Nicholas L. Andrews

Membrane Receptor Mapping: The Membrane Topography of FceRI Signaling

Ligand binding to membrane receptors initiates cascades of biochemical events leading to physiological responses. Hundreds of proteins and lipids are implicated in signaling networks and programs in genomics and proteomics are continuously adding new components to the signaling “parts lists”. Here, we generate high resolution maps of signaling networks using cytoplasmic face-up membrane sheets that can be labeled with inununogold probes (3–10 nm) and imaged in the transmission electron microscope. Our model system is the mast cell and we focus on mapping the topography of the high affinity IgE receptor, FceRI, its associated tyrosine kinases, Lyn and Syk, and the signaling proteins that propagate signals from these kinases. Crosslinked receptors and their signaling partners segregate during signaling to multiple, dynamic membrane domains, including a transient FceRI-Lyn domain and at least two other distinct domains, one characterized by the presence of receptor, Syk and multiple signaling proteins, but not Lyn (primary signaling domains), and one characterized by the presence of LAT and PLCγl but not receptor (secondary signaling domains). PI 3-kinase associates with both primary and secondary signaling domains and may help to recruit specific signaling proteins through the local remodeling of inositol phospholipids. The lipid raft markers, GM1 and Thy-1, fail to localize in native membrane sheets either with each other or with signaling domains. We introduce new probes to localize multiple signaling molecules on the same membrane sheet and new computational tools to capture and analyze their topographical relationships. In the future, we expect that high resolution maps of signaling networks will be integrated with chemical kinetic analyses, with cell fractionation data and with a range of real-time fluorescence measurements, into mathematical models with power to predict mechanisms that regulate the efficiency, specificity, amplitude and duration of signaling pathways.

Synthesizing Biofunctionalized Nanoparticles to Image Cell Signaling Pathways

This minireview outlines the synthetic efforts, from our research group, to produce nanomaterials for use as imaging agents to study cell signaling pathways. An overview of our approach to the synthesis and biofunctionalization of metal, semiconductor, and ceramic nanomaterials is presented. The probes investigated include coinage metals, Cd-based, Gedeg, naturally occurring fluorescent (NOF) minerals, and Ln-based nanoparticles which were synthesized from novel metal alkoxide, amide, and alkyl precursors. We illustrate the applications of some of these materials as imaging probes to detect signaling pathway components and cellular responses to signals (apoptosis and degranulation) in inflammatory and cancer cells

Crystallographic characterization of the esterification pathway of Group V alkoxides

Abstract The products isolated from the stoichiometric reaction mixtures of [M(μ-OEt)(ONep)4]2 where M=Nb (1) or Ta (2) and ONep=OCH2CMe3, and a series of sterically demanding carboxylic acids (HORc where ORc=O2CCMe3 (OBc) or O2CCH2CMe3 (ONc)) led to the isolation and characterization of a new family of compounds at varied degrees of esterification. The number of oxo ligands formed from the esterification pathway was found to be dictated by the temperature of the reaction mixture, as well as the steric bulk of the carboxylate ligand. For the Nb systems, independent of the temperature of the reaction, [Nb(μ-OBc)(ONep)3]2(μ-O) (3) and [Nb2(μ-O)2(μ-ONc)2(ONep)4]2 (4) were isolated. For the room temperature reactions of the Ta systems, [Ta(μ-OBc)(ONep)3]2(μ-O) (5) and [Ta(μ-ONc)(ONep)4]2 (6) were formed but upon heating, [Ta2(μ-O)2(μ-OBc)2(ONep)4]2 (7) and [Ta(μ-ONc)(ONep)3]2(μ-O) (8) were isolated. [Ta2(μ-O)2(μ-ONc)2(ONep)4]2 (9) was also identified from a concentrated heated reaction mixture of 2 and HONc. The arrangements observed for 3, 5, 6, and 8 were dinuclear symmetric structures, wherein each metal was isolated in an octahedral (Oh) geometry with bridging ORc ligands. For 3, 5, and 8, an oxo ligand bridges the two metal centers which was not observed for 6. For 4, 7, and 9 the general structure consists of two “M2(μ-O)2(μ-ORc)2(ONep)4” moieties that are linked by oxo ligands forming an [Mue5f8O]4 eight-member ring as the central core. Each Oh bound metal center possesses two bridging ORc and two terminal ONep ligands. Compounds 3–8 were further characterized by solid-state MAS NMR wherein the bulk powders are in agreement with the single crystal structures. Solution NMR studies indicated that the solid-state arrangements are retained in solution. Combined, the structural information garnered from these compounds define the step-wise esterification mechanism of Group V metal alkoxides.

