Molecular mobility in a homologous series of amorphous solid glucose oligomers

The molecular mobility of amorphous solid biomaterials influences the stability of dried foods and pharmaceuticals, the viability of seeds and spores, and the desiccation-tolerance of organisms during anhydrobiosis. Current understanding of how structure correlates with molecular mobility in the glassy state is inadequate. We used phosphorescence from vanillin dispersed in amorphous films to study the effect of temperature on molecular mobility in the homologous series of oligosaccharides glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose. Phosphorescence emission spectra and intensity decays were collected from −10 to as high as 130 °C. Emission peak energy, a measure of the extent of dipolar relaxation around the excited state prior to emission, decreased monotonically with temperature, decreasing more significantly in the glassy state in larger sugars (higher degree of polymerisation). The intensity decays were well fitted with sums of either four (glucose, maltose, maltotriose) or three exponentials (maltotetraose, maltopentaose, maltohexaose, maltoheptaose); fit lifetimes at each temperature varied over nearly two orders of magnitude, suggesting a comparable range in matrix dynamic heterogeneity. The lifetimes decreased monotonically with temperature, while the lifetime amplitudes favoured the long lifetime components at lower and short lifetime components at higher temperatures near T_g. Arrhenius analysis indicated that the rate of non-radiative decay, which reflects coupling of probe vibrations with matrix motions and thus provides an estimate of the matrix molecular mobility, increased with molecular size in the glassy state. Both apparent activation energy and activation entropy increased systematically with temperature in all sugars. These data provide additional evidence that the rate and extent of molecular mobility in glassy state carbohydrates is higher in sugars of greater molecular size (mass) and thus higher glass transition temperature and provides additional insight into the molecular dynamics of the glassy state in carbohydrates.     

Phosphorescence of erythrosin B as a robust probe of molecular mobility in amorphous solid sucrose

Luminescence from the triplet probe erythrosin B (tetra-iodo fluorescein, Ery B) provides spectroscopic characteristics such as lifetime and emission energy that are sensitive to molecular mobility of the local environment in amorphous solids. This study investigated how variations in the local concentration of Ery B free acid as well as the presence of the dispersing solvent affect the spectroscopic measurements of solid matrix properties (the free acid of Ery B is poorly soluble in water and thus must be introduced via an organic solvent). The emission energy of Ery B from 5 to 100 degrees C in thin films of amorphous sucrose at various probe and solvent (N,N-dimethyl formamide, DMF) concentrations was determined using excitation at 500 nm and emission over the range 520-750 nm. The emission lifetime was determined over the same temperature range using a stretched exponential analysis of intensity decays collected using excitation at 530 nm and emission at 680 nm. Variations in the probe/sucrose mole ratio (concentration) over the range from 0.5 to 10 x 10(-4) and 10-fold variations in the amount of DMF used to disperse the probe did not affect the emission energy, the shape of the emission spectra, or the measured lifetimes of Ery B in amorphous sucrose. These results thus indicate that erythrosin B introduced into amorphous solids can provide a robust measure of the intrinsic mobility of the solid matrix that is relatively insensitive to final probe concentration or presence of residual solvent.     

Influence of lipid composition on pediocin PA-1 binding to phospholipid vesicles

Pediocin PA-1 bound to anionic lipid vesicles with saturated or unsaturated fatty acid chains in a lipid concentration-dependent fashion. Little change in binding parameters was observed for zwitterionic lipid vesicles. Decreasing the anionic lipid content of the vesicles gave a higher relative dissociation constant for the peptide-lipid interactions and further supports the electrostatic interaction model of binding.     

