Solid-solid interface adsorption of proteins and enzymes in nanophase-separated amphiphilic conetworks

Amphiphilic polymer conetworks (APCNs) are materials with a very large interface between their hydrophilic and hydrophobic phases due to their nanophase-separated morphologies. Proteins were found to enrich in APCNs by up to 2 orders of magnitude when incubated in aqueous protein solutions, raising the question of the driving force of protein uptake into APCNs. The loading of poly(2-hydroxyethyl acrylate)-linked by-poly(dimethylsiloxane) (PHEA-l-PDMS) with heme proteins (myoglobin, horseradish peroxidase, hemoglobin) and lipases was studied under variation of parameters such as incubation time, pH, concentration of the protein solution, and conetwork composition. Adsorption of enzymes to the uncharged interface is the main reason for protein uptake, resulting in protein loading of up to 23 wt %. Experimental results were supported by computation of electrostatic potential maps of a lipase, indicating that hydrophobic patches are responsible for the adsorption to the interface. The findings underscore the potential of enzyme-loaded APCNs in biocatalysis and as sensors.     

Starch-related carbon fluxes in roots and leaves of Arabidopsis thaliana

Both photoautotrophic and heterotrophic tissues from plants are capable of synthesizing and degrading starch. To analyze starch metabolism in the two types of tissue from the same plant, several starch-related mutants from Arabidopsis thaliana were grown hydroponically together with the respective wild-type control. Starch contents, patterns of starch-related enzymes and the monomer patterns of the cytosolic starch-related heteroglycans were determined. Based on the phenotypical data obtained, three comparisons were made: First, data from leaves and roots of the mutants were compared with the respective wild-type controls. Secondly, data from leaves and roots from the same plant were compared. Third, we included data obtained from soil-grown plants and compared them with those from hydroponically grown plants. Thus, phenotypical features reflecting altered gene expression can be distinguished from those that are due to the specific growth conditions. Implications on the carbon fluxes in photoautotrophic and heterotrophic cells are discussed.Key words: starch metabolism, cytosolic heteroglycans, cytosolic glucosyl transferases, carbon fluxes     

Brown adipose tissue activity controls triglyceride clearance

Brown adipose tissue (BAT) burns fatty acids for heat production to defend the body against cold and has recently been shown to be present in humans. Triglyceride-rich lipoproteins (TRLs) transport lipids in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase (LPL). Peripheral organs such as muscle and adipose tissue take up the fatty acids, whereas the remaining cholesterol-rich remnant particles are cleared by the liver. Elevated plasma triglyceride concentrations and prolonged circulation of cholesterol-rich remnants, especially in diabetic dyslipidemia, are risk factors for cardiovascular disease. However, the precise biological role of BAT for TRL clearance remains unclear. Here we show that increased BAT activity induced by short-term cold exposure controls TRL metabolism in mice. Cold exposure drastically accelerated plasma clearance of triglycerides as a result of increased uptake into BAT, a process crucially dependent on local LPL activity and transmembrane receptor CD36. In pathophysiological settings, cold exposure corrected hyperlipidemia and improved deleterious effects of insulin resistance. In conclusion, BAT activity controls vascular lipoprotein homeostasis by inducing a metabolic program that boosts TRL turnover and channels lipids into BAT. Activation of BAT might be a therapeutic approach to reduce elevated triglyceride concentrations and combat obesity in humans.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Unbiased screening of polymer libraries to define novel substrates for functional hepatocytes with inducible drug metabolism

Maintaining stable differentiated somatic cell function in culture is essential to a range of biological endeavors. However, current technologies, employing, for example, primary hepatic cell culture (essential to the development of a bio-artificial liver and improved drug and toxicology testing), are limited by supply, expense, and functional instability even on biological cell culture substrata. As such, novel biologically active substrates manufacturable to GMP standards have the potential to improve cell culture-based assay applications. Currently hepatic endoderm (HE) generated from pluripotent stem cells is a genotypically diverse, cheap, and stable source of "hepatocytes"; however, HE routine applications are limited due to phenotypic instability in culture. Therefore a manufacturable subcellular matrix capable of supporting long-term differentiated cell function would represent a step forward in developing scalable and phenotypically stable hESC-derived hepatocytes. Adopting an unbiased approach we screened polymer microarrays and identified a polyurethane matrix which promoted HE viability, hepatocellular gene expression, drug-inducible metabolism, and function. Moreover, the polyurethane supported, when coated on a clinically approved bio-artificial liver matrix, long-term hepatocyte function and growth. In conclusion, our data suggest that an unbiased screening approach can identify cell culture substrate(s) that enhance the phenotypic stability of primary and stem cell-derived cell resources.     

Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells

The culture of human embryonic stem (hES) cells in defined and xenogeneic-free conditions will contribute substantially to future biotechnological and medical applications. To achieve this goal, we developed the first fully defined synthetic polymer coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) that sustains long-term growth of hES cells in different culture media. Here we describe a detailed protocol for the reproducible fabrication of PMEDSAH coating on tissue culture polystyrene dishes, and for the feeder-free culture of hES cells on PMEDSAH coating in defined culture medium. This culture system represents a key step toward the fully defined and xenogeneic-free culture of hES cells.     

Genotyping of environmental and clinical Stenotrophomonas maltophilia isolates and their pathogenic potential

Stenotrophomonas maltophilia is a highly versatile species with useful biotechnological potential but also with pathogenic properties. In light of possible differences in virulence characteristics, knowledge about genomic subgroups is therefore desirable. Two different genotyping methods, rep-PCR fingerprinting and partial gyrB gene sequencing were used to elucidate S. maltophilia intraspecies diversity. Rep-PCR fingerprinting revealed the presence of 12 large subgroups, while gyrB gene sequencing distinguished 10 subgroups. For 8 of them, the same strain composition was shown with both typing methods. A subset of 59 isolates representative for the gyrB groups was further investigated with regards to their pathogenic properties in a virulence model using Dictyostelium discoideum and Acanthamoeba castellanii as host organisms. A clear tendency towards accumulation of virulent strains could be observed for one group with A. castellanii and for two groups with D. discoideum. Several virulent strains did not cluster in any of the genetic groups, while other groups displayed no virulence properties at all. The amoeba pathogenicity model proved suitable in showing differences in S. maltophilia virulence. However, the model is still not sufficient to completely elucidate virulence as critical for a human host, since several strains involved in human infections did not show any virulence against amoeba.     

Zur Frage einer Evolution der Menschheit während des Eiszeitalters II. Teil

Ohne Zusammenfassung     

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.