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Amyloidoses are diseases, including some currently prominent such as Alzheimer's disease, bovine spongiform encephalophaty (BSE) and Type II diabetes, in which soluble proteins are deposited in a specific, highly stable, fibrillar form. The amyloid fibrils are made up of protofilaments whose molecular structure is composed of pairs of beta-sheets in a helical form that allows them to be continuously hydrogen-bonded along the length of the fibril. The observation that similar fibrils are generated from different proteins indicates that fibril formation is accompanied by structural conversion. The transmissible spongiform encephalopathies, such as BSE and kuru, involve an infectious agent identified with the prion protein. The properties of the agent are more consistent with prion amyloid than the protein itself, suggesting infectivity in these diseases in equivalent to the 'seeding' of amyloid fibrils at a new site.

The effect of glutaraldehyde (GTA) on the course of collagen gel formation was studied by measuring the absorbance against time. It was found that the t1/2 of fibril formation decreased with the addition of GTA and reached a minimum at a concentration of 6 microliters of GTA per g of collagen. For GTA concentrations, [GTA], above this value, t1/2 increased again and fibril formation was inhibited at concentrations of about 50-60 microliters of GTA per g of collagen. Thermal analysis showed that the denaturation temperature was the highest for the gels formed with [GTA] of 6 microliters/g, the transition peak also being the sharpest. At this [GTA], the compressive rigidity of the gels was also the highest. For low [GTA], above and below the optimum value, the fibrils formed had the normal collagen periodicity when observed in the electron microscope. This study shows that collagen gels which find applications as biomaterials can be effectively crosslinked at the gelation stage itself by the addition of low concentrations of GTA.

Polymerization of collagen in vitro has been studied with the electron microscope at early time points of fibril assembly. We have found morphologically distinct stages of aggregation, which we suggest represent successive steps in fibril formation. Linear growth of the fibril appears to occur by the tandem addition of aggregates to each other and subsequently to the ends of a subfibril; lateral growth occurs by the entwining, like a rope, of these subfibrils. Fibrillogenesis is also accompanied by extensive development of suprafibrillar order in which various patterns of parallel, spiral, and orthogonal sets of fibrils were frequently observed.

溶液中のコラーゲン分子は,適当な環境条件下でフィブリルを再構成する.本研究では,この再構成コラーゲンフィブリルの熱変性挙動を示差走査熱量測定(DSC)により,分子内に存在する架橋(少量の分子間架橋も含む)やフィブリルの集合状態との関連で検討した.すなわち豚皮より抽出した酸可溶性コラーゲン(ASC),中性塩可溶性コラーゲン(NSC)およびこれらのペプシン処理物(PASCおよびPNSC)の分子分散溶液から,種々の条件でフィプリルを再構成し,そのDSCを行なった.いずれのフィブリルも,そのDSCパターンにおいて明らかに熱変性による2つの吸熱ピークを示した.分子内架橋の少ないPASCやPNSCおよびNSCの吸熱ピークは,架橋の多いASCより約10°C低温度領域に検知された.フィブリルの熱変性処理条件をコントロールすることにより,低温側吸熱ピーク成分と高温側ピーク成分を分離して両者の分子内架橋の量を調べたが,相違は認められなかった.低温側ピ-ク成分のみを熱変性させて除き,高温側ピーク成分のみを未変性のまま再び酢酸に溶解後,フィブリルを再構成してみると,DSC曲線に再び2つの吸熱ピークが現われた。このことは,DSCにおける2つの吸熱ピークが,熱変性温度の異なる2つの成分の存在を反映しているのではなく,フィブリルのマクロな集合状態の差を反映していることを示唆している.比較的希薄な濃度の溶液からASCフィブリルを調製したところ,吸熱曲線は約10°C低温側にシフトするとともに,単一のピークを示すことを認めた,以上のことから,フィブリル化したコラーゲンの熱的構造安定性には,分子内架橋のような分子自体の構造の差異に加えて,フィブリルの集合状態のようなマクロな高次構造の差異が大きな影響を及ぼすと考えられる.

