Reconstitution of membranes simulating "glycosignaling domain" and their susceptibility to lyso-GM3

GM3 ganglioside at the surface of mouse melanoma B16 cells is clustered and organized with signal transducer molecules c-Src, Rho A, and focal adhesion kinase (FAK) to form a membrane unit separable from caveolae, which are enriched in cholesterol and caveolin but do not contain GM3 or the above three signal transducers. The GM3-enriched membrane units are involved in GM3-dependent cell adhesion coupled with activation of c-Src, Rho A, and FAK and are termed the "glycosphingolipid signaling domain" or the "glycosignaling domain" (GSD). In order to assess the essential components that display GSD function, membranes with properties similar to those of GSD were reconstituted using GM3, sphingomyelin, and c-Src, with or without other lipid components. The reconstituted membrane thus prepared displayed GM3-dependent adhesion to plates coated with Gg3 or anti-GM3 antibody, resulting in enhanced c-Src phosphorylation (c-Src phosphorylation response). This response in reconstituted membrane depends on GM3 concentration and was not observed when GM3 was absent or replaced with other gangliosides GM1 or GD1a, or with LacCer. The GM3-dependent c-Src phosphorylation response was enhanced when cholesterol and phosphatidylcholine were added. Although GM3, sphingomyelin, and c-Src are essential for GSD function, a small quantity of cholesterol and phosphatidylcholine may act as an auxiliary factor to stabilize membrane. GSD function in terms of GM3-dependent adhesion and signaling was blocked in the presence of lyso-GM3 or its analogue but not psychosine, lactosyl-sphingosine, or lyso-phosphatidylcholine. Such susceptibility of reconstituted GSD to lyso-GM3 and other lyso compounds is the same as GSD of original B16 cells. Thus, functional organization of the reconstituted membrane closely simulates that of GSD in B16 cells, which is based on clustered GM3 organized with c-Src as the essential components.     

Ultraweak sugar-sugar interactions for transient cell adhesion

Carbohydrate-carbohydrate interactions are rarely considered in biologically relevant situations such as cell recognition and adhesion. One Ca(2+)-mediated homotypic interaction between two Lewis(x) determinants (Le(x)) has been proposed to drive cell adhesion in murine embryogenesis. Here, we confirm the existence of this specific interaction by reporting the first direct quantitative measurements in an environment akin to that provided by membranes. The adhesion between giant vesicles functionalized with Le(x) was obtained by micropipette aspiration and contact angle measurements. This interaction is below the thermal energy, and cell-cell adhesion will require a large number of molecules, as illustrated by the Le(x) concentration peak observed at the cell membranes during the morula stage of the embryo. This adhesion is ultralow and therefore difficult to measure. Such small interactions explain why the concept of specific interactions between carbohydrates is often neglected.     

Experimental assessment of the influence of beam hardening filters on image quality and patient dose in volumetric 64-slice X-ray CT scanners

Beam hardening filters have long been employed in X-ray Computed Tomography (CT) to preferentially absorb soft and low-energy X-rays having no or little contribution to image formation, thus allowing the reduction of patient dose and beam hardening artefacts. In this work, we studied the influence of additional copper (Cu) and aluminium (Al) flat filters on patient dose and image quality and seek an optimum filter thickness for the GE LightSpeed VCT 64-slice CT scanner using experimental phantom measurements. Different thicknesses of Cu and Al filters (0.5–1.6 mm Cu, 0.5–4 mm Al) were installed on the scanner’s collimator. A planar phantom consisting of 13 slabs of Cu having different thicknesses was designed and scanned to assess the impact of beam filtration on contrast in the intensity domain (CT detector’s output). To assess image contrast and image noise, a cylindrical phantom consisting of a polyethylene cylinder having 16 holes filled with different concentrations of K₂HPO₄ solution mimicking different tissue types was used. The GE performance and the standard head CT dose index (CTDI) phantoms were also used to assess image resolution characterized by the modulation transfer function (MTF) and patient dose defined by the weighted CTDI. A 100 mm pencil ionization chamber was used for CTDI measurement. Finally, an optimum filter thickness was determined from an objective figure of merit (FOM) metric. The results show that the contrast is somewhat compromised with filter thickness in both the planar and cylindrical phantoms. The contrast of the K₂HPO₄ solutions in the cylindrical phantom was degraded by up to 10% for a 0.68 mm Cu filter and 6% for a 4.14 mm Al filter. It was shown that additional filters increase image noise which impaired the detectability of low density K₂HPO₄ solutions. It was found that with a 0.48 mm Cu filter the 50% MTF value is shifted by about 0.77 lp/cm compared to the case where the filter is not used. An added Cu filter with approximately 0.5 mm thickness accounts for 50% reduction in radiation-absorbed dose as measured by the weighted CTDI. The FOM results indicate that with an additional filter of 0.5 mm Cu or minimum 4 mm Al, a good compromise between image quality and patient dose is achieved for CT images acquired at tube voltages of 120 and 140 kVp. The results seem to indicate that an optimum filter for high kVp acquisitions, routinely used in cardiovascular imaging, should be 0.5 mm copper or 4 mm aluminium minimum.     

