/ultrastructure , T-Lymphocytes/immunologyABSTRACT
Myelin basic protein (MBP) and myristoylated alanine-rich C-kinase substrate (MARCKS) are similar in terms of having extended conformations regulated by their environment (i.e., solubilised or lipid-associated), N-terminal modifications, a dual nature of interactions with lipids, binding to actin and Ca2+-calmodulin, and being substrates for different kinds of protein kinases. The further sequence similarities of segments of MBP with lipid effector regions of MARCKS, and numerous reports in the literature, support the thesis that some developmental isoform of MBP functions in signal transduction.
