Pepsin−colloidal gold conjugates were prepared by a simple protein-friendly process and the enzymatic
activity of the bioconjugates is reported. The pepsin−gold conjugates are obtained by mixing colloidal gold
and protein solutions at pH = 3 and, thereafter, centrifugation, washing, and redispersion of the pepsin−gold conjugate material in water. The bioconjugates in solution were characterized by UV−vis spectroscopy,
fluorescence spectroscopy, and biocatalytic activity measurements while films of the bioconjugate material
obtained by solvent evaporation on suitable substrates were further analyzed by scanning electron microscopy
(SEM), energy dispersive analysis of X-rays (EDAX), transmission electron spectroscopy (TEM), Fourier
transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). While TEM and SEM
measurements showed aggregates of the enzyme/colloidal gold conjugates, the intactness of secondary and
tertiary structures of the enzyme, as determined by FTIR and fluorescence spectroscopies and confirmed
by biocatalytic activity measurements, clearly indicates that the enzyme is stable in its natural state and
is possibly stabilized by the colloidal gold particles. The enzyme in the pepsin−Au bioconjugate retained
substantial biocatalytic activity and was more stable than the free enzyme in solution.
Autophagy is markedly impaired in Alzheimer’s disease (AD). Here we reveal unique autophagy dysregulation within neurons in five AD mouse models in vivo and identify its basis using a neuron-specific transgenic mRFP-eGFP-LC3 probe of autophagy and pH, multiplex confocal imaging and correlative light electron microscopy. Autolysosome acidification declines in neurons well before extracellular amyloid deposition, associated with markedly lowered vATPase activity and build-up of Aβ/APP-βCTF selectively within enlarged de-acidified autolysosomes. In more compromised yet still intact neurons, profuse Aβ-positive autophagic vacuoles (AVs) pack into large membrane blebs forming flower-like perikaryal rosettes. This unique pattern, termed PANTHOS (poisonous anthos (flower)), is also present in AD brains. Additional AVs coalesce into peri-nuclear networks of membrane tubules where fibrillar β-amyloid accumulates intraluminally. Lysosomal membrane permeabilization, cathepsin release and lysosomal cell death ensue, accompanied by microglial invasion. Quantitative analyses confirm that individual neurons exhibiting PANTHOS are the principal source of senile plaques in amyloid precursor protein AD models.
The controlled assembly of nanoparticles in thin film form on solid supports, both as monolayers and as superlattice structures, is a problem of considerable topical interest. Among the many interactions used to program the assembly of nanoparticles, electrostatic forces are particularly interesting for many reasons. This Account deals with assembling surface-modified nanoparticles in thin film form using electrostatic interactions at the air-water interface and in thermally evaporated lipid films. The generality of the electrostatic assembly protocol is demonstrated in the immobilization of DNA and proteins in lipid films.
The assembly of aqueous gold nanoparticles on the surface of polyurethane (PU) spheres
leading to [gold nanoparticle shell]−[polyurethane core] structures is demonstrated. The
assembly of gold nanoparticles on the polymer microspheres occurs through interaction of
the nitrogens in the polymer with the nanoparticles. Such direct assembly obviates the need
to perform additional surface modification of the polymer microspheres, which is an important
step in other polymer-based core−shell structure protocols. The nanogold−PU material is
then conjugated with the enzyme pepsin, leading to the formation of a new class of biocatalyst.
In relation to the free enzyme in solution, the new bioconjugate material exhibited a slightly
higher biocatalytic activity and significantly enhanced pH and temperature stability. The
use of gold nanoparticle-labeled polymer microspheres in pepsin bioconjugation enables easy
separation from the reaction medium and reuse of the bioconjugate over six reaction cycles.
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