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At least 343 records · Page 19Linked to original sources
PubMed2000

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy.

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

Amino Acid Sequence↗
PubMed2005

Confocal microscopy of the peripheral gustatory system: comparison between healthy subjects and patients suffering from taste disorders during radiochemotherapy.

OBJECTIVES: Laser-scanning microscopy (LSM) was used to compare taste buds and epithelia of fungiform papillae of healthy subjects with those of patients suffering from taste disorders during/after radiochemotherapy (RCT). Aim of the study was to investigate effects responsible for taste loss at a microscopic level. STUDY DESIGN: Prospective study. METHODS: Data from 12 healthy subjects (mean age 52.4, SD 9.5 years) were compared with those of 12 patients (mean age 54.7, SD 8.5 years) with head and neck cancer suffering from taste disorders during RCT. Four parameters from LSM were selected for analysis: 1) distance between the pore of the taste buds of fungiform papillae and the crest of the papillary vessels; 2) epithelial cells of each taste bud at 34 mum; 3), cell density, and 4) area of the taste pore at 4 mum. These data were correlated to measures of gustatory sensitivity obtained with both the validated "taste strips" test kit and electrogustometry. RESULTS: Patients complaining from taste disorders during RCT exhibited a significant decrease of taste function assessed with both natural and electric stimuli. In these patients, we found thicker epithelia and smaller areas of the taste pores compared with healthy subjects. In 30% of those patients, no taste pores were detectable; in deeper sections, however, normal taste buds were present. CONCLUSIONS: In conclusion, in RCT patients with taste disorders, LSM indicates changes of epithelia of fungiform papilla but no changes of the taste bud structure. Damage of the chorda tympani nerve by scattered rays, direct or indirect mucotoxic effects of chemotherapeutic agents, and covering of taste pores by epithelial cells are likely reasons for taste loss during RCT.

Adult↗
PubMed2005

HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET.

The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Psi) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Psi+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Psi- RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Psi does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Psi+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Psi thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.

Animals↗