1. 1. The rate of fibrillogenesis by soluble collagen is reduced by ascorbic acid and dihydroxymaleic acid but not by dehydroascorbic acid. 2. 2. Upon incubation for 5 days with the inhibitors followed by dialysis, the ability to form gels is lost. Dehydroascorbic acid reduced the rate of fibrillogenesis. 3. 3. The normal electrophoretic pattern of collagen subunits was unaltered by treatment with dehydroascorbic acid for 5 days but was lost by treatment with the other two substances. 4. 4. In place of two distinct protein bands (ga 1 and ga 2 -chains) a diffuse region of stained material was observed. 5. 5. It is concluded that the ene-diol group is involved in both phenomena.

The effect of selenium (Se) on collagen characteristics and glutathione peroxidase (GSH-Px) activity in the skin of Japanese quails Coturnix coturnix japonica fed a formulated, semipurified, low-Se diet (basal) (0.05 ppm) was investigated. The quails exhibited severe Se-deficiency symptoms and significant reduction in skin GSH-Px activity at the end of 30 d. Selenium supplementation at a 2-ppm level restored the normal skin conditions and enhanced skin GSH-Px activity significantly. But a dietary Se level of 0.1 ppm was found to be inadequate in restoring the general skin conditions and GSH-Px activity. A markedly low total collagen content of about 23% was observed in the skin of quails fed the basal diet, compared to 39% of total collagen content in the skin of the 2-ppm Se-supplemented group. Molecular organization of skin collagen of quails on the basal and 0.1-ppm Se diet showed an abundance of monomeric forms with less crosslinks, compared to the presence of polymeric forms with more crosslinks, indicating enhanced stability in the skin collagen of quails on the 2-ppm diet. The delay in the in vitro fibril formation of collagen from the basal and 0.1-ppm Se groups, compared to a relatively faster rate in the case of the 2-ppm Se group, indicates a disturbance in the aggregation phenomenon of collagen. The increase in skin GSH-Px activity and concurrent increase in polymeric collagen on increasing the dietary Se level suggest a possible role for Se in collagen metabolism. ? 1986 The Humana Press Inc.

Having relatively similar melt viscosities, polyoxymethylene (POM), and copolyamide (CPA) on mixing (over the entire range of composition) form mixtures which differ from the initial polymers in viscosity and elastic properties of melts. The type of viscosity-composition curve is determined by shear stress. For extruded mixtures a 3–10 fold swelling is observed in the flow after emerging from the capillary. Results are due to specific fibre formation, in which POM forms hundreds of thousands of ultra-thin fibres in the matrix of CPA, which are oriented along the axis during extrusion of these mixtures through a capillary.

Acid-soluble collagens isolated from young and old rat tail tendon were fluorescent-labeled with dansyl hydrazine, which is capable of reacting with aldehyde groups in collagen. The dansyl fluorescence of aged collagen exhibited a weak peak at 525 nm, whereas that of young collagen had a stronger broad peak at 500 nm. Fibril formation in vitro was partially inhibited in these dansylated collagens. During the turbidity lag phase, the dansyl fluorescence was found to increase (30–50%), also shifting to 485 nm. These changes reveal the telopeptide conformation changes occurring during this period. A new fluorescence peak at 420 nm also increased during fibril formation. When the dansylated collagen was irradiated in air with uv light (340 nm), a rapid decrease of the dansyl fluorescence with a concurrent shift to 490 nm occurred. Also, the formation of fibrils was further inhibited. With increasing temperature, the dansyl fluorescence of young collagen decreased, whereas that of old collagen substantially increased, particularly at the denaturation temperature around 38°C. After denaturation, both fluorescences became similar in their intensity and position (490 nm). These findings are discussed in connection with both age-related structural changes of collagen and the mechanism of fibril formation.

