Fluorescence Quantum Yield of Thioflavin T in Rigid Isotropic Solution and Incorporated into the Amyloid Fibrils

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates

The regulatory subunit of PKA-I remains partially structured and undergoes β-aggregation upon thermal denaturation

Background: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RIα and a truncated RIα(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. Methodology/Principal Findings: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RIα proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly α-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of β-structure. A similar α→β transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-β-intermolecular aggregation and formation of non-fibrillar oligomers. Conclusions/Significance: Thermal denaturation of RIα leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the β-sandwiches in these domains favors inter-molecular β-aggregation, which is suppressed in the ligand-bound states of RIα under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation. © 2011 Dao et al

Non-Conjugated Small Molecule FRET for Differentiating Monomers from Higher Molecular Weight Amyloid Beta Species

Background: Systematic differentiation of amyloid (Aβ) species could be important for diagnosis of Alzheimer's disease (AD). In spite of significant progress, controversies remain regarding which species are the primary contributors to the AD pathology, and which species could be used as the best biomarkers for its diagnosis. These controversies are partially caused by the lack of reliable methods to differentiate the complicated subtypes of Aβ species. Particularly, differentiation of Aβ monomers from toxic higher molecular weight species (HrMW) would be beneficial for drug screening, diagnosis, and molecular mechanism studies. However, fast and cheap methods for these specific aims are still lacking. Principal Findings: We demonstrated the feasibility of a non-conjugated FRET (Förster resonance energy transfer) technique that utilized amyloid beta (Aβ) species as intrinsic platforms for the FRET pair assembly. Mixing two structurally similar curcumin derivatives that served as the small molecule FRET pair with Aβ40 aggregates resulted in a FRET signal, while no signal was detected when using Aβ40 monomer solution. Lastly, this FRET technique enabled us to quantify the concentrations of Aβ monomers and high molecular weight species in solution. Significance: We believe that this FRET technique could potentially be used as a tool for screening for inhibitors of Aβ aggregation. We also suggest that this concept could be generalized to other misfolded proteins/peptides implicated in various pathologies including amyloid in diabetes, prion in bovine spongiform encephalopathy, tau protein in AD, and α-synuclein in Parkinson disease.National Institute on Aging (K25AG036760

RNA Oxidation Adducts 8-OHG and 8-OHA Change with Aβ42 Levels in Late-Stage Alzheimer's Disease

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain regions (Brodmann's Area 9, hippocampus, inferior parietal lobule, and the superior and middle temporal gyri), we found that the RNA adduct 8-hydroxyguanine (8-OHG) decreased, while 8-hydroxyadenine (8-OHA) increased in AD. The cerebellum, which is generally spared in AD, did not show disease related changes, and no RNA adducts correlated with the number of plaques or tangles. Multiple regression analysis revealed that SDS-soluble Aβ42 was the best predictor of changes in 8-OHG, while formic acid-soluble Aβ42 was the best predictor of changes in 8-OHA. This study indicates that although there is a connection between AD related neuropathology and RNA oxidation, this relationship is not straightforward

Hypoxia Due to Cardiac Arrest Induces a Time-Dependent Increase in Serum Amyloid β Levels in Humans

Amyloid β (Aβ) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular mechanisms underlying cerebral Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 elevations were observed in all patients. Elevations ranged from approximately 80% to over 70-fold, with most elevations in the range of 3–10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD

Mutation Analysis of BRAF, MEK1 and MEK2 in 15 Ovarian Cancer Cell Lines: Implications for Therapy

Among gynecologic cancers, ovarian cancer is the second most common and has the highest death rate. Cancer is a genetic disorder and arises due to the accumulation of somatic mutations in critical genes. An understanding of the genetic basis of ovarian cancer has implications both for early detection and for therapeutic intervention in this population of patients.Fifteen ovarian cancer cell lines, commonly used for in vitro experiments, were screened for mutations using bidirectional direct sequencing in all coding regions of BRAF, MEK1 and MEK2. BRAF mutations were identified in four of the fifteen ovarian cancer cell lines studied. Together, these four cell lines contained four different BRAF mutations, two of which were novel. ES-2 had the common B-Raf p.V600E mutation in exon 15 and Hey contained an exon 11 missense mutation, p.G464E. The two novel B-Raf mutants identified were a 5 amino acid heterozygous deletion p.N486-P490del in OV90, and an exon 4 missense substitution p.Q201H in OVCAR 10. One of the cell lines, ES-2, contained a mutation in MEK1, specifically, a novel heterozygous missense substitution, p.D67N which resulted from a nt 199 G-->A transition. None of the cell lines contained coding region mutations in MEK2. Functional characterization of the MEK1 mutant p.D67N by transient transfection with subsequent Western blot analysis demonstrated increased ERK phosphorylation as compared to controls.In this study, we report novel BRAF mutations in exon 4 and exon 12 and also report the first mutation in MEK1 associated with human cancer. Functional data indicate the MEK1 mutation may confer alteration of activation through the MAPK pathway. The significance of these findings is that BRAF and MEK1/2 mutations may be more common than anticipated in ovarian cancer which could have important implications for treatment of patients with this disease and suggests potential new therapeutic avenues

The effects of thermal capsulorrhaphy of medial parapatellar capsule on patellar lateral displacement

<p>Abstract</p> <p>Background</p> <p>The effectiveness of thermal shrinkage on the medial parapatellar capsule for treating recurrent patellar dislocation is controversial. One of reasons why it is still controversial is that the effectiveness is still qualitatively measured. The purpose of this study was to quantitatively determine the immediate effectiveness of the medial parapatellar capsule shrinkage as in clinical setting.</p> <p>Methods</p> <p>Nine cadaveric knees were used to collect lateral displacement data before and after medial shrinkage or open surgery. The force and displacement were recorded while a physician pressed the patella from the medial side to mimic the physical exam used in clinic. Ten healthy subjects were used to test the feasibility of the technique on patients and establish normal range of lateral displacement of the patella under a medial force. The force applied, the resulting displacement and the ratio of force over displacement were compared among four data groups (normal knees, cadaveric knees before medial shrinkage, after shrinkage and after open surgery).</p> <p>Results</p> <p>Displacements of the cadaveric knees both before and after thermal modification were similar to normal subjects, and the applied forces were significantly higher. No significant differences were found between before and after thermal modification groups. After open surgery, displacements were reduced significantly while applied forces were significantly higher.</p> <p>Conclusion</p> <p>No immediate difference was found after thermal shrinkage of the medial parapatellar capsule. Open surgery immediately improved of the lateral stiffness of the knee capsule.</p

Different Factors Affecting Human ANP Amyloid Aggregation and Their Implications in Congestive Heart Failure

Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis.A significant prevalence (85\%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric β-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing β-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA.We established a link between IAA and CHF by ex vivo examination and assessed that β-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that β-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis