Fibril incubation

Preparation of mixed peptide fibrils was similar for both of these labelled peptides 1% (by weight) was incubated at 37 °C with the other, unlabelled peptide in 10% CH3CN/H20atpH2.ForTTRio i9with l%dansyl-TTRio5 n5, a blue shift and dansyl anisotropy increase were observed, indicating the inclusion of dansyl-TTRios-i 15 into fibrils. CD spectroscopy proved that the stmcture was primarily p-sheet. 

They have many of the morphological and ultrastructural characteristics of disease filaments [11, 12] (Fig. 45-5). Assembly is a nucleation-dependent process that occurs through its amino-terminal repeats. The carboxy-terminal region, in contrast, is inhibitory. Assembly is accompanied by the transition from random coil to a [3-pleated sheet. By electron diffraction, a-synuclein filaments show a conformation characteristic of amyloid fibers. Under the conditions of these experiments, P- and y-synucleins failed to assemble, consistent with their absence from the filamentous lesions of the human diseases. When incubated with a-synuclein, P- and y-synucleins inhibit the fibrillation of a-synuclein, suggesting that they may indirectly influence the pathogenesis of Lewy body diseases and multiple system atrophy. 

In this model (Fay et al., 2005), portions of the N- and C-terminal regions, specifically residues 6 and 137, are in close proximity in the fibril, and the C-terminal domain retains a native-like structure. There is evidence that this non-amyloid-like fibril can convert to the cross-/ -containing filament with heat treatment, incubation at low pH (Bousset et al., 2003), or extensive drying (Fay et al., 2005), but it is unclear what sort of structural change might link the two fibril types. 

The ubiquity of this power-law behaviour in SCG tests on PE has been the subject of considerable discussion, usually based on the assumption of a fibril creep failure mechanism [43, 45, 46, 47, 76, 79]. At high and intermediate K, after a certain induction period, steady-state crack advance is generally observed to occur by a stick-slip mechanism all or part of the fibrillar zone breaks down rapidly after an incubation time during which fibril creep takes place. The crack-tip then advances rapidly over a short distance and a new fibrillar zone stabilises, as sketched in Fig. 12. 

Dianilino-l,T-binaphthyl-5,5 -disulfonate (bis-ANS) is a probe that has been shown to increase in fluorescence with soluble Ap in acidic buffer solutions.24 Briefly, Ap 42 was incubated for 30 minutes at room temperature in the presence of different polyphenols. Bis-ANS fluorescence (excitation = 360 nm, emission = 485 nm) was then measured by dilution of 100-pl aliquots in a final volume of 300 pi of citrate buffer (30 mM, pH 2.4) containing bis-ANS (25 pM), using a fluorescence multiwell plate reader (Bio-Tek Instruments , Inc.). To determine amyloid fibril formation, the thioflavin T (Th-T) fluorescence method was performed as previously described.24 Briefly, a fresh solution of Ap 42 was incubated at 37°C for 24 hours in phosphate-buffered saline (pH 7.4). After incubation, a 100-pl aliquot of solution was added in a final volume of 300 pi of phosphate buffer (50 mM, pH 6.0) containing 5 pM Th-T in the presence of different drugs. Fluorescence was then monitored at excitation and emission wavelengths of 450 and 485 nm, respectively. 

Fig. 15.2. Direct observation of (32-m amyloid fibril growth obtained by TIRFM. Adapted from ref. [13] with permission. Incubation times are 0, 30, 60, and 90 min...

For purposes of this manuscript, we wish to concentrate only on the steps leading to the formation of desmosines, amino acids found predominantly in elastin. With respect to their formation, the following suggests their spontaneous formation from peptidyl lysine and the oxidation product, peptidyl allysine. Narayanan et al. (28,29) have shown that when purified lysyl oxidase and non-crosslined elastin, specifically tropoelastin, are incubated together, the desmosines are formed. Desmosine formation, however, only occurs at temperatures that favor fibrillar arrangements of tropoelastin. Subsequently, it is felt that the maturation of non-crosslinked elastin into cross-linked elastin appears to involve only two major steps, namely insolublization through the formation of fibrils and fixation of the fibrils by crosslinking. 

Recrystallized trypsin (Armour) had no effect on the purified fibers, but commercial grade trypsin effected the dissolution of the fibers when incubated overnight at 37°C. This was apparently due to the presence in the commercial trypsin preparations of pancreatic elastase (Bald and Banga, 1949). When treated for a short time with a partly purified preparation of elastase, obtained from pancreatin, the elastic fibers were eroded to a degree that the smooth cylindrical structure was lost and under the light microscope the fiber appeared to be composed of a pair of intertwined fibrils each approximately 2 n ln diameter. The process of dissolution then accelerated, and in favorable specimens each fibril was seen to resolve into a pair of similarly intertwined members, each with a diameter of approximately 1 ft. These 4 strands were very friable and under Brownian motion rapidly broke up into short beadlike segments and soon disappeared. 

The seven-residue peptide A -acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH2 is shown by electron microscopy to form highly ordered fibrils upon incubation of aqueous solutions. X-ray powder diffraction and optical birefringence measurements have confirmed that these are amyloid fibrils. The peptide conformation and supramolecular organization in fibrils were investigated by solid state NMR. ... 

In vitro studies indicate that synthetic A 3i 2 (A(342) can form insoluble aggregates and cause neurotoxicity after incubation for several days (Forloni et al., 1996). These A(342 assemblies may be stained by Congo red and Thioflavin S, similar to those observed in the AD brain (Jarrett and Lansbury, 1993). Both APi o (A(34o) and A 42 are formed intracellularly, but exert damaging effects when transported outside of cells. A 42 is more hydrophobic and toxic than AP4o, and its fibrils clump together to form amyloid plaques (Lue et al., 1999). Although A. 42 is the abundant Ap form in neuritic plaques, AP40 is more abundant in cerebrovascular plaques (Lue et al., 1999). 

FIGURE 9.7 Site-directed mutagenesis. Fibril formation was inhibited by incubation of 1 100 molar ratios of BChE or BSP41 with A(3. The BSP41 inactive mutant W8R was ineffective. Data is presented as percentage of the basal rate obtained with A(3 alone over time. 

Formation of big combined biomolecular structures in a buffer solution was verified by photoluminescence experiments during which amyloid specific dye (thioflavin T) was mixed into the colloidal solution. Thioflavin T related increase in the intensity of fluorescence was detected for the solutions with both GDH-Ap40 and Trx-Ap40 hybrid proteins compared to the solutions with single biomolecular components. The intensity increase was obtained after incubation of protein solutions at 310 K in PBS buffer (pH = 7.4) for two days. It was supposed that this period was required for formation of fibrils because the increase in the intensity of fluorescence was comparable with the results obtained for the solution with lysozyme fibrils. 

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