The effects of various proteoglycan samples, isolated from human articular cartilage of different ages, on the rate of the lateral growth phase of the fibril formation of collagen type II were studied by turbidimetry. In general, proteoglycan aggregates accelerate fibrillogenesis, whereas non-aggregating proteoglycans retard this process. The only exception were non-aggregating proteoglycans from very young cartilage, which stimulated the fibril formation strongly. The extent of stimulation by proteoglycans from hip and knee cartilage were compared. The effects of non-aggregating proteoglycans dominate those of aggregated proteoglycans. Chondroitinase ABC digestion of proteoglycan samples did not change the effects on the fibrillogenesis of collagen type II, when these samples were isolated from 18 years-old knee cartilage. The collagen fibril formation was less stimulated in the presence of ABC-ase digested proteoglycan samples from 0-3 month-old knee cartilage, suggesting a primary role for keratan sulphate and a possible influence of chondroitin sulphate when keratan sulphate is not present. Only proteoglycans from very old cartilage were able to reduce the amount of collagen fibrils formed in vitro. Proteoglycans could not be detected bound to the fibril pellet despite the fact that part of the pellet was not dissolvable in acetic acid. It is concluded that proteoglycans may play a regulatory role in collagen type II fibril formation in articular cartilage.

The silica valve structures related to β -chitin fibril formation were investigated in the centric diatom algae Cyclotella cryptica, Cyclotella nana, Cyclotella meneghiniana , and Thalassiosira fluviatilis with transmission and scanning electron microscopy. The results for T. fluviatilis are given in detail: The silica valves are characterized by a marginal ring of up to 86 pores and a central, less regular set of pores. The pores are surrounded by a system of chambers and supporting structures. The β -chitin fibrils originate from these pore apparatuses. All pores may function in fibril formation. Normally one, but sometimes two, fibril is seen to be attached in a pore. Each pore may be the source of numerous, sequentially formed fibrils, as judged from the huge number of chitin fibrils, relative to the number of cells, when the fibrils are continually detached from the cells by aerating the cultures. In the investigated Cyclotella species, the situation is very similar with respect to pores and chitin fibrils, with species-specific variations in the architecture of the pore apparatus and the valves. It is concluded that the pores of the investigated centric diatoms are structures specialized for the formation of β -chitin fibrils.

Alkylureas inhibit the rate of in vitro fibril formation at 10 m m range of concentrations. There is a direct correlation between the extent of inhibition and the length of the alkyl chain (degree of hydrophobicity). When the alkylureas are added during the lag phase, the extent of inhibition depends on the time, after the onset of polymerization, in which the alkylurea is added. The effect of alkylureas is reversible since after dialysis the rate of fibril formation is normal. In conditions in which the lag phase is very short or not observable, the rate of fibril formation is not affected by the alkylureas. Ethylurea inhibits the rate of fibril formation but the extent of polymerization appears to be unaffected. In the presence of alkylurea there is an increase in the activation energy. It is concluded that hydrophobic interactions are significantly involved in the stabilization of intermediates formed during the lag phase.

Sodium dodecyl sulphate (SDS) was used to weaken both the electrostatic and the hydrophobic interactions during collagen fibrillogenesis in vitro. The rate and extent of fibril formation as well as fibril morphology were affected by SDS concentration. Both the formation of large fibrils at 0.3 mM-SDS and the complete cessation of fibril formation at 0.5 mM-SDS were considered to be the result of SDS-induced conformational changes in the non-helical telopeptides. A possible mechanism of SDS interaction with the N-terminal and the distal region of the C-terminal telopeptides is offered.

The deformation of four types of thin films of polyoxymethylene is discussed. Failure, even at elevated temperatures, occurred by micronecking or in a brittle manner. Slip bands were observed in some of the samples. Moire patterns from portions of the sample indicated little internal distortion of the lamellae during the brittle failure. Micronecking with fibril formation occurred primarily during draw at temperatures near the α transition temperature. The results are in general agreement with concepts of the deformation process previously developed on the basis of work with thin films of polyethylene but cannot be applied directly to the mechanism of deformation of bulk samples. Die Verformung von vier auf verschiedene Weise hergestellten Typen von dunnen Polyoxymethylenfilmen wurde untersucht. Die Filme zerresen, sogar bei erhohter Temperatur, unter mikroskopischer Schulter-Hals-Bildung oder durch sproden Bruch. In einigen Proben wurden Gleitbander beobachtet. Moire-Figuren an verschiedenen Stellen der Proben deuten auf Verzerrungen innerhalb der Lamellen wahrend des sproden Zerreisens. Schulter-Hals-Bildung verbunden mit Fibrillenbildung erfolgt besonders dann, wenn die Dehnung im Bereich der α-Ubergangs-Temperatur erfolgt. Die Ergebnisse sind in Ubereinstimmung mit Vorstellungen uber den Deformationsvorgang, die fruher an Hand von Untersuchungen mit Polyathylenfilmen entwickelt wurden; sie erlauben aber keine direkten Aussagen uber den Mechanismus der Deformation groserer Probekorper.

