Full-length native rat amylin 1-37 has previously been widely shown to be unable to form fibrils and to lack the toxicity of the human amylin form leading to its use as a non-amyloidogenic control peptide. A recent study has suggested that rat amylin 1-37 forms amyloidogenic beta-sheet structures in the presence of the human amylin form and suggested that this property could promote toxicity. Using TEM analysis we show here fibril formation by synthetic rat amylin 1-37 and 8-37 peptides when the lyophilized HPLC purified peptides are initially dissolved in 20 mM Tris-HCl. Dissolution of synthetic rat amylin 1-37 and 8-37 peptides in H2O or phosphate buffered saline failed to produce fibrils. Addition of 20 mM Tris-HCl to synthetic rat amylin 1-37 and 8-37 peptides initially dissolved in H2O also failed to induce fibril formation. The rat amylin fibrils have a uniform structure and bind Congo red suggesting that they are amyloid fibrils. The rat amylin fibrils also bind catalase, which could be inhibited by Amyloid-beta 31-35 and a catalase amyloid-beta binding domain-like peptide (R9). The rat amylin 1-37 and 8-37 fibrils are toxic in both human pancreatic islet and neuronal cell culture systems. The toxicity of rat amylin fibrils can be inhibited by an amylin receptor antagonist (AC187) and a caspase inhibitor (zVAD-fmk) in a similar manner to previous observations for human amylin toxicity. Chemically induced rat amylin fibril formation of uniform structured fibrils provides a potentially novel anti-amyloid drug discovery tool. (c) 2012 Elsevier Ltd. All rights reserved.

Collagen is the most abundant protein in the human body, and has primary roles in the formation of tendons, cartilage and bone, it provides mechanical strength to skin and indeed almost every organ and muscle is associated with a layer of collagen. It is thus a key component of the extracellular matrix. Here we have studied the in vitro fibrillogenesis of acetic acid-soluble collagen type I under physiological and varying non-physiological conditions by TEM from negatively stained specimens. At pH 2.5 the collagen heterotrimer remains soluble at increasing buffer concentrations and in the presence of increasing NaCl concentrations. At pH 4.5 molecular aggregates form at low NaCl concentrations, but at higher NaCl concentrations fibrils with a diffuse similar to 11 nm banding are formed. At pH 7.0, initial molecular aggregates form at low NaCl concentrations that progressively form characteristic similar to 67 nm D-banded collagen fibrils at intermediate NaCl concentrations that cluster to form thicker multi-fibril D-banded fibres in higher NaCl concentrations. By contrast, increasing concentrations of sodium phosphate at pH 7.0 leads to the formation of flexuous, unbanded fibrils at higher concentrations from the initial, loosely aggregated form of collagen. At higher pHs, the formation of D-banded fibrils is less efficient, particularly at pH 9.0. Thus at neutral pH, the presence of chloride anions, rather than sodium cations, is required for the production of D-banded collagen fibrils; higher than normal physiological chloride concentrations in the form of NaCl or Tris-HCl at neutral pH, but not phosphate buffer, can also lead to the efficient in vitro formation of D-banded collagen fibrils. (C) 2013 Elsevier Ltd. All rights reserved.

Caf1 of the plague bacterium, Yersinia pestis is a polymeric virulence factor and vaccine component. formed from monomers by a donor strand exchange (DSE) mechanism. Here. EM images of Caf1 reveal flexible polymers up to 1 5 mu m long (4 MDa). The bead-like structures along the polymer are 58 +/- 1 nm long and correspond to single Caf1 proteins. Short polymers often form circles, presumably by DSE. We also provide the first images of proteins bound to alhydrogel adjuvant. Call, hemocyanin and anthrax PA are all resolved clearly and Caf1 exhibits adjuvant bound stretches with long intervening loops draped from the edges. (C) 2010 Elsevier Ltd. All rights reserved.

The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A novel form of macroscopic 3D crystals of turnip yellow mosaic virus has been grown in the negative staining solution. On the basis of the experimental results, the hypothesis is advanced that microscopic arrays might be planar crystallisation nuclei. The formation of 2D crystals and the enhancing effect of polyethylene glycol on the self-organisation of virions at the air/water interface are discussed. Synopsis: The formation of 2D arrays of icosahedral viruses was investigated by spectroscopic and transmission electron microscopic methods. (c) 2007 Elsevier Ltd. All rights reserved.

