Structural characterization of polyurethane foam and implications of aging

Polyurethane (PU) foam is used as a shock mitigation material in the national stockpile, which degrades over time. A replacement foam, ReCrete was shown to exhibit acceptable mechanical properties after aging, but fractured with impact testing. Previously, processing ReCrete foam at elevated temperature resulted in a decrease of the modulus (stiffness), which was believed to be related to chemical changes during processing. In this thesis, chemical and structural analysis of ReCrete processed at 25°C and 85°C were performed using photoacoustic infrared spectroscopy, IR imaging, and thermal analysis. The change in modulus was related to thermal decomposition of uretoneimine linkages in the diisocyanate starting material by monitoring the change in intensities of diisocyanate, carbodiimide, and uretoneimine bands in the IR spectra. Thermal analysis (DSC and TMA) were consistent with IR finding, where endothermic and exothermic events could be associated with chemical changes in the foam

Band 3 diffusion on healthy and diseased red blood cells and implications for RBC membrane structure

Band 3 which is the one of the most abundant membrane protein in red blood cell membrane. It is bound to the spectrin network via other proteins such as ankyrin, 4.1 or junctional complex. In addition to that literature suggests the presence of different sub populations of band 3. The spectrin mesh leads to the hexagonal compartments in red blood cell membrane. The arrangement of this skeleton network affects the stability and the size of the compartments. We discovered that spectrin compartment size and the band3 diffusion vary from normal to disease patient’s blood such as HbSS (Sickle cell anemia), HbSC (Sickle hemoglobin C disease), HbSB° (Sickle cell zero-beta-thalasamia), HbSB+ (Sickle cell beta-plus-thalasamia), HS (Hereditary Spherocytosis), and HPP (Hereditary Pyropoikilocytosis). HbSS has comparatively smaller compartment size and slow diffusion coefficient than normal blood cells. We have been able to show that by Single particle tracking (SPT) studies, which is investigated by video-enhanced TIR microscopy with 8ms temporal resolution, by labeling the band 3 with DIDS-Biotin linker bound to streptavidin Q-dots. Specificity and binding ability of linker to band 3 were investigated; (i) whole red blood cells were labeled with a synthesized compound called DIDS-biotin linker (Figure 1). Labeled red blood cells were co-precipitated with avidin beads to indicate the presence of band3. (ii) Fluorescence microscopy images were collected by incubating the cells in the presence and the absence of linker. RBC appears only in the fluorescents field of the specimen which is incubated with linker. (iii) Flow cytometry study of binding assay indicates the concentration dependency of DIDS-biotin linker to red blood cells. The 50% saturation was attained at ∼1-2E-5M. (iv) The cells were labeled with 1E-11M concentration of linker to obtain the one or few Q-dots (0-3) per cell. The SPT data suggest the presence of mobile and immobile populations of band 3 in red blood cell membranes. Based on these studies variation of distribution of these populations on different blood samples suggest the changes in structural attachment of band 3 to the skeleton. This may also help to explain why the HS and HPP red blood cells are fragile compared to rigid sickle cells

Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes

<div><p>Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells</p></div

Microscopic and macroscopic diffusion coefficient data for various healthy and sickle cell erythrocytes populations.

<p>Microscopic and macroscopic diffusion coefficient data for various healthy and sickle cell erythrocytes populations.</p

Distribution of the compartment sizes in intact healthy and sickle erythrocytes.

<p>Compartment sizes were determined by analysis of individual trajectories of labeled band 3 molecules in intact unfixed normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and HbSC cells.</p

Distributions of the logarithms of the microscopic (D_μ) and macroscopic (D_M) diffusion coefficients of band 3 in healthy and sickle erythrocytes.

<p>Diffusion coefficients were determined by analysis of individual trajectories of labeled band 3 molecules in intact fixed normal cells, unfixed normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and HbSC erythrocytes.</p

Identification of Contact Sites between Ankyrin and Band 3 in the Human Erythrocyte Membrane

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175–185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63–73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3–ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D₃D₄-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69–ankyrin E645, (ii) cdB3 E72–ankyrin K611, and (iii) cdB3 D183–ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail

Identification of Contact Sites between Ankyrin and Band 3 in the Human Erythrocyte Membrane

The red cell membrane is stabilized by a spectrin/actin-based cortical cytoskeleton connected to the phospholipid bilayer via multiple protein bridges. By virtue of its interaction with ankyrin and adducin, the anion transporter, band 3 (AE1), contributes prominently to these bridges. In a previous study, we demonstrated that an exposed loop comprising residues 175–185 of the cytoplasmic domain of band 3 (cdB3) constitutes a critical docking site for ankyrin on band 3. In this paper, we demonstrate that an adjacent loop, comprising residues 63–73 of cdB3, is also essential for ankyrin binding. Data that support this hypothesis include the following. (1) Deletion or mutation of residues within the latter loop abrogates ankyrin binding without affecting cdB3 structure or its other functions. (2) Association of cdB3 with ankyrin is inhibited by competition with the loop peptide. (3) Resealing of the loop peptide into erythrocyte ghosts alters membrane morphology and stability. To characterize cdB3–ankyrin interaction further, we identified their interfacial contact sites using molecular docking software and the crystal structures of D₃D₄-ankyrin and cdB3. The best fit for the interaction reveals multiple salt bridges and hydrophobic contacts between the two proteins. The most important ion pair interactions are (i) cdB3 K69–ankyrin E645, (ii) cdB3 E72–ankyrin K611, and (iii) cdB3 D183–ankyrin N601 and Q634. Mutation of these four residues on ankyrin yielded an ankyrin with a native CD spectrum but little or no affinity for cdB3. These data define the docking interface between cdB3 and ankyrin in greater detail