Biophysical characterization of the structure of the amino-terminal region of gp41 of HIV-1. Implications on viral fusion mechanism.
A peptide of 51 amino acids corresponding to the NH2-terminal region (5-55) of the glycoprotein gp41 of human immunodeficiency virus type 1 was synthesized to study its conformation and assembly. Nuclear magnetic resonance experiments indicated the sequence NH2-terminal to the leucine zipper-like domain of gp41 was induced into helix in the micellar solution, in agreement with circular dichroism data. Light scattering experiment showed that the peptide molecules self-assembled in water into trimeric structure on average. That the peptide molecules oligomerize in aqueous solution was supported by gel filtration and diffusion coefficient experiments. Molecular dynamics simulation based on the NMR data revealed a flexible region adjacent to the hydrophobic NH2 terminus of gp41. The biological significance of the present findings on the conformational flexibility and the propensity of oligomerization of the peptide may be envisioned by a proposed model for the interaction of gp41 with membranes during fusion process. (+info
Aggregation of deoxyhemoglobin S at low concentrations.
The self-association of deoxyhemoglobin S was measured in dilute solutions (0 to 5 g/dl) by Rayleigh light scattering at 630 nm and osmometry in 0.05 M potassium phosphate buffer (pH 7.35). Weight and number average molecular weights (Mw and Mn, respectively) and the second or higher virial coefficients, B' were determined. No experimentally significant differences were observed between oxy- and deoxy-Hb S up to the concentration of 2 g/dl; their apparent average molecular weights were within experimental error. Above that concentration, both Mn and Mw of deoxy-Hb S were significantly different from that of oxy-Hb S. The negative second viral coefficent of deoxy-Hb S, observed by both techniques, is consistent with the self-association of this protein. The lack of effect of 0.4 M propylurea on the state of aggregation and the significant influence of 0.1 M NaCl suggests that polar interactions are involved in formation of these aggregates. (+info
The effect of the antiscatter grid on full-field digital mammography phantom images.
Computer Analysis of Mammography Phantom Images (CAMPI) is a method for making quantitative measurements of image quality. This article reports on a recent application of this method to a prototype full-field digital mammography (FFDM) machine. Images of a modified ACR phantom were acquired on the General Electric Diagnostic Molybdenum Rhodium (GE-DMR) FFDM machine at a number of x-ray techniques, both with and without the scatter reduction grid. The techniques were chosen so that one had sets of grid and non-grid images with matched doses (200 mrads) and matched gray-scale values (1500). A third set was acquired at constant 26 kVp and varying mAs for both grid conditions. Analyses of the images yielded signal-to-noise-ratio (SNR), contrast and noise corresponding to each target object, and a non-uniformity measure. The results showed that under conditions of equal gray-scale value the grid images were markedly superior, albeit at higher doses than the non-grid images. Under constant dose conditions, the non-grid images were slightly superior in SNR (7%) but markedly less uniform (60%). Overall, the grid images had substantially greater contrast and superior image uniformity. These conclusions applied to the whole kVp range studied for the Mo-Mo target filter combination and 4 cm of breast equivalent material of average composition. These results suggest that use of the non-grid technique in digital mammography with the GE-DMR-FFDM unit, is presently not warranted. With improved uniformity correction procedure, this conclusion would change and one should be able to realize a 14% reduction in patient dose at the same SNR by using a non-grid technique. (+info
pH-dependent conformational change of gastric mucin leads to sol-gel transition.
We present dynamic light scattering (DLS) and hydrophobic dye-binding data in an effort to elucidate a molecular mechanism for the ability of gastric mucin to form a gel at low pH, which is crucial to the barrier function of gastric mucus. DLS measurements of dilute mucin solutions were not indicative of intermolecular association, yet there was a steady fall in the measured diffusion coefficient with decreasing pH, suggesting an apparent increase in size. Taken together with the observed rise in depolarized scattering ratio with decreasing pH, these results suggest that gastric mucin undergoes a conformational change from a random coil at pH >/= 4 to an anisotropic, extended conformation at pH < 4. The increased binding of mucin to hydrophobic fluorescent with decreasing pH indicates that the change to an extended conformation is accompanied by exposure of hydrophobic binding sites. In concentrated mucin solutions, the structure factor S(q, t) derived from DLS measurements changed from a stretched exponential decay at pH 7 to a power-law decay at pH 2, which is characteristic of a sol-gel transition. We propose that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated. (+info
Effect of salt addition on the fractal structure of aggregates formed by heating dilute BSA solutions.