There is increasing interest in the role of brain cholesterol in Alzheimer's disease and the contribution of cholesterol to the formation of amyloid plaques. This paper presents a TEM study showing the binding of soluble similar to 10 nm diameter cholesterol-PEG 600 micelles to amyloid-beta(1-42) (A beta(1-42)) fibrils formed either in the presence of this cholesterol derivative or to preformed fibrils generated under four different fibrillogenesis conditions. Specimens negatively stained with uranyl acetate revealed that during 24 h fibrillogenesis at 37 degrees C the cholesterol-PEG micelles bound periodically to A beta(1-42) protofibrils and apparently also formed a thin smooth unbroken coating on mature double helical fibrils. Preformed protofibrils, generated in water alone or in the presence of 0.1 mM cupric sulphate, also exhibited periodic binding of cholesterol-PEG micelles, indicating the inherently helical nature of the protofibril. Double helical mature A beta(1-42) fibrils, generated in the presence of cholesterol microcrystals or hydrogen peroxide (I mM), bound cholesterol-PEG micelles with no immediately apparent regularity and without creating a smooth coating. The differing capacities of the A beta(1-42) protofibrils and mature double helical fibrils to bind cholesterol-PEG 600 may indicate differences in the accessibility of the micellar cholesterol to the purported A beta(17-21) hydrophobic cholesterol-binding motif on the fibril surfaces. (C) 2008 Elsevier Ltd. All rights reserved.

Collagen fibres have been shown by transmission electron microscopy to progressively bind the polyoxomolybdate ring-complex, termed molybdenum blue. Nucleation of cuboidal molybdenum blue microcrystals occurs on the surface of the collagen fibres, leading eventually to extensive coating of the fibres with microcrystals.

Aluminium-based vaccine adjuvants have been in use since the 1920s. Aluminium hydroxide (alum) that is the chemical basis of Alhydrogel, a widely used adjuvant, is a colloid that binds proteins to the particular surface for efficient presentation to the immune system during the vaccination process. Using conventional TEM and cryo-TEM we have shown that Alhydrogel can be finely dispersed by ultrasonication of the aqueous suspension. Clusters of ultrasonicated aluminium hydroxide micro-fibre crystals have been produced (similar to 10-100 nm), that are significantly smaller than those present the untreated Alhydrogel (similar to 2-12 mu m). However, even prolonged ultrasonication did not produce a homogenous suspension of single aluminium hydroxide micro-fibres. The TEM images of unstained and negatively stained air-dried Alhydrogel are very similar to those obtained by cryo-electron microscopy. Visualization of protein on the surface of the finely dispersed Alhydrogel by TEM is facilitated by prior ultrasonication. Several examples are given, including some of medical relevance, using proteins of widely ranging molecular mass and oligomerization state. Even with the smaller mass proteins, their presence on the Alhydrogel surface can be readily defined by TEM. It has been found that low quantities of protein tend to cross-link and aggregate the small Alhydogel clusters, in a more pronounced manner than high protein concentrations. This indicates that complete saturation of the available Alhydrogel surface with protein may be achieved, with minimal cross-linkage, and future exploitation of this treatment of Alhydrogel is likely to be of immediate value for more efficient vaccine production. (C) 2011 Elsevier Ltd. All rights reserved.

The Alzheimer's amyloid-beta (A beta) peptide exists as a number of naturally occurring forms due to differential proteolytic processing of its precursor molecule. Many of the A beta peptides of different lengths form fibrils in vitro, which often show polymorphisms in the fibril structure. This study presents a TEM based analysis of fibril formation by eighteen different A beta peptides ranging in length from 5 to 43 amino acids. Spectrophotometric analysis of Congo red binding to the fibrillar material has been assessed and the binding of human erythrocyte catalase (HEC) to A beta fibrils has also been investigated by TEM. The results show that a diverse range of A beta peptides form fibrils and also bind Congo red. The ability of both A beta 1-28 and A beta 29-40 to form fibrils indicates that there are at least two fibril-forming domains within the full-length A beta 1-40 sequence, the ability of many A beta peptides to form Congo red-binding aggregates suggests that there may be up to 4 possible aggregation promoting domains. The binding of HEC was limited to A beta forms containing residues 29-32. The differing capacities of fibrillar and ribbon-like structures may reflect the accessibility of the 29-32 region and suggest that HEC may be able to discriminate between different forms of A beta fibrils. (C) 2009 Elsevier Ltd. All rights reserved.