No abstract

Fluorescence spectroscopy was used to determine whether quinacrine and ethidium, two high-affinity noncompetitive inhibitors of the Torpedo acetylcholine receptor (AcChR), bind to the same loci. The ability of three nitroxide spin-labels, 5-doxylstearate (5-SAL), spin-labeled androstane (ASL), and TEMPO, to quench receptor-bound quinacrine and ethidium fluorescence was measured. When bound to a phencyclidine-displaceable site on the AcChR, quinacrine was 16.9 and 19 times more efficiently quenched than ethidium by the highly lipophilic 5-SAL and ASL, respectively. TEMPO, which has a limited ability to partition into Torpedo plasma membranes (< 1%), was only twice as efficient at quenching receptor-bound quinacrine than ethidium fluorescence. The relative sensitivity of quinacrine and ethidium fluorescence to paramagnetic quenching was examined in three solvents, 1-butanol, sodium phosphate buffer, and acetonitrile, with TEMPO as a quencher. The results from the different solvents demonstrate that quinacrine fluorescence is intrinsically 1.4-3.6 times more sensitive than ethidium fluorescence to quenching by nitroxide spin-labels. Examination of the effect of high concentrations of 5-SAL on ethidium and quinacrine dissociation constants showed that quinacrine but not ethidium binding was competitively inhibited. Together, these results indicate that although quinacrine and ethidium bind in a mutually exclusive manner, the two inhibitors interact at different loci on the AcChR. Whereas the ethidium binding site is at a distance from membrane lipids, probably in or near the lumen, the quinacrine binding site appears to be at a lipid-protein interface in the transmembrane domain and at a distance from the lumen.

Quantitative fluorescence spectroscopy was used to develop a structural picture of the effects of two monoclonal antibodies (mAbs) on the conformation of the Torpedo nicotinic acetylcholine receptor (nAChR). The two mAbs (A6 and B1) examined selectively blocked ligand binding to either the high-affinity (A) or the low-affinity (B) binding sites for agonists/competitive antagonists. The distances between dansyl-C6-choline bound to the unblocked agonist/competitive antagonist binding site and one of two lipophilic probes (C12-eosin or C18-rhodamine) partitioned into the lipid membrane were estimated by using fluorescence resonance energy transfer. Control experiments demonstrated that both mAbs decreased the affinity and fluorescence lifetime of receptor-bound dansyl-C6-choline. The binding of the B1 mAb to the B site did not significantly change the calculated distance between the unblocked A binding site and the membrane surface. However, the binding of the A6 mAb to the A site induced the B site to move into close proximity to the lipid membrane. This conformational change was confirmed by a 45-fold increase in the paramagnetic quenching of the B-site-bound dansyl-C6-choline fluorescence by lipid-intercalated 5-doxylstearate. The results indicate that these mAbs not only selectively block ligand binding to either the A or the B acetylcholine sites but also, in the case of the A6 mAb, induce global conformational changes of the receptor, which appear to involve a movement of the B binding site into close proximity of the lipid membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

The purpose of this study was to determine whether the institutional presence of public health faculty within colleges and universities influenced operational plans for the fall semester of 2020. Using cross-sectional data collected by the College Crisis Initiative of Davidson College, six levels of instructional modalities (ranked from least to most restrictive) were compared between Council on Education of Public Health (CEPH)-accredited and non-CEPH-accredited 4-year institutions. Institutions with CEPH-accredited schools and programs were more likely to select some restrictive teaching modalities: 63.8% more likely to use hybrid/hyflex or more restrictive and 66.9% more likely to be primarily online (with some in person) or more restrictive. However, having CEPH-accredited programs did not push institutions to the most restrictive modalities. COVID-19 cases in county, enrollment, and political affiliation of the state governor were also found to influence instructional modality selection. While any ecological study has certain limitations, this study demonstrates that college and university fall plans appear to have been influenced by the presence of CEPH-accredited schools and programs of public health, and/or the input of their faculty. The influence of relevant faculty expertise on institutional decision-making can help inform college and university responses to future crises.