The triphenylmethane dye brilliant blue G is only moderately effective at inhibiting amyloid formation by human amylin or at disaggregating amylin amyloid fibrils, but interferes with amyloid assays; Implications for inhibitor design.

The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer's Aβ peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aβ also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aβ amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aβ amyloid formation and inhibit Aβ induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured β-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aβ and h-amylin amyloid formation, illustrates the limitation of using Aβ inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation

Can High-risk Older Drivers be Identified in a DMV Setting with a Brief Battery of Functional Tests?

Recent research has indicated that some laboratory measures of functional ability may discriminate between crash-involved and crash-free older adults. However, the ability of these tests to provide the same level of discriminability in a real-world setting such as a Department of Motor Vehicles remains to be established. Therefore, in conjunction with the Maryland Department of Motor Vehicles and the National Highway Traffic Safety Administration, a brief battery of tests was developed and evaluated. The battery contained a number of cognitive tests (e.g., UFOV® subtest 2, the closure subtest of the Motor Free Visual Perception Test [MVPT], Trails A and B, etc.) and physical measures (e.g., Rapid Pace Walk, Head and Neck Rotation, etc.) that prior literature had indicated might be related to crash risk in older adults. Motor Vehicle Administration staff were trained to administer the test battery. Older adults (N=4,173, mean age =69 years) were approached by the staff after license renewal and asked to help evaluate the brief battery. Of the 4,173 older adults approached at the field sites, 2,112 individuals aged 55-96 years of age participated. The primary outcome of interest for this study was the occurrence of an at-fault Motor Vehicle Collision (MVC) following assessment. For members of this sample, the outcome period ranged from 2-3 years. Rate Ratios were determined for each functional variable based upon at-fault crashes adjusted for driving exposure over this period. Univariate analyses revealed that five variables (Age, Walk Time, MVPT, Trails A and UFOV®) were significantly related to crash frequency. These significant variables overlapped with one another to a certain degree, indicating that impaired older drivers score poorly on multiple cognitive assessments. The UFOV® subtest 2 appears to be the most strongly associated within this analysis (RR=3.78, p\u3c .05) and Rapid Pace Walk (RR=1.96, p \u3c .05) remained uniquely related to the frequency of state-reported, at-fault crashes. The role of such a screening battery in field settings such as a DMV will be discussed

Crystallization of a designed peptide from a molten globule ensemble

Backgound:The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles.Results & Conclusions:Crystals diffracting to 3.5å resolution have been grown of a 16-residue peptide (α1A) designed to form a tetramer of α-helices. In addition, a closely related peptide, α1, has previously been shown to yield crystals that diffract to 1.2å resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form α-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins

Linking Gas-Phase and Solution-Phase Protein Unfolding via Mobile Proton Simulations

Native mass spectrometry coupled to ion mobility (IM-MS) combined with collisional activation (CA) of ions in the gas phase (in vacuo) is an important method for the study of protein unfolding. It has advantages over classical biophysical and structural techniques as it can be used to analyze small volumes of low-concentration heterogeneous mixtures while maintaining solution-like behavior and does not require labeling with fluorescent or other probes. It is unclear, however, whether the unfolding observed during collision activation experiments mirrors solution-phase unfolding. To bridge the gap between in vacuo and in-solution behavior, we use unbiased molecular dynamics (MD) to create in silico models of in vacuo unfolding of a well-studied protein, the N-terminal domain of ribosomal L9 (NTL9) protein. We utilize a mobile proton algorithm (MPA) to create 100 thermally unfolded and coulombically unfolded in silico models for observed charge states of NTL9. The unfolding behavior in silico replicates the behavior in-solution and is in line with the in vacuo observations; however, the theoretical collision cross section (CCS) of the in silico models was lower compared to that of the in vacuo data, which may reflect reduced sampling

Methods for Planning the Great Lakes MegaRegion

http://deepblue.lib.umich.edu/bitstream/2027.42/110967/1/glmegaregion_methodsmanual_finalcorrect2006.pd

Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Forster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions

Through a Wider Lens: Re-envisioning the Great Lakes Mega Region

http://deepblue.lib.umich.edu/bitstream/2027.42/110966/1/throughwiderlens2006.pd