Understanding the structure and formation of amyloid fibrils, the filamentous aggregates of proteins and peptides, is crucial in preventing diseases caused by their deposition and, moreover, for obtaining further insight into the mechanism of protein folding and misfolding. We have combined solid-state NMR, x-ray fiber diffraction, and atomic force microscopy to reveal the 3D structure of amyloid protofilament-like fibrils formed by a 22-residue K3 peptide (Ser 20 -Lys 41 ) of ␤2-microglobulin, a protein responsible for dialysis-related amyloidosis. Although a uniformly 13 C, 15 N-labeled sample was used for the NMR measurements, we could obtain the 3D structure of the fibrils on the basis of a large number of structural constraints. The conformation of K3 fibrils was found to be a ␤-strand-loop-␤-strand with each K3 molecule stacked in a parallel and staggered manner. It is suggested that the fibrillar conformation is stabilized by intermolecular interactions, rather than by intramolecular hydrophobic packing as seen in globular proteins. Together with thermodynamic studies of the full-length protein, formation of the fibrils is likely to require side chains on the intermolecular surface to pack tightly against those of adjacent monomers. By revealing the structure of ␤2-microglobulin protofilament-like fibrils, this work represents technical progress in analyzing amyloid fibrils in general through solid-state NMR.2,2,2-trifluoroethanol ͉ amyloid fibril ͉ dialysis-related amyloidosis ͉ protein misfolding ͉ x-ray fiber diffraction

The molecular mechanism responsible that determines cell fate after mitotic slippage is unclear. Here we investigate the post-mitotic effects of different mitotic aberrations—misaligned chromosomes produced by CENP-E inhibition and monopolar spindles resulting from Eg5 inhibition. Eg5 inhibition in cells with an impaired spindle assembly checkpoint (SAC) induces polyploidy through cytokinesis failure without a strong anti-proliferative effect. In contrast, CENP-E inhibition causes p53-mediated post-mitotic apoptosis triggered by chromosome missegregation. Pharmacological studies reveal that aneuploidy caused by the CENP-E inhibitor, Compound-A, in SAC-attenuated cells causes substantial proteotoxic stress and DNA damage. Polyploidy caused by the Eg5 inhibitor does not produce this effect. Furthermore, p53-mediated post-mitotic apoptosis is accompanied by aneuploidy-associated DNA damage response and unfolded protein response activation. Because Compound-A causes p53 accumulation and antitumour activity in an SAC-impaired xenograft model, CENP-E inhibitors could be potential anticancer drugs effective against SAC-impaired tumours.

PPE remains the most important strategy for protecting HCW from potentially fatal pathogens. Further research into optimal PPE design and use to improve the safety of HCWs is urgently needed.

Amyloidosis accompanies the deposition of normally soluble proteins into insoluble amyloid fibrils (1-3). Among various amyloidogenic proteins, ␤ 2 -microglobulin (␤2-m) 3 is a target of extensive study because of its clinical importance and a suitable size for examining the relation between protein folding and amyloid fibril formation (4 -11). ␤2-m, a typical immunoglobulin domain made of seven ␤-strands and one intramolecular disulfide bond (12, 13), is present as the non-polymorphic light chain of the class I major histocompatibility complex (14) (Fig. 1). As a part of its normal catabolic cycle, ␤2-m dissociated from the complex is transported in serum to the kidneys where the majority (95%) of it is degraded (15). Renal failure disrupts the clearance of ␤2-m from the serum and moreover ␤2-m does not pass through the dialysis membrane, resulting in an increase in the ␤2-m concentration by up to 50-fold in the blood circulation (15). When a high blood level is retained for more than 10 years, ␤2-m then self-associates to form amyloid fibrils, causing dialysis-related amyloidosis (15, 16).The results so far obtained with various approaches suggest a picture of ␤2-m amyloid fibrils with an increased amount of ␤-structure in comparison with the native structure, including the transformation of native ␤-turns, but with disordered Nand C-terminal regions (6 -9, 17). However, details remain unknown. To address the structure of amyloid fibrils, we have taken advantage of the unique properties of Trp fluorescence. One important feature of immunoglobulin domains is the presence of a buried Trp residue located close to the conserved disulfide bonds (18), leading to a strong quenching of Trp fluorescence (19,20). We noticed that, although ␤2-m has two Trp residues (i.e. Trp 60 and Trp 95 ), neither of these corresponds to the buried Trp common to other immunoglobulin domains. In the x-ray crystallographic structure of wild-type ␤2-m, while Trp 60 located on the ␤-turn connecting ␤-strands D and E is exposed to solvent, Trp 95 at the end of ␤-strand G is partially buried (Fig. 1). We designed and expressed a series of single Trp mutants in which Trp residues of wild-type ␤2-m have been deleted and a new Trp residue was introduced at various positions. Examination of the fluorescence spectra of mutants in the native and amyloid states revealed a notable conformational change upon the formation of amyloid fibrils. EXPERIMENTAL PROCEDURES␤2-m Amyloid Fibril Formation-The expression and purification of human recombinant ␤2-m and the single Trp mutants were achieved as described previously (21). Acidic pH fibrils were prepared by a repeated seed-dependent extension with human recombinant ␤2-m expressed in Escherichia coli (21). or yeast Pichia pastoris (22). Neutral pH fibrils were prepared at pH 7.0 by a repeated seeding starting with the acidic pH fibrils * This work was supported in part by the Takeda Science Foundation, and by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology o...

beta(2)-Microglobulin (beta2-m), a light chain of the major histocompatibility complex class I, forms amyloid fibrils in patients undergoing long-term haemodialysis, causing dialysis-related amyloidosis. Based on a comparison of the X-ray structure obtained at pH 5.7 and that of beta2-m in the histocompatibility complex, it has been proposed that the continuous D-strand observed in the crystal structure at pH 5.7 increases the propensity of beta2-m to self-associate via edge-to-edge interactions, thus initiating the formation of fibrils. To obtain further insight into the mechanism by which amyloid fibrils form, we determined the crystal structure of beta2-m at pH 7.0 at a resolution of up to 1.13 A. The crystal structure at pH 7.0 was basically the same as that at pH 5.6, suggesting that the conversion of the beta-bulge in strand D into a contiguous beta-strand is not unique to the crystals formed under slightly acidic conditions. In other words, although the formation of beta2-m fibrils was enhanced under acidic conditions, it remains unknown if it is related to the increased propensity for the disappearance of the beta-bulge in strand D. We consider that the enhanced fibrillation is more directly coupled with the decreased stability leading to the increased propensity of exposing amyloidogenic regions.

No abstract

An 85-year-old man underwent endoscopic submucosal dissection for a large superficial esophageal epithelial neoplasm, which required removal of 95% of the circumference of the esophageal mucosa. Steroids were given orally to prevent esophageal stricture starting on day 3 postoperatively. In the 6th week of steroid treatment, he developed high fever without other symptoms. Chest computed tomography revealed a nodular lesion in the lung. Sputum sample showed Gram-positive, branching, filamentous bacteria, and a diagnosis of nocardiosis was suspected. Brain magnetic resonance imaging revealed multiple focal lesions which indicated dissemination of nocardiosis. Trimethoprim-sulfamethoxazole was immediately started, which led to the disappearance of pulmonary and cerebral nocardiosis with alleviation of fever. Recently, oral steroid treatment has been widely used for the prevention of esophageal stricture. However, the present case indicates the risk of life-threatening infection and the importance of close monitoring of this treatment.

The hand hygiene adherence in Japanese teaching hospitals in our sample was low, even lower than reported mean values from other international studies. Greater adherence to hand hygiene should be encouraged in Japan.