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Red Cell Aggregation and Yield Stress
Published in Gordon D. O. Lowe, Clinical Blood Rheology, 2019
Because the fibrinogen effects discussed above result from a weak, nonspecific adsorption to the red cell and from the length of the molecule allowing it to cross-link between adjacent cells, it is reasonable to suppose that other large plasma proteins might also be able to induce such aggregation. Chien et al.39 produced viscometric evidence in favor of this by studying cells suspended in serum. However Schmid-Schönbein et al.8 showed the aggregating effect of serum more directly using the rheoscope, where rouleaux could be seen microscopically. They also attempted to define the serum proteins that were responsible, by separating them by gel filtration chromatography and free flow electrophoresis, and then testing the fractions generated for aggregating ability. They concluded that such activity was associated with α2 macroglobulin and IgM, but that transferrin, ceruloplasmin, haptoglobin, and albumin were without effect. They were able to show that α2 macroglobulin would produce aggregation at a concentration above 200 mg/dℓ, while IgM required concentrations in excess of 500 mg/dℓ. In another study using purified preparations, Rovel et al.40 showed that IgG also led to changes consistent with aggregation, provided that the concentration was in excess of 1500 mg/dℓ.
Mechanisms of Cholestasis
Published in Robert G. Meeks, Steadman D. Harrison, Richard J. Bull, Hepatotoxicology, 2020
Although there is much evidence supporting the basolateral localization of Na+, K+-ATPase, Leffert et al. (1985) have provided immunologic evidence, using monoclonal antibodies against Na+, K+-ATPase, for its localization in the canalicular membrane as well. A recent report by Sutherland et al. (1988) suggests that the more rigid apical (canalicular) membrane prevents expression of Na+, K+-ATPase in vitro since increasing the fluidity of this membrane with agents such as 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C), benzyl alcohol, and Triton WR-1339 increased enzyme activity to 75% of that in basolateral membranes. The method used to prepare the canalicular membranes is apparently important to the detection of Na+, K+-ATPase activity. Sellinger et al. (1990) counter that use of freeflow electrophoresis to further purify canalicular liver plasma membranes originally separated by sucrose density centrifugation separated canalicular membranes into six fractions. Fractions enriched in canalicular markers were devoid of Na+,K+-ATPase activity, whereas this activity could be detected in fractions containing small amounts of canalicular markers but also containing glucagon-stimulated adenyl cyclase, a basolateral marker enzyme. Addition of A2C to all subfractions did not reveal any further Na+, K+-ATPase activity. In contrast, free-flow electrophoresis of canalicular membranes isolated by the Mg2+-precipitation technique used by Sutherland et al. (1988) could not separate Na+,K+-ATPase activity from canalicular membrane marker enzymes. Thus, Sellinger et al. (1990) argue that any Na+,K+-ATPase activity detected in canalicular membranes represents contamination by basolateral membranes.
Current knowledge about the impact of microgravity on the proteome
Published in Expert Review of Proteomics, 2019
Sebastian M. Strauch, Daniela Grimm, Thomas J. Corydon, Marcus Krüger, Johann Bauer, Michael Lebert, Petra Wise, Manfred Infanger, Peter Richter
The free-flow electrophoresis technique was useful for the preparation of peptide mixtures because of its improved capabilities in particle electrophoresis and the enhanced resolution in protein separation [102]. In addition, free-flow IEF proved to be helpful when proteins of low solubility, obtained e.g. from cell membranes, were investigated [102].
Understanding Leishmania parasites through proteomics and implications for the clinic
Published in Expert Review of Proteomics, 2018
Several online 2-DE derived databases have further assisted in building the foundation of proteome mapping through data mining and management. One of the major advances in this technique has been the use of an immobilized pH gradient (IPG) matrix [58,59], a modification of this system using zoom-gels, in which fractionation of the sample into narrow pH ranges with low resolution is followed by high-resolution separation by 2-D polyacrylamide gel electrophoresis (PAGE). Another modification of 2-D PAGE is DIGE [60], in which differences in protein profiles of cells in different functional states are examined by labelling the samples with different fluorescent dyes. DIGE from VL sera has provided important clues concerning the prognosis of VL [61]. The 2-DE-based protein array has also been reported to provide a framework for inter-species profiling of Leishmania proteins [62]. The major shortcoming associated with conventional 2-DE is the under representation of basic proteins [63], especially in the context of intracellular pathogens that have been predicted to bear larger arrays of basic proteomes than their free-living counterparts [64,65]. There is evidence for liquid phase isoelectric focusing (IEF) prior to 2-D gel separation with increased resolution of basic proteins but no clear identification of proteins [66], which led to the emergence of free-flow electrophoresis (FFE) that was later miniaturized approximately 1994. FFE has offered possibilities due to its rapid separation protocol and requirement for low sample volumes (in the microliter range). This technique employs electrophoretic separation of analytes by flowing through a planar flow channel and electric field perpendicular to the flow that diverts the analytes based on their mobility. This combination of IEF-FFE and 2-DE-based high-resolution separation technology has paved the way for the discovery of novel basic proteins from Leishmania [67] that could be of therapeutic importance. These microfluidic FFE systems and their different modifications, viz. free-flow zone electrophoresis (FFZE), free-flow IEF, free-flow ITP, and free-flow field-step electrophoresis (FFFSE), might find applications in so-called lab-on-a-chip devices for real-time monitoring and separation applications. Cup-loading at the anode has been another useful tool for basic proteome mapping [68]. The limited throughput associated with 2-DE has led to difficulties in-large scale comparative protein expression assays, resulting in the introduction of three-dimensional geometry gel electrophoresis in which a large number of samples are analyzed simultaneously. IEF followed by the use of several IPG strips arrayed on the surface of a 3-D gel body and the application of electro-kinetic field-mediated transfer to the gel ensures constant thermal conditions that provide data concerning the comparability of separation patterns. This format uses a laser-based fluorescence detection system for Cy3 dyes, and images are acquired, processed and recorded as a stack of 3-D images by the digital camera, thus offering wide range of applications [69]. The use of fluorescent dyes (Cy and Sypro Ruby dyes) has added quantification feature in 2-DE for analyzing quantitative differences among the spots on the 2-D gel [69–71].