About 50% of collagen in calf skin was solubilized by repeatedly changing the pH of an extraction medium from acid to neutral and neutral to acid and keeping temperature at 23°-25°C during acid extraction. This method is called 'pH relaxation extraction'.Addition of glucose (10%) to neutral buffer solubilized a large amount of collagen from calf skin.It was found that the collagen solubilized by these methods contained more β component than α and showed normal capability for fibril formation. The rate of fibril formation was higher than that of so-called acid soluble collagen. Amino and composition of the pH relaxation soluble collagen was little different from that of the acid soluble collagen.The mechanism of collagen solubilizatron by pH relaxation extraction is discussed in relation to the associating forces of insoluble collagen.

In electron microscope examinations of leg muscles from fetal and postnatal rats the development of SR tubules precedes the development of Z lines and the formation of fibrils, thus showing that a function of SR in Z line development is possible. Early stages of fibril formation reveal very small Z lines (0.06 μ thick) which are encircled by SR tubules separated from the Z lines by a space about 100 A wide, and this space is traversed by electron-opaque strands which appear to be connections between SR tubules and Z lines. The encircling SR tubules are observed from the beginning to the completion of Z line development. The small Z lines contain dense lines transversely oriented between thin filament tips that resemble transversely oriented dense lines found in fully developed Z lines. It is concluded that Z line enlargement involves growth in circumference of encircling SR tubules as the tubules incorporate additional layers of thin filament tips. It is suggested that the substance represented by electronopaque strands connecting SR tubules and Z lines might provide precursors for the substance represented by transversely oriented dense lines between thin filament tips.

Based on recent studies of the morphology and of shear bands and crazes in amorphous polymers it is suggested that shear is the basic mechanism of plastic deformation. Three stages are proposed for the formation of crazes, shear yielding at a defect or craze tip due to stress concentration, nodular movement resulting in fibril formation, followed by void formation between the fibrils.

The effect in vitro of nitrogen mustard (HN2) on physical parameters of rat tail tendon collagen and rabbit skin collagen was studied. Intrinsc viscosities, helix-coil transition temperatures (by viscosimetry and rates of fibril formation (by turbidimetry) of HN2-treated (1 mg/ml HN2, 0·.9% saline, 2 hr at 25°) and control collagens were compared. Helix-coil transition temperatures were not affected by HN 2 treatment. Fibril formation by both collagens was inhibited by HN 2 , but apparently this effect was nonspecific since it was reversible by dialysis. Rat tail tendon collagen aggregates in solution were disaggregated by HN 2 treatment. Only 0·5 per cent of the hydroxyproline of collagen became dialyzable after HN 2 treatment.

The formation of protein fibrils, and in particular amyloid fibrils, underlies many human diseases. Understanding fibril formation mechanisms is important for understanding disease pathology, but fibril formation kinetics can be complicated, making the relationship between experimental observables and specific mechanisms unclear. Here we examine one often-proposed fibril formation mechanism, nucleated polymerization with off-pathway aggregation. We use the characteristics of this mechanism to derive three tests that can be performed on experimental data to identify it. We also find that this mechanism has an especially striking feature: although increasing protein concentrations generally cause simple nucleated polymerizations to reach completion faster, they cause nucleated polymerizations with off-pathway aggregation to reach completion more slowly when the protein concentration becomes too high.