The fractal dimension, Df, of aggregates in a dilute BSA system with added salt was evaluated by static light scattering (SLS). A fractal structure was observed for the system with NaCl addition. The values of Df increased with increasing heating time and ionic strength. The values of Df were larger than those (Df = 1.8 or 2.1) predicted by the conventional cluster-cluster aggregation model, probably due to a "restructuring" of aggregates during the aggregation process. On the other hand, a fractal structure was not apparent for the system with added CaCl2. (+info
17beta-estradiol reduces tumor necrosis factor-alpha-mediated LDL accumulation in the artery wall.
Estrogens have direct effects on the vascular wall that may prevent the development of atherosclerosis. In particular, estrogens, such as 17beta-estradiol (estradiol), are known to have potent antioxidant activity. Tumor necrosis factor-alpha (TNF) is found in human atheroma and produces oxygen-derived free radicals. These oxygen-derived free radicals may modify low density lipoproteins (LDL) and increase LDL binding in the artery wall. We asked: 1) does TNF increase LDL accumulation in the artery wall and 2) can the TNF-mediated increase in LDL accumulation be prevented by the antioxidant activity of estradiol? Carotid arteries from ovariectomized 3-month-old rats were removed and perfused with fluorescently labeled LDL and arterial LDL flux was measured using quantitative fluorescence microscopy. In six arteries, addition of TNF (10 ng/ml) to the perfusate resulted in a 2.3-fold increase in the rate of LDL accumulation (1.50 +/- 0.37 ng/min per cm2 vs. 3.38 +/- 0.48 ng/min per cm2; P < 0.01). Estradiol (65 pg/ml) and alpha-tocopherol (6 mg/L) both attenuated TNF-mediated LDL accumulation (P < 0.05), indicating that TNF may exert its effects on LDL accumulation through cellular production of oxygen-derived free radicals. These results support an antioxidant role for estradiol in the protection against LDL accumulation in the artery wall and subsequent progression of atherosclerosis. (+info
Directional and spectral reflectance of the rat retinal nerve fiber layer.
PURPOSE: To measure and describe the reflectance properties of a mammalian retinal nerve fiber layer (RNFL) and to determine the mechanisms responsible for the RNFL reflectance. METHODS: An isolated rat retina suspended across a slit in a black membrane and mounted in a black perfusion chamber provided high quality images of the RNFL. Imaging microreflectometry was used to measure RNFL reflectance at wavelengths from 400 nm to 830 nm and as a function of illumination angle. RESULTS: The directional reflectance of rat RNFL at all wavelengths was consistent with the theory of light scattering by cylinders; each nerve fiber bundle scattered light into a conical sheet coaxial with the bundle. There was no evidence of a noncylindrical component at any wavelength. Measured reflectance spectra were consistent between animals, similar to ones previously measured in macaque, and varied with scattering angle. All spectra could be described by a two-mechanism cylindrical scattering model with three free parameters. CONCLUSIONS: At all wavelengths the reflectance of rat RNFL arises from light scattering by cylindrical structures. The highly directional nature of this reflectance can be an important source of measurement variability in clinical assessment of the RNFL. The reflectance spectra reveal a combination of mechanisms: At wavelengths shorter than approximately 570 nm the reflectance comes from cylinders with diameters much smaller than the wavelength, but at wavelengths longer than approximately 680 nm the reflectance comes from cylinders with effective diameters of 350 nm to 900 nm. (+info
Single-polymer dynamics in steady shear flow.
The conformational dynamics of individual, flexible polymers in steady shear flow were directly observed by the use of video fluorescence microscopy. The probability distribution for the molecular extension was determined as a function of shear rate, gamma;, for two different polymer relaxation times, tau. In contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition. Large, aperiodic temporal fluctuations were observed, consistent with end-over-end tumbling of the molecule. The rate of these fluctuations (relative to the relaxation rate) increased as the Weissenberg number, gamma;tau, was increased. (+info