Characterization of skeletal muscle actin labeled with the triplet probe erythrosin-5-iodoacetamide

We have labeled rabbit skeletal muscle actin with the triplet probe erythrosin-5-iodoacetamide and characterized the labeled protein. Labeling decreased the critical concentration and lowered the intrinsic viscosity of F-actin filaments; labeled filaments were motile in an in vitro motility assay but were less effective than unlabeled F-actin in activating myosin S1 ATPase activity. In unpolymerized globular actin (G-actin), both the prompt and delayed luminescence were red-shifted from the spectra of the free dye in solution and the fluorescence anisotropy of the label was high (0.356); filament formation red shifted all excitation and emission spectra and increased the fluorescence anisotropy to 0.370. The erythrosin phosphorescence decay was at least biexponential in G-actin with an average lifetime of 99 microseconds while in F-actin the decay was approximately monoexponential with a lifetime of 278 microseconds. These results suggest that the erythrosin dye was bound at the interface between two actin monomers along the two-start helix. The steady-state phosphorescence anisotropy of F-actin was 0.087 at 20 degrees C and the anisotropy increased to approximately 0.16 in immobilized filaments. The phosphorescence anisotropy was also sensitive to binding the physiological ligands phalloidin, cytochalasin B and tropomyosin. This study lays a firm foundation for the use of this triplet probe to study the large-scale molecular dynamics of F-actin.     

Differential activity of P-glycoprotein in normal blood lymphocyte subsets

To better understand the phenomenon of P-glycoprotein (P-170) expression we investigated lymphocyte subpopulations for P-170 function in healthy volunteers. Studies were based on three-colour flow cytometry including the fluorescent probe rhodamine 123 (Rh123), which is transported by P-170. Marked Rh123 efflux was detected in CD8+ T lymphocytes with CD8+/CD45RA+ T cells (naive cells) showing significantly higher P-170 activity as compared with CD8+/CD45RA- cells (P<0.04). Vice versa, CD8+/CD45RO+ T cells (memory cells) demonstrated less P-170 activity than CD8+/CD45RO- cells (P<0.04). P-170 function was less prominent in CD4+ T cells, however, Rh123 efflux was higher in the CD4+/CD45RA+ and CD4+/CD45RO- subpopulations (P<0.025) corresponding to the CD8+ results. Dye efflux differed significantly between activated and non-activated CD8+ and CD4+ as well as CD8+/CD11b+ and CD8+/CD11b- T lymphocytes. Since CD16+ natural killer cells (NK) expressed the highest level of P-170, the NK cytotoxicity against 51Cr-labelled K562 target cells was assayed in the presence or absence of P-170 inhibitors. NK related cytotoxicity was significantly reduced in the presence of R-verapamil and dexnigaldipine-HCP in a dose-dependent manner. The differential expression of P-170 activity in naive and memory T cells together with the reduced NK related cytotoxicity in the presence of MDR-modulators suggest a physiological role of P-170 in immunological functions of these lymphocyte subsets. Consequently, the addition of MDR modulators to conventional chemotherapy as a strategy to overcome drug resistance should consider possible adverse immunosuppressive effects.     

Analysing the Combined Health,Social and Economic Impacts of the Corovanvirus Pandemic Using Agent-Based Social Simulation

Minds and Machines - During the COVID-19 crisis there have been many difficult decisions governments and other decision makers had to make. E.g. do we go for a total lock down or keep schools open?...     

Effect of temperature on molecular mobility,oxygen permeability,and dynamic site heterogeneity in amorphous α-lactalbumin films

We investigated molecular mobility and oxygen diffusion in amorphous solid films of α-lactalbumin (α-La) using phosphorescence from the xanthene probe erythrosin B in order to elucidate the molecular mechanism(s) that control oxygen permeability in amorphous solid proteins. Emission peak energy and bandwidth were determined by fitting spectra to a lognormal bandwidth function; intensity decays were fit to a stretched exponential function to determine the lifetime and the stretching exponent. Peak emission energy decreased gradually over the range from −20 to over 120 °C, indicating a gradual increase in the matrix dipolar relaxation rate. Bandwidths were constant at low temperature but increased dramatically above ∼50 °C, indicating that the dynamic heterogeneity of the protein matrix increased at high temperature. Emission lifetimes decrease gradually at low and more steeply at high temperature, indicating that the rate of matrix collisional quenching increased with temperature. Arrhenius analysis of the rate constant for non-radiative decay showed a gradual increase in quenching indicative of matrix softening. Comparison of lifetimes in air and N₂ (±oxygen) monitored oxygen permeability. Oxygen permeability became detectable at about 0 °C and appeared to correlate with matrix mobility. The emission spectrum shifted to higher energy as a function of time following excitation, whereas the phosphorescence lifetime decreased with increasing emission wavelength; both behaviors provided strong evidence for distinct sites within the protein matrix varying in molecular mobility. Phosphorescence spectroscopy thus provides a simple tractable tool for monitoring conditions that activate oxygen diffusion in amorphous solid foods.     