Small, mobile FcεRI aggregates are signaling competent

Crosslinking of IgE-bound FcepsilonRI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that FcepsilonRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-FcepsilonRI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. FcepsilonRI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.

N-Methyl­imidazolidinetrione

In the crystal structure of the title compound, C4H4N2O3, the packing is dominated by interxadmolecular carbonxadyl–carbonxadyl interxadactions and N—H⋯O hydrogen bonds.

Analytical investigation of AlCl[3]/SO[2]Cl[2] catholyte materials for secondary fuze reserve batteries.

Exploration of the fundamental chemical behavior of the AlCl{sub 3}/SO{sub 2}Cl{sub 2} catholyte system for the ARDEC Self-Destruct Fuze Reserve Battery Project under accelerated aging conditions was completed using a variety of analytical tools. Four different molecular species were identified in this solution, three of which are major. The relative concentrations of the molecular species formed were found to depend on aging time, initial concentrations, and storage temperature, with each variable affecting the kinetics and thermodynamics of this complex reaction system. We also evaluated the effect of water on the system, and determined that it does not play a role in dictating the observed molecular species present in solution. The first Al-containing species formed was identified as the dimer [Al({mu}-Cl)Cl{sub 2}]{sub 2}, and was found to be in equilibrium with the monomer, AlCl{sub 3}. The second species formed in the reaction scheme was identified by single crystal X-ray diffraction studies as [Cl{sub 2}Al({mu}-O{sub 2}SCl)]{sub 2} (I), a scrambled AlCl{sub 3}{center_dot}SO{sub 2} adduct. The SO{sub 2}(g) present, as well as CL{sub 2}(g), was formed through decomposition of SO{sub 2}CL{sub 2}. The SO{sub 2}(g) generated was readily consumed by AlCl{sub 3} to form the adduct 1 which was experimentally verified when 1 was also isolated from the reaction of SO{sub 2}(g) and AlCl {sub 3}. The third species found was tentatively identified as a compound having the general formula {l_brace}[Al(O)Cl{sub 2}][OSCl{sub 2}]{r_brace}{sub n}. This was based on {sup 27}Al NMR data that revealed a species with tetrahedrally coordinated Al metal centers with increased oxygen coordination and the fact that the precipitate, or gel, that forms over time was shown by Raman spectroscopic studies to possess a component that is consistent with SOCl{sub 2}. The precursor to the precipitate should have similar constituents, thus the assignment of {l_brace}[Al(O)Cl{sub 2}][OSCl{sub 2}]{r_brace}{sub n}. The precipitate was further identified by solid state {sup 27}Al MAS NMR data to possess predominantly octahedral A1 metal center which implies {l_brace}[Al(O)Cl{sub 2}][OSCl{sub 2}]{r_brace}{sub n} must undergo some internal rearrangements. A reaction sequence has been proposed to account for the various molecular species identified in this complex reaction mixture during the aging process. The metallurgical welds were of high quality. These results were all visually determined there was no mechanical testing performed. However, it is recommended that the end plate geometry and weld be changed. If the present weld strength, based on .003 - .005 penetration, is sufficient for unit performance, the end plate thickness can be reduced to .005 instead of the .020 thickness. This will enable the plug to be stamped so that it can form a cap rather than a plug and solve existing problems and increase the amount of catholyte which may be beneficial to battery performance.