Isolation of alveolar plates from Coleps hirtus

In the ciliate Coleps hirtus, the alveoli contain rigid alveolar plates that are almost unstudied so far. Neither the exact composition nor the genesis and function are known. A necessary step to study the alveolar plates is to isolate these structures in an adequate amount. Therefore, culture conditions of C. hirtus were optimized to obtain an axenic and dense long-time culture. The protocol we developed to isolate C. hirtus alveolar plates is presented and clean alveolar plates were documented via scanning electron microscopy. The described procedure delivers alveolar plates of very good structure and integrity with preserved filigree details in sufficient amount. They can be analysed via a range of different material and biological characterisations. Since there are indications of a mineral phase within the alveolar plates, the presented results will allow to study C. hirtus alveolar plates also in the context of biomineralisation.     

Phosphorylation Drives a Dynamic Switch in Serine/Arginine-Rich Proteins

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Arabidopsis TWISTED DWARF1 Functionally Interacts with Auxin Exporter ABCB1 on the Root Plasma Membrane

Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding protein42 TWISTED DWARF1 (TWD1), although underlying mechanisms are unclear. By genetic manipulation of TWD1 expression, we show here that TWD1 affects shootward root auxin reflux and, thus, downstream developmental traits, such as epidermal twisting and gravitropism of the root. Using immunological assays, we demonstrate a predominant lateral, mainly outward-facing, plasma membrane location for TWD1 in the root epidermis characterized by the lateral marker ABC transporter G36/PLEIOTROPIC DRUG-RESISTANCE8/PENETRATION3. At these epidermal plasma membrane domains, TWD1 colocalizes with nonpolar ABCB1. In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)–TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein–protein interaction at the plasma membrane, minimizing reflux from the root apoplast into the cytoplasm.     

Soil Food Web Changes during Spontaneous Succession at Post Mining Sites: A Possible Ecosystem Engineering Effect on Food Web Organization?

Parameters characterizing the structure of the decomposer food web, biomass of the soil microflora (bacteria and fungi) and soil micro-, meso- and macrofauna were studied at 14 non-reclaimed 1– 41-year-old post-mining sites near the town of Sokolov (Czech Republic). These observations on the decomposer food webs were compared with knowledge of vegetation and soil microstructure development from previous studies. The amount of carbon entering the food web increased with succession age in a similar way as the total amount of C in food web biomass and the number of functional groups in the food web. Connectance did not show any significant changes with succession age, however. In early stages of the succession, the bacterial channel dominated the food web. Later on, in shrub-dominated stands, the fungal channel took over. Even later, in the forest stage, the bacterial channel prevailed again. The best predictor of fungal bacterial ratio is thickness of fermentation layer. We argue that these changes correspond with changes in topsoil microstructure driven by a combination of plant organic matter input and engineering effects of earthworms. In early stages, soil is alkaline, and a discontinuous litter layer on the soil surface promotes bacterial biomass growth, so the bacterial food web channel can dominate. Litter accumulation on the soil surface supports the development of the fungal channel. In older stages, earthworms arrive, mix litter into the mineral soil and form an organo-mineral topsoil, which is beneficial for bacteria and enhances the bacterial food web channel.