ZIP, Biotinylated

Biotinylated, highly luminescent CdSe-ZnS quantum dot (QD) conjugates were prepared and used in immunofiltration assays. Water-soluble quantum dot surfaces having a homogeneous negative charge density at basic pH were initially coated with a two-domain recombinant maltose-binding protein appended with a positively charged leucine zipper. Biotin functionalization of these electrostatically stabilized QD-protein complexes was then carried out using amine-reactive NHS biotin. These protein-coated biotin-functionalized quantum dot conjugates were incorporated into flow immunofiltration/displacement assays employing Affi-gel agarose resin for antibody immobilization, analyte capture, and immune complex formation with a second biotinylated antibody. A key component of the assay was the use of tetranitromethane-modified NeutrAvidin, used to link the biotinylated QDs to the immune complexes and facilitate their release at basic pH for subsequent quantification. This assay methodology was used to detect as little as 10 ng/mL staphylococcal enterotoxin type-B.

Hormonal control of transepithelial sodium (Na(+)) transport utilizes phosphatidylinositide 3'-kinase (PI3K) and Raf-MAPK/ERK kinase (MEK)-ERK-dependent signaling pathways, which impact numerous cell functions. How signals transmitted by these pathways are sorted and appropriately transmitted to alter Na(+) transport without altering other physiologic processes is not well understood. Here, we report the identification of a signaling complex that selectively modulates the cell surface expression of the epithelial sodium channel (ENaC), an ion channel that is essential for fluid and electrolyte balance in mammals. Raf-1 and the ubiquitin ligase, Nedd4-2, are constitutively-expressed inhibitory components of this ENaC regulatory complex, which interact with, and decrease the expression of, cell surface ENaC. The activities of Nedd4-2 and Raf-1 are inhibited cooperatively by the PI3K-dependent kinase serum- and glucocorticoid-induced kinase 1 (SGK1), and the Raf-1-interacting protein glucocorticoid-induced leucine zipper (GILZ1), which are aldosterone-stimulated components of the complex. Together, SGK1 and GILZ1 synergistically stimulate ENaC cell surface expression. Interestingly, GILZ1 and SGK1 do not have synergistic, and in fact have opposite, effects on an unrelated activity, FKHRL1-driven gene transcription. Together, these data suggest that GILZ1 and SGK1 provide a physical and functional link between the PI3K- and Raf-1-dependent signaling modules and represent a unique mechanism for specifically controlling Na(+) transport without inappropriately activating other cell functions.

Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin alpha5beta1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri- and tetrameric coiled coils and thus self-assembling, multimeric integrin alpha5beta1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate 'anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin alpha5beta1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin alpha5beta1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro.

We describe the preparation and characterization of bioinorganic conjugates in which luminescent semiconductor CdSe-ZnS core-shell nanocrystal quantum dots (QDs) were coupled to antibodies through the use of an avidin bridge adsorbed to the nanocrystal surface via electrostatic self-assembly. Avidin, a highly positively charged protein, was found to adsorb tightly to QDs modified with dihydrolipoic acid, which gives their surface a homogeneous negative charge. QD conjugation to biotinylated antibodies subsequently is readily achieved. Fluoroimmunoassays utilizing these antibody conjugated QDs were successful in the detection of protein toxins (staphylococcal enterotoxin B, cholera toxin). QD-antibody conjugates formed in such a facile manner permit their use as a common immuno reagent, and in the development of multianalyte detection.