Anticalins versus antibodies: made-to-order binding proteins for small molecules

Engineering proteins to bind small molecules presents a challenge as daunting as drug discovery, for both hinge upon our understanding of receptor-ligand molecular recognition. However, powerful techniques from combinatorial molecular biology can be used to rapidly select artificial receptors. While traditionally researchers have relied upon antibody technologies as a source of new binding proteins, the lipocalin scaffold has recently emerged as an adaptable receptor for small molecule binding. 'Anticalins', engineered lipocalin variants, offer some advantages over traditional antibody technology and illuminate features of molecular recognition between receptors and small molecule ligands.     

Elucidation of the Mechanism of Enhanced Insulin Uptake and Release from pH Responsive Hydrogels

Summary: Environmentally responsive hydrogels composed of poly(methacrylic acid-g-ethylene glycol) (P(MAA-g-EG)) have shown promise for oral insulin delivery due to their pH responsive complexation behavior. A series of hydrogel formulations were polymerized with varying amounts of crosslinker and varying monomer volume fraction. The mesh size of the network depended primarily on pH, varying from 8.0 to 27.2 nm. Insulin loading efficiency varied directly with crosslink density, ranging from 42.7 to 84.9% of available insulin loaded into the hydrogels. The release of insulin was performed with each polymer formulation at 5 pH levels ranging from 2.7 to 6.8. Insulin release was less than 20% for all formulations tested with insulin for the duration of the 3 hour release study for all pH levels considered except when the pH was 6.8, at which point the release occurred as a burst. Loading studies performed with insulin glargine, an insulin analog with an increased pI, showed the same trends as native insulin. However, the release of insulin glargine only occurred at a pH level above that of the pI of the protein. These results indicate that hydrogen bonds and ionic interactions between the protein and P(MAA-g-EG) may strongly influence its loading and release behavior in vitro.     

The kinetics of electroreduction of europium(III) cations at bismuth single-crystal electrode

Electroreduction of Eu(ClO₄)₃ and Eu₂(SO₄)₃ has been studied at electrochemically polished \textBi( 01[`1] ) {\text{Bi}}\left( {01\bar{1}} \right) single-crystal electrode in an acidic HClO₄ or H₂SO₄ (pH ~3) aqueous solution with LiClO₄ or Na₂SO₄ additions as a surface inactive electrolyte. The Eu³⁺ cations electroreduction rate depends on the electrode potential applied and the concentration of the supporting electrolyte as well as the concentration of the Eu³⁺ ions. At the more negative electrode potentials than the zero charge potential (zcp), the diffusion current plateaus were observed. The values of the rate constant for the heterogeneous reaction calculated at zcp are independent of the base electrolyte concentration studied. Analysis of the kinetic data corrected for the electrical double-layer effect shows that the coincidence of the corrected Tafel plots can be achieved, assuming that the effective charge of a reactant (+1.6) is significantly lower than it would be expected (z _A = +3).     

Green fluorescent protein in inertially injected aqueous nanodroplets

We inertially inject and study the contents of optically trappable aqueous nanodroplets (hydrosomes) emulsified in a perfluorinated matrix. A new piezoelectric actuated device for production of single hydrosomes on demand is introduced. Hydrosomes containing enhanced green fluorescent protein (EGFP) were injected, optically trapped, and held at the focus of an excitation laser in a confocal microscope, and single-molecule photobleaching events were observed. The rotational diffusion time of EGFP in trapped hydrosomes was measured using time-resolved fluorescence anisotropy. In free solution, the mean rotational diffusion time was determined to be 13.8 +/- 0.1 ns at 3 microM and 14.0 +/- 0.2 ns at 10 microM. In hydrosomes, the mean rotational diffusion time was similar and determined to be 12.6 +/- 1.0 ns at 3 microM and 15.5 +/- 1.6 ns at 10 microM. We conclude that the rotational motion inside the nanodroplets is consistent with rotation in free solution and that the protein therefore does not aggregate at the water-oil interface. Protein can be confined in hydrosomes with high efficiency using this technique, which provides an alternative to surface attachment or lipid encapsulation and opens up new avenues of research using single molecules contained in fluid nanovolumes.     

Book Review: Biomimetic Materials and Design

Biomimetic materials and design;Edited by Angela K. Dillow and Anthony M. Lowman Catalysis in application;Edited by S.D. Jackson, J.S.J. Hargreaves and D. Lennon This revised version was published online in June 2006 with corrections to the Cover Date.