Peptide backbone modification in the bend region of amyloid-ß inhibits fibrillogenesis but not oligomer formation.

Current evidence suggests that oligomers of the amyloid-ß (Aß) peptide are involved in the cellular toxicity of Alzheimer's disease, yet their biophysical characterization remains difficult because of lack of experimental control over the aggregation process under relevant physiologic conditions. Here, we show that modification of the Aß peptide backbone at Gly29 allows for the formation of oligomers but inhibits fibril formation at physiologic temperature and pH. Our results suggest that the putative bend region in Aß is important for higher-order aggregate formation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Prevalence of Chest Injury With the Presence of NEXUS Chest Criteria: Data to Inform Shared Decisionmaking About Imaging Use.

STUDY OBJECTIVE: The NEXUS chest decision instrument identifies a very-low-risk population of patients with blunt trauma for whom chest imaging can be avoided. However, it requires that all 7 National Emergency X-Ray Utilization Study (NEXUS) chest criteria be absent. To inform patient and physician shared decisionmaking about imaging, we describe the test characteristics of individual criteria of the NEXUS chest decision instrument and provide the prevalence of injuries when 1, 2, or 3 of the 7 criteria are present. METHODS: We conducted this secondary analysis of 2 prospectively collected cohorts of patients with blunt trauma who were older than 14 years and enrolled in NEXUS chest studies between December 2009 and January 2012. Physicians at 9 US Level I trauma centers recorded the presence or absence of the 7 NEXUS chest criteria. We calculated test characteristics of each criterion and combinations of criteria for the outcome measures of major clinical injuries and thoracic injury observed on chest imaging. RESULTS: We enrolled 21,382 patients, of whom 992 (4.6%) had major clinical injuries and 3,135 (14.7%) had thoracic injuries observed on chest imaging. Sensitivities of individual test characteristics ranged from 15% to 56% for major clinical injury and 14% to 53% for thoracic injury observed on chest imaging, with specificities varying from 71% to 84% for major clinical injury and 67% to 84% for thoracic injury observed on chest imaging. Individual criteria were associated with a prevalence of major clinical injury between 1.9% and 3.8% and of thoracic injury observed on chest imaging between 5.3% and 11.5%. CONCLUSION: Patients with isolated NEXUS chest criteria have low rates of major clinical injury. The risk of major clinical injury for patients with 2 or 3 factors range from 1.7% to 16.6%, depending on the combination of criteria. Criteria-specific risks could be used to inform shared decisionmaking about the need for imaging by patients and their physicians.

Chaperone-like N-methyl peptide inhibitors of polyglutamine aggregation.

Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into beta-sheet-rich insoluble aggregates associated with Huntington's disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe(2) [Anth-K-Q-Q(Me(2))-Q-Q(Me(2))-Q-CONH(2), where Anth is N-methylanthranilic acid and Q(Me(2)) is side chain N-methyl Q], has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe(2) can completely prevent fibrillation of a synthetic aggregating peptide, YAQ(12)A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K(d) with very fast k(on) and k(off) values. Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe(2) is predominantly or entirely monomeric at concentrations of

Site-specific effects of peptide lipidation on beta-amyloid aggregation and cytotoxicity.

Beta-amyloid (Abeta) aggregates at low concentrations in vivo, and this may involve covalently modified forms of these peptides. Modification of Abeta by 4-hydroxynonenal (4-HNE) initially increases the hydrophobicity of these peptides and subsequently leads to additional reactions, such as peptide cross-linking. To model these initial events, without confounding effects of subsequent reactions, we modified Abeta at each of its amino groups using a chemically simpler, close analogue of 4-HNE, the octanoyl group: K16-octanoic acid (OA)-Abeta, K28-OA-Abeta, and Nalpha-OA-Abeta. Octanoylation of these sites on Abeta-(1-40) had strikingly different effects on fibril formation. K16-OA-Abeta and K28-OA-Abeta, but not Nalpha-OA-Abeta, had increased propensity to aggregate. The type of aggregate (electron microscopic appearance) differed with the site of modification. The ability of octanoyl-Abeta peptides to cross-seed solutions of Abeta was the inverse of their ability to form fibrils on their own (i.e. Abeta approximately Nalpha-OA-Abeta>>K16-OA-Abeta>>K28-OA-Abeta). By CD spectroscopy, K16-OA-Abeta and K28-OA-Abeta had increased beta-sheet propensity compared with Abeta-(1-40) or Nalpha-OA-Abeta. K16-OA-Abeta and K28-OA-Abeta were more amphiphilic than Abeta-(1-40) or Nalpha-OA-Abeta, as shown by lower "critical micelle concentrations" and higher monolayer collapse pressures. Finally, K16-OA-Abeta and K28-OA-Abeta are much more cytotoxic to N2A cells than Abeta-(1-40) or Nalpha-OA-Abeta. The greater cytotoxicity of K16-OA-Abeta and K28-OA-Abeta may reflect their greater amphiphilicity. We conclude that lipidation can make Abeta more prone to aggregation and more cytotoxic, but these effects are highly site-specific.

Immunolocalization of KATP channel subunits in mouse and rat cardiac myocytes and the coronary vasculature.

BACKGROUND: Electrophysiological data suggest that cardiac KATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other KATP channel subunits) is poorly defined. We examined the localization of each of the KATP channel subunits in the mouse and rat heart. RESULTS: Immunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular KATP channels. CONCLUSIONS: Collectively, our data demonstrate unique cellular and subcellular KATP channel subunit expression patterns in the heart. These results suggest distinct roles for KATP channel subunits in diverse cardiac structures.