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The amyloid beta (Aβ) peptide is regarded as a causative agent of Alzheimer's disease. In this study, fluorescent and luminescent fusion proteins were constructed to analyze Aβ aggregation. A system was developed to monitor changes in luminescence that provides information about Aβ aggregation. In the presence of monomeric Aβ, the fusion protein exhibits higher luminescence intensity, and the luminescence intensity is diminished after aggregation of the fusion protein and Aβ. In contrast, the fluorescence is sustained in the presence of Aβ. In the absence of Aβ, the fusion protein self-aggregates, and its luminescence and fluorescence are quenched, thus decreasing the background fluorescence and enhancing the detection of Aβ inside and outside the cells. The ratio of the luminescence intensity to the fluorescence intensity would allow the aggregation degrees of Aβ to be distinguished. This study would be a promising method for analyzing the aggregation state of a particular amyloid protein/peptide (monomer, oligomer, or fibril), as well as the distribution of the amyloid protein/peptide within and at the cell surface, by using a single fusion protein. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

We report the assembly of seven different antibodies (and two antigens) into functional supramolecular structures that are specifically designed to facilitate integration into devices using entirely biologically based bottom-up fabrication. This is enabled by the creation of an engineered IgG-binding domain (HG3T) with an N-terminal hexahistidine tag that facilitates purification and a C-terminal enzyme-activatable pentatyrosine “pro-tag” that facilitates covalent coupling to the pH stimuli-responsive polysaccharide, chitosan. Because we confer pH-stimuli responsiveness to the IgG-binding domain, it can be electrodeposited or otherwise assembled into many configurations. Importantly, we demonstrate the loading of both HG3T and antibodies can be achieved in a linear fashion so that quantitative assessment of antibodies and antigens is feasible. Our demonstration formats include: conventional multiwell plates, micropatterned electrodes, and fiber networks. We believe biologically based fabrication (i.e., biofabrication) provides bottom-up hierarchical assembly of a variety of nanoscale components for applications that range from point-of-care diagnostics to smart fabrics. Biotechnol. Bioeng. 2009;103: 231–240. © 2008 Wiley Periodicals, Inc.

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

Bilirubin oxidase (EC1.11.1.7) was used to catalyze the oxidative polymerization of 1,5-dihydroxynaphthalene to its polymer in a mixed solvent composed of dioxane, ethyl acetate, and acetate buffer. In an aqueous solution, the enzymatic oxidative polymerization hardly occurred and resulted in negligible yield mainly due to the poor solubility of 1,5-dihydroxynaphthalene. In the mixed solvent the conversion proceeded with a yield of ca. 70%. The polymer yield was studied with respect to reaction time and solvent components. Elemental analysis, UV-visible, fluorescent, and FT-IR spectroscopic analyses, proton NMR and electrochemical studies, and solubility in various organic solvents revealed that 1,5-dihydroxynaphthalene is polymerized by the CC coupling. The molecular weight of the polymeric products solubilized with DMF varied from low molecular weight product to high molecular weight polymer. From the chromatographic studies, the organic solvent–insoluble residue was suggested to be highly polymerized material. Based on these findings a possible mechanism for enzymatic polymerization of 1,5-dihydroxynaphthalene is presented: less stable intermediates produced enzymatically from 1,5-dihydroxynaphthalene undergo coupling and polymerization to ortho-1,5-dihydroxynaphthalene polymer, thereby resulting in a regioselective polymerization of 1,5-dihydroxynaphthalene. © 1993 John Wiley & Sons, Inc.

A novel modulating method of the expression of Saccharomyces PHO 5 gene, responsible for acid phosphatase (APase), is proposed. The method is based on electrochemical modulation of an effector (inorganic phosphate) concentration, as the gene expression is initiated below a threshold concentration of phosphate and is terminated above the threshold value. By positioning the yeast in the close neighborhood of a conducting polymer, the authors show the effectiveness of the electrochemical approach toward PHO 5 induction. Based on the approach, phosphate concentration is easily modulated at the boundary concentration by taking advantage of anion doping–undoping at a conducting polymer and the resulting anion localization–delocalization in the polymer, as the local enrichment of phosphate in the polymer results in the lowering of phosphate in the vicinity of polypyrrole. External phosphate concentration is thus electrochemically modulated when the conducting polymer is positioned in the close neighborhood of the yeast cells; thereby the PHO gene is induced. Here an electrochemical approach for the APase expression as a strategy of selective induction of specific genetic information is described. © 1993 John Wiley & Sons, Inc.

A cell-free protein-synthesizing system for the purpose of specific genetic information translation was constructed: ribosome, t-RNA, and enzymes extracted from yeast cells were combined with an immobilized mRNA. The soluble fraction mixed with energy sources and amino acids was incubated with the immobilized mRNA such as poly(U), yeast mRNA, and myeloma mRNA to incorporate [(3)H]phenylalanine into polypeptides of de novo synthesis. By supplying amino acids to these protein-synthesizing systems, amino acid incorporation was ascertained. Lower efficiency of the incorporation in the immobilized system than that of the homogeneous system was mainly attributed to the heterogeneous reaction where the mass-transfer process is diffusion limited. Results obtained show the possibility of a system for specific translation of a desired genetic code.