A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta-19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta-F/F) approximate to 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta-F agreed well with those for epibatidine-induced currents, but were shifted approximate to 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha-gamma-binding site than to the alpha-delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta-F monitors an event linked to binding specifically at the alpha-delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta-F occurs within milliseconds, i.e., during activation. At low [ACh] (less than or equal to 10 muM), a phase of Delta-F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta-F is complete before the slowest phase of recovery from desensitization (time constant approximate to 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha-delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes

Use of Semi-Structured Interviews to Explore Competing Demands in a Prostate Cancer Prevention Intervention Clinical Trial (PCPICT)

In this paper we report on findings from the first known study using qualitative methods to explore factors influencing physicians’ participation in an ongoing federally-funded prostate cancer chemoprevention clinical trial. We sought to identify ways to improve collaboration between researchers and physicians and enhance the success of future projects and employed purposive sampling to recruit physician/investigators who were involved or invited to participate in the trial. Using the data from open-ended semi-structured interviews, we examined patterns in their languaging and created themes. We found that individual and structural factors served as barriers and facilitators to participation. Willingness and desire to participate in the trial (individual factors) were not always enough to result in actual participation due to practice environment (structural) constraints. Our research provides a better understanding of the complex intersection of factors in this setting and through our findings we extend the theory of competing demands into the arena of prostate cancer prevention clinical trials, moving the science towards solutions to current challenges in recruitment to this type of trial

A mapping approach to synchronization in the "Zajfman trap": stability conditions and the synchronization mechanism

We present a two particle model to explain the mechanism that stabilizes a bunch of positively charged ions in an "ion trap resonator" [Pedersen etal, Phys. Rev. Lett. 87 (2001) 055001]. The model decomposes the motion of the two ions into two mappings for the free motion in different parts of the trap and one for a compressing momentum kick. The ions' interaction is modelled by a time delay, which then changes the balance between adjacent momentum kicks. Through these mappings we identify the microscopic process that is responsible for synchronization and give the conditions for that regime.Comment: 12 pages, 9 figures; submitted to Phys Rev

Transcatheter edge-to-edge repair in acute mitral regurgitation following acute myocardial infarction: Recent advances

Acute mitral regurgitation (MR) is not a rare finding following acute myocardial infarction (AMI). It may develop due to papillary muscle rupture (primary MR) or due to rapid remodeling of the infarcted areas leading to geometric changes and leaflets tethering (secondary or functional MR). The clinical presentation can be catastrophic with pulmonary edema and refractory cardiogenic shock. Acute MR is a potentially life-threating complication and is linked to worse clinical outcomes. Until recently, medical treatment or mitral valve surgery were the only established treatment options for these patients. However, there is growing evidence for the benefits of safe and effective trans-catheter interventions in this condition, specifically transcatheter edge-to-edge repair (TEER). We aimed to review the current role of TEER in post-MI acute MR patients, focusing on different etiologies

gp25L/emp24/p24 Protein Family Members of the cis-Golgi Network Bind Both COP I and II Coatomer

Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties

Single NanoParticle Photothermal Tracking (SNaPT) of 5 nm gold beads in live cells

Tracking individual nano-objets in live cells during arbitrary long times is an ubiquitous need in modern biology. We present here a method for tracking individual 5 nm gold nanoparticles on live cells. It relies on the photothermal effect and the detection of the Laser Induced Scattering around a NanoAbsorber (LISNA). The key point for recording trajectories at video rate is the use of a triangulation procedure. The effectiveness of the method is tested against Single fluorescent Molecule Tracking in live COS7 cells on subsecond time scales. We further demonstrate recordings for several minutes of AMPA receptors trajectories on the plasma membrane of live neurons. SNaPT has the unique potential to record arbitrary long trajectory of membrane proteins using non-fluorescent nanometer sized labels