Silver is widely used as a biocidal agent in ointments and wound dressings. However, it has also been associated with tissue toxicity and impaired healing. In vitro characterization has also revealed that typical loadings of silver employed in ointments and dressings (∼ 100 μg/cm2) lead to cytotoxicity. In this paper, we report the results of an initial study that sought to determine if localization of carefully controlled loadings of silver nanoparticles within molecularly thin films immobilized on surfaces can lead to antimicrobial activity without inducing cytotoxicity. Polymeric thin films of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) were prepared by layer-by-layer deposition and loaded with ∼0.4 μg/cm2 to ∼23.6 μg/cm2 of silver nanoparticles. Bacterial killing efficiencies of the silver-loaded films were investigated against Staphylococcus epidermidis, a gram-positive bacterium, and it was determined that as little as ∼0.4 μg/cm2 of silver in the polymeric films caused a reduction of 6 log10 CFU/mL (99.9999%) bacteria in suspensions incubated in contact with the films (water-borne assays). Significantly, whereas the antibacterial films containing high loadings of silver were found to be toxic to a murine fibroblast cell line (NIH-3T3), the polymeric films containing ∼0.4 μg/cm2 of silver were not toxic and allowed attachment, and growth of the mammalian cells. Thus, the results of this study go beyond prior reports by identifying silver-impregnated, polymeric thin films that are compatible with in vitro mammalian cell culture yet exhibit antibacterial activity. These results support the hypothesis that localization of carefully controlled loadings of silver nanoparticles within molecularly thin polymeric films can lead to antimicrobial activity without cytotoxicity. More broadly, this strategy of modifying surfaces with minimal loadings of bioactive molecules indicates the basis of approaches that may permit management of microbial burden in wound beds without impairment of wound healing.

The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3-ankyrin-␤-spectrin and glycophorin C-protein 4.1-␤-spectrin. 1-7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C-protein 4.1 interaction has little effect on membrane stability. 8 We demonstrate the existence of a novel band 3-adducin-spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3-adducinspectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane. (Blood. 2009;114: 1904-1912 IntroductionThe model of the erythrocyte membrane presented in cell biology, hematology, and biochemistry textbooks shows 2 major protein bridges that span between the phospholipid bilayer and the spectrin/actin skeleton. [1][2][3][4][5][6][7] The more prominent bridge, a linkage from the integral membrane protein, band 3, to spectrin via ankyrin, is composed of multiple high-affinity protein-protein interactions. [9][10][11] Defects or deficiencies in either band 3 or ankyrin lead to a decrease in cohesion between the lipid bilayer and membrane skeleton, resulting in loss of membrane surface area and a pathology termed hereditary spherocytosis. [12][13][14] Manual rupture of this bridge by addition of competing fragments of either band 3 or ankyrin, or by addition of competing monoclonal antibodies, or mutation of the ankyrin binding site on band 3 induces spontaneous membrane vesiculation and fragmentation. [14][15][16] Spontaneous mutations in the ankyrin-bridging function in other cells can also lead to serious pathologies. [17][18][19][20] Taken together, these data support the importance of the ankyrin-spectrin bridge in maintaining membrane integrity.The second bridge connecting the membrane bilayer to the spectrinactin skeleton consists of the membrane-spanning protein, glycophorin C (GPC), tethered to spectrin via the adapter protein 4.1. [21][22][23] The complex of cytoskeletal proteins at this nexus (primarily actin, dematin, tropomyosin, adducin, protein 4.1, and tropomodulin) forms a junctional complex from which spectrin tetramers extend radially into a 2-dimensional lattice that provides mechanical stability to the overlying membrane. Based on the finding that GPC-deficient red cells exhibit decreased membrane mechanical stability, it has been inferred that the GPC-protein 4.1 bridge is essential to erythrocyte integrity. 24,25 However...