Influence of tightly bound Mg2+ and Ca2+, nucleotides, and phalloidin on the microsecond torsional flexibility of F-actin

To better understand the relationship between structure and molecular dynamics in F-actin, we have monitored the torsional flexibility of actin filaments as a function of the type of tightly bound divalent cation (Ca2+ or Mg2+) or nucleotide (ATP or ADP), the level of inorganic phosphate and analogues, KCl concentration, and the level of phalloidin. Torsional flexibility on the microsecond time scale was monitored by measuring the steady-state phosphorescence emission anisotropy (rFA) of the triplet probe erythrosin-5-iodoacetamide covalently bound to Cys-374 of skeletal muscle actin; extrapolations to an infinite actin concentration corrected the measured anisotropy values for the influence of variable amounts of rotationally mobile G-actin in solution. The type of tightly bound divalent cation modulated the torsional flexibility of F-actin polymerized in the presence of ATP; filaments with Mg2+ bound (rFA = 0.066) at the active site cleft were more flexible than those with Ca2+ bound (rFA = 0.083). Filaments prepared from G-actin in the presence of MgADP were more flexible (rFA = 0.051) than those polymerized with MgATP; the addition of exogenous inorganic phosphate or beryllium trifluoride to ADP filaments, however, decreased the filament flexibility (increased the anisotropy) to that seen in the presence of MgATP. While variations in KCl concentration from 0 to 150 mM did not modulate the torsional flexibility of the filament, the binding of phalloidin decreased the torsional flexibility of all filaments regardless of the type of cation or nucleotide bound at the active site. These results emphasize the dynamic malleability of the actin filament, the role of the cation-nucleotide complex in modulating the torsional flexibility, and suggest that the structural differences that have previously been seen in electron micrographs of actin filaments manifest themselves as differences in torsional flexibility of the filament.     

Models and mechanisms for bacteriocin action and application

There is considerable research on bacteriocin genetics, purification, and properties. Less is known about the mechanism(s) by which bacteriocins kill pathogens, the physical chemistry of the bacteriocin/pathogen interaction, and of the variables which influence bacteriocins' efficacy in foods. Such knowledge is prerequisite to the wider applications of bacteriocins and to increasing their efficacy by genetic engineering. Mechanistic studies using spores as bacteriocin targets are relatively few. Empirical challenge studies in a variety of foods have had mixed results. Working with well defined model foods, we have determined that increasing protein or phospholipid concentrations decrease nisin 's effectiveness against Clostridium botulinum growth from spore inocula. Nisin is also less effective at abuse compared to refrigerated temperatures. This may be a general characteristic of bacteriocins since increasing temperature decreases many bacteriocins' inhibition of Listeria monocytogenes in foods. L. monocytogenes vegetative cells provide a better target for bacteriocin action than do C. botulinum spores. Bacteriocins dissipate proton motive force (PMF) in L. monocytogenes, C. sporogenes and vegetative cells of other sensitive species. The cytoplasmic membrane is generally considered to be the site at which bacteriocins act. We have adopted fluorescence spectroscopy to characterize the interaction of bacteriocins with liposomes comprised of lipids extracted from L. monocytogenes membranes. The regulatory status of bacteriocins, various models for bacteriocin action, and future prospects for their application are also discussed.