Reversible in vitro growth of Alzheimer disease beta-amyloid plaques by deposition of labeled amyloid peptide.

Abstract

The salient pathological feature of Azhelmer (1-4).Radloiodination. Peptides containing tyrosine were radiolabeled by oxidative radioiodination using Na125I and chloramine T and separated from free iodide by reverse-phase adsorption. Peptides not containing tyrosine were first acylated with the N-hydroxysuccinimide ester of 4-hydroxyphenylpropionic acid and then oxidatively radioiodinated as above. Labeled peptides containing methionine were then reduced from sulfoxide to native form with 2-mercaptoethAbbreviations: AD, Alzheimer disease; ,BA4, ,B-amyloid peptide; RP-HPLC, reversed-phase HPLC; 125I-PA4-(1-40), 1251-labeled ,BA4-(1-40). tTo whom reprint requests should be addressed. 5462The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Blood–brain barrier delivery of protein and non-viral gene therapeutics with molecular Trojan horses

“…Conjugation of FGF-2 to the TfRMAb results in an 80% reduction in stroke volume in the permanent MCAO model following delayed intravenous administration [21]. The Aβ 1-40 peptide rapidly binds pre-existing amyloid plaque [23], and Aβ 1-40 could be used as a peptide radiopharmaceutical for imaging brain amyloid with standard external detection modalities such as positron emission tomography (PET) or single photon computed emission tomography (SPCET). However, Aβ does not cross the BBB [24].…”

In Situ Formation of Protease-resistant Prion Protein in Transmissible Spongiform Encephalopathy-infected Brain Slices

“…35 S-PrP C (20 -50,000 cpm/reaction) from either mouse or hamster was then layered over the tissue and incubated at 37°C for 18 h. The supernatants were removed, and tissue sections were briefly rinsed and then treated with PK (0 to 200 g/ml) in reaction buffer lacking BSA, followed by incubation at 37°C for 1 h. Tissue sections were washed in reaction buffer containing 2 mM phenylmethylsulfonyl fluoride for 15 min. Conversion products were detected by solubilizing the tissue in SDS-PAGE sample loading buffer and analyzing for PK-resistant 35 SPrP by SDS-PAGE and fluorography. Alternatively, tissue sections were left on the slide and exposed directly to X-Omat AR film for autoradiography or dipped in NTB-2 liquid emulsion (Eastman Kodak Co.) and developed according to manufacturer's directions.…”

“…Cell-free Conversion Reaction-The cell-free conversion reaction was performed by incubating ϳ1.6 g of brain-derived PrP-res (prepared as described previously (26) except the proteinase K digestion step was omitted) with 35 S-PrP C (20,000 cpm, ϳ3 ng) lacking its glycophosphatidylinositol C-terminal anchor that was purified from murine fibroblast cells transfected with the Syrian hamster PrP gene as described previ-ously (19,20,22). The mixture (total volume of 16 l) was incubated for various lengths of time (0 -24 h), digested with PK at 100 g/ml for 1 h at 37°C, and analyzed by SDS-PAGE and fluorography.…”

“…Both the brain-derived PrP-res and nascent 35 S-PrP-res are reduced in molecular mass by 6 -7 kDa after PK digestion, whereas any unconverted 35 S-PrP C is completely degraded. The formation of 35 S-PrP-res cell-free conversion products requires the presence of PrP-res aggregates (20). Irreversible denaturation of PrP-res structure results in the coincident loss of conversion activity, resistance to PK, and a decrease in TSE infectivity (21).…”

“…The mechanism of PrP-res formation has been investigated in a cell-free conversion reaction in which [ 35 S]methioninelabeled PrP C ( 35 S-PrP C ) is converted into proteinase K (PK)-resistant 35 S-PrP ( 35 S-PrP-res) in the presence of TSE brainderived PrP-res (19). Both the brain-derived PrP-res and nascent 35 S-PrP-res are reduced in molecular mass by 6 -7 kDa after PK digestion, whereas any unconverted 35 S-PrP C is completely degraded.…”