Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V‐genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V‐genes were shuffled at random and cloned for display as single‐chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V‐genes, with high specificities of binding to human self‐antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)‐10(6) M‐1s‐1, k(off) = 10(−2)s‐1 and Ka = 10(7) M‐1). The kinetics of association are typical of known Ab‐protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.

These authors contributed equally to this study.Abbreviations used: 3-MA, 3-methyladenine; FACS, fluorescenceactivated cell sorting; GFP, green fluorescent protein; LC3, microtubuleassociated protein 1 light chain 3; MEF, mouse embryonic fibroblasts; mTOR, mammalian target of rapamycin; PrP, prion protein; PrP AbstractLithium is used for several decades to treat manic-depressive illness (bipolar affective disorder). Recently, it was found that lithium induces autophagy, thereby promoting the clearance of mutant huntingtin and a-synucleins in experimental systems. We show here for the first time that lithium significantly reduces the amount of pathological prion protein (PrP Sc ) in

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We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP 90 -232 ) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP 90 -232 showed an infrared absorbance spectrum typical of proteins with a predominant ␣-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3-2 M guanidine hydrochloride), the secondary structure of SHaPrP 90 -232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel ␤-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm ؊1 . Kinetic analysis of the transition process showed that the decrease in ␣-helical structures and the increase in ␤-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No ␤-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP 90 -232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP 90 -232 . A new model for the structural transition and aggregation process of the prion protein is described.The prion protein (PrP) 1 is, following the protein-only hypothesis, the sole agent causing a group of neurodegenerative disorders (1, 2), the so-called prion diseases or prionoses (3). The most important ones among them are bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and Creutzfeldt-Jakob disease in humans.The crucial step in transmission and manifestation of prion diseases is the conversion of benign monomeric cellular prion protein (PrP C ), which has a mainly ␣-helical secondary structure, to pathogenic multimeric scrapie prion protein (PrP Sc ), which is predominantly folded into ␤-sheets (4, 5). It is noteworthy that PrP C and PrP Sc do not differ in their amino acid sequence.Similar mechanisms play an essential role in a number of other neurodegenerative disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Therefore, the coupled processes of protein misfolding and aggregation, the kinetics of these processes, and the molecular species involved are of fundamental interest.Late products of the conversion are amyloid fibrils and amyloid plaques, which are widely considered to be direct effectors of the above mentioned disorders. However, evidence is accumulating that intermediates or by-product...

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