Erythropoietin and stem cell factor are the key cytokines that regulate early stages of erythroid differentiation. However, it remains undetermined whether additional cytokines also play a role in the differentiation program. Here, we report that osteopontin (OPN) is highly expressed and secreted by erythroblasts during differentiation. We also demonstrate that OPNdeficient human and mouse erythroblasts exhibit defects in F-actin filaments, and addition of exogenous OPN to OPN-deficient erythroblasts restored the F-actin filaments in these cells. Furthermore, our studies demonstrate that OPN contributes to erythroblast proliferation. OPN knock-out male mice exhibit lower hematocrit and hemoglobin levels compared with their wild-type counterparts. We also show that OPN mediates phosphorylation or activation of multiple proteins including Rac-1 GTPase and the actin-binding protein, adducin, in human erythroblasts. In addition, we show that the OPN effects include regulation of intracellular calcium in human erythroblasts. Finally, we demonstrate that human erythroblasts express CD44 and integrins ␤ 1 and ␣ 4 , three known receptors for OPN, and that the integrin ␤ 1 receptor is involved in transmitting the proliferative signal. Together these results provide evidence for signal transduction by OPN and contribution to multiple functions during the erythroid differentiation program in human and mouse.Early stages of erythroid cell differentiation are regulated by multiple growth factors including interleukin-3, erythropoietin (EPO), 4 and stem cell factor (SCF) (1, 2). EPO and SCF have distinct functions. The predominant role of EPO is to deliver survival signals and maintain cell viability (3, 4), whereas SCF provides signals for cell proliferation (4 -6). Together these two growth factors guide the erythroid differentiation program from the early basophilic stage through the late polychromatic stage of maturation. However, the effects of these cytokines explain only the early stages of erythropoiesis. We were interested in identifying additional cytokines and/or factors involved in the erythroid differentiation program, especially factors that regulate the remodeling of the cytoskeleton. To achieve these objectives we developed methods to obtain extremely pure primary erythroblasts that synchronously differentiate into reticulocytes. Utilizing these cells, we screened a cDNA microarray and identified OPN as one of the cytokines that is highly expressed by erythroblasts during differentiation.OPN is a multifunctional cytokine that is highly expressed during bone remodeling and has pro-inflammatory effects (7-12). OPN has anti-apoptotic, chemotactic, and proliferative properties, depending on the cell type and context. It also plays a vital role in the delayed-type immune response and is known to be secreted by activated T cells and macrophages (13). OPN knock-out mice are viable and live a normal life span but suffer from bone defects and problems with wound and fracture healing (14). To date, OPN has not been show...

The principal bridge connecting the erythrocyte membrane to the spectrin-based skeleton is established by band 3 and ankyrin; mutations leading to reduced bridge formation or increased bridge rupture result in morphological and mechanical abnormalities. Because membrane mechanical properties are determined in part by the protein interactions that stabilize the membrane, we have evaluated the rates of rupture and reattachment of band 3-ankyrin bridges under both resting and mechanically stressed conditions. To accomplish this, we have examined the rate of ankyrin displacement from inside-out vesicles by the hexahistidine-tagged cytoplasmic domain of band 3, cdb3-(His) 6 and the rate of substitution of cdb3-(His) 6 into endogenous band 3-ankyrin bridges in resealed erythrocytes in the presence and absence of shear stress. We demonstrate that 1) exogenous cdb3-(His) 6 displaces endogenous ankyrin from IOVs with a half-time and first order rate constant of 42 ؎ 14 min and 0.017 ؎ 0.0058 min ؊1 , respectively; 2) exogenous cdb3-(His) 6 substitutes endogenous band 3 in its linkage to ankyrin in resealed cells with a half-time and first order rate constant of 12 ؎ 3.6 min and 0.060 ؎ 0.019 min ؊1 , respectively; 3) cdb3-(His) 6 -mediated rupture of the band 3-ankyrin bridge in resealed cells results in decreased membrane mechanical stability, decreased deformability, abnormal morphology, and spontaneous vesiculation of the cells; and 4) the above on/off rates are not significantly accelerated by mechanical shear stress. We conclude that the off rates of the band 3-ankyrin interaction are sufficiently slow to allow sustained erythrocyte deformation without loss of elasticity.In its simplest representation, the human erythrocyte membrane is thought to be comprised of a two-dimensional cortical membrane skeleton connected to a lipid bilayer by multiple protein-protein and protein-lipid interactions (1). Several lines of evidence suggest that the protein-protein interactions within the membrane skeleton are essential for membrane stability, including data demonstrating that rupture of individual protein linkages can lead to membrane instability (2-5) and deficiencies in specific skeletal components can yield misshapen and poorly deformable cells (6 -10). Similar evidence supporting the importance of membrane-to-skeleton bridges derives from mechanical studies showing that deletion, reduction, or mutation of major bridging components can result in mechanically and morphologically compromised cells (2, 4). In virtually all well characterized membrane mechanical defects, a reduction in either the number or affinity of critical protein-protein associations has been documented.Although the equilibrium binding constants for most protein-protein interactions in the human red cell membrane have been reported, little information is available on the dynamics of the same protein-protein associations. Such dynamics are, however, very important because the "on" and "off " rates of major protein interactions determine the elasticity ver...

This paper introduces novel technologies that will be instrumental in the development of new methods for the Raman spectrochemical imaging of painted works of art. We show that second-derivative preprocessing diminishes interference from fluorescence. Normalization and mean-centering of such second-derivative spectra cast them in the form of unit vectors, the displacement of which from library standards serves as a basis for the instantaneous on-line identification of artistic pigments and binders. Multivariate measures of displacement are shown to function well in difficult cases such as spectrally similar fatty acid media and pigments diluted to varying degrees in uncharacterized binders. A self-calibrating technique for background subtraction extracts pure component spectra from pigment-acrylic mixtures.