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Cryogenic transmission electron microscopy (cryo-TEM) is utilized to determine the second virial coefficient of osmotic pressure of PbSe quantum dots (QDs) dispersed in apolar liquid. Cryo-TEM images from vitrified samples provide snapshots of the equilibrium distribution of the particles. These snapshots yield radial distribution functions from which second virial coefficients are calculated, which agree with second virial coefficients determined with analytical centrifugation and small-angle X-ray scattering. The size dependence of the second virial coefficient points to an interparticle interaction that is proportional to the QD surface area. A plausible cause for this attraction is the interaction between the surface ions on adjacent QDs.

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We report a study of mixtures of initially oppositely charged particles with similar size. Dispersions of silica spheres (negatively charged) and alumina-coated silica spheres (positively charged) at low ionic strength, mixed at various volume ratios, exhibited a surprising stability up to compositions of 50% negative colloids as well as spontaneous repeptization of particles from the early-stage formed aggregates. The other mixtures were found to contain large heteroaggregates, which were imaged using cryogenic electron microscopy. Electrophoretic mobility, electrical conductivity, static and dynamic light scattering and sedimentation were studied as a function of volume fraction of the mixed dispersions to investigate particle interactions and elucidate the repeptization phenomenon.

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T cell factor / lymphocyte enhancer factor (Tcf/Lef) transcription factors complex with the transcriptional co-activator beta-catenin to transduce Wnt signals in a variety of developmental systems. The prototypic family member Tcf-1 is highly expressed in T lineage cells. Tcf1-/- mice are defective in cell cycling of early thymocyte stages. Here, we show that the interaction of beta-catenin with Tcf-1 is required for full thymocyte development. This interaction may be established by signals mediated by Wnt1 and Wnt4, leading to increased Tcf-dependent transcriptional activity in thymocytes, as demonstrated in Tcf-LacZ reporter mice. Transduction of fetal thymocytes with Wnt1 and Wnt4 results in increased survival in an in vitro cell culture system. Retroviral expression of soluble Wnt receptor mutants that block Wnt signaling inhibits thymocyte development. These results imply an important role for the Wnt cascade in thymocyte development.

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Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their alpha-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

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The use of catheters is often complicated by infection, mainly due to Staphylococcus epidermidis. Recently, a novel poly(vinylpyrrolidone)-grafted silicone elastomer catheter (SEpvp) was introduced. Less bacteria adhered to SEpvp than to conventional SE catheters in vitro. The frequency of S. epidermidis infection associated with SEpvp and SE was assessed in a rabbit model. Unexpectedly, abscesses were induced by the injection of low numbers of S. epidermidis along subcutaneously inserted SEpvp. No abscesses were seen around SE, even when very high numbers of S. epidermidis were injected. This bioincompatibility reaction observed around the SEpvp was independent of the host, bacterial strain, and method of inoculation. Abscesses were also induced by nonviable S. epidermidis and by bacterial cell wall components. Because these incompatibility reactions were not observed in the absence of bacteria, biocompatibility testing should include experiments in which the inflammatory effects of the combination of catheter and (non)viable bacteria are tested.

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In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.

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In Escherichia coli K-12, temperature-sensitive mutations in the secA gene have been shown to interfere with protein export. Here we show that the effect of a secA mutation is strongly pleiotropic on membrane biogenesis. Freeze-fracture experiments as well as cryosections of the cells revealed the appearance of intracytoplasmic membranes upon induction of the SecA phenotype. The permeability barrier of the outer membrane to detergents was lost. Two alterations in the outer membrane may be responsible for this effect, namely the reduced amounts of outer membrane proteins, or the reduction of the length of the core oligosaccharide of the lipopolysaccharide, which was observed in phage-sensitivity experiments and by SDS-polyacrylamide gel electrophoresis. Phospholipid analysis of the secA mutant, grown under restrictive conditions, revealed a lower content of the negatively charged phospholipid cardiolipin and of 18:1 fatty acid compared to those of the parental strain grown under identical conditions. These results are in line with the hypothesis that protein export and lipid metabolism are coupled.

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