The early stages of peptide aggregation are currently not accessible by experimental techniques at atomic resolution. In this article, we address this problem through the application of a mixed simulation scheme in which a preliminary coarse-grained Monte Carlo analysis of the free-energy landscape is used to identify representative conformations of the aggregates and subsequent all-atom molecular dynamics simulations are used to analyze in detail possible pathways for the stabilization of oligomers. This protocol was applied to systems consisting of multiple copies of the model peptide GNNQQNY, whose detailed structures in the aggregated state have been recently solved in another study. The analysis of the various trajectories provides dynamical and structural insight into the details of aggregation. In particular, the simulations suggest a hierarchical mechanism characterized by the initial formation of stable parallel β-sheet dimers and identify the formation of the polar zipper motif as a fundamental feature for the stabilization of initial oligomers. Simulation results are consistent with experimentally derived observations and provide an atomically detailed view of the putative initial stages of fibril formation.

Aging and apolipoprotein E4 (apoE4) expression are strong risk factors for the development of Alzheimer's disease (AD); however, their pathological roles remain to be clarified. In the process of AD development, the conversion of the nontoxic amyloid beta-protein (Abeta) monomer to its toxic aggregates is a fundamental process. We previously hypothesized that Abeta aggregation is accelerated through the generation of GM1 ganglioside (GM1)-bound Abeta which acts as a seed for Abeta fibril formation. Here we report that GM1 level in detergent-resistant membrane microdomains (DRMs) of synaptosomes increased with age and that this increase was significantly pronounced in the apoE4- than the apoE3-knock-in mouse brain. Furthermore, we show that Abeta aggregation is markedly accelerated in the presence of the synaptosomes of the aged apoE4-knock-in mouse brain. These observations suggest that aging and apoE4 expression cooperatively accelerate Abeta aggregation in the brain through an increase in the level of GM1 in neuronal membranes.

To examine the effect of aggregation sequence QGGYQQQYNP from yeast Sup35 on fibril formation of sperm whale apomyoglobin (apoMb), we constructed several mutants via substitution. Urea-induced unfolding of apoMb confirms that the substitution of the aggregation sequence does not significantly affect the stability of the mutants compared to wild type (WT) at pH 4.2. Under this condition, however, despite the difference in rate most apoMb mutants form fibrils more readily than WT with distinct morphology. These results suggest that the aggregation sequence facilitates fibril assembly of apoMb at acidic pH in vitro and this facilitation depends on the regions replaced.

SummaryThe inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway.

Background : Brain amyloid plaque, a diagnostic feature of Alzheimer's disease (AD), contains an insoluble fibrillar core that is composed primarily of variants of the β-amyloid protein (Aβ). As Aβ amyloid fibrils may initiate neurodegeneration, the inhibition of fibril formation is a possible therapeutic strategy. Very little is known about the early steps of the process, however. Results : Atomic force microscopy was used to follow amyloid fibril formation in vitro by the Aβ variants Aβ1-40 and Aβ1-42. Both variants first form small ordered aggregates that grow slowly and then rapidly disappear, while prototypical amyloid fibrils of two discrete morphologies appear. Aβ1-42 aggregates much more rapidly than Aβ1-40, which is consistent with its connection to early-onset AD. We propose that the metastable intermediate species be called Aβ amyloid protofibrils. Conclusions : Aβ protofibrils are likely to be intermediates in the in vitro assembly of Aβ amyloid fibrils, but their in vivo role has yet to be determined. Numerous reports of a nonfibrillar form of Aβ aggregate in the brains of individuals who are predisposed to AD suggest the existence of a precursor form, possibly the protofibril. Thus, stabilization of Aβ protofibrils may be a useful therapeutic strategy.

This report provides a brief preliminary account of the solution crystallization of isotactic polystyrene under the influence of velocity gradients. During crystallizations from 1,3,5-trimethylbenzene and cyclohexanol solutions, the polymer appears in a number of morphological forms which are composed of microfibrillar units. In isotactic polystyrene produced by heterogeneous Ziegler–Natta catalysis, similar microfibrillar units are observed. It is suggested, therefore, that these two apparently different processes have a common underlying mechanism in the formation of the microfibrils. The possible mechanisms of fibril formation are discussed.