Synthesis and evaluation of alpha-LNA

Three different synthetic routes to the alpha-configured LNA thymine monomer starting from D-allose or D-arabinose were investigated. The introduction of one or four alpha-LNA monomers into alpha-DNA had a destabilizing effect on the duplexes. However, a fully modified alpha-LNA sequence displayed strong recognition of complementary RNA, but no transition with DNA.     

Low Physical Activity Level and Short Sleep Duration Are Associated with an Increased Cardio-Metabolic Risk Profile: A Longitudinal Study in 8-11 Year Old Danish Children

Background

As cardio-metabolic risk tracks from childhood to adulthood, a better understanding of the relationship between movement behaviors (physical activity, sedentary behavior and sleep) and cardio-metabolic risk in childhood may aid in preventing metabolic syndrome (MetS) in adulthood.

Objective

To examine independent and combined cross-sectional and longitudinal associations between movement behaviors and the MetS score in 8-11 year old Danish children.

Design

Physical activity, sedentary time and sleep duration (seven days and eight nights) were assessed by accelerometer and fat mass index (fat mass/height²) was assessed using Dual-energy X-ray absorptiometry. The MetS-score was based on z-scores of waist circumference, mean arterial blood pressure, homeostatic model assessment of insulin resistance, triglycerides and high density lipoprotein cholesterol. All measurements were taken at three time points separated by 100 days. Average of the three measurements was used as habitual behavior in the cross-sectional analysis and changes from first to third measurement was used in the longitudinal analysis.

Results

723 children were included. In the cross-sectional analysis, physical activity was negatively associated with the MetS-score (P<0.03). In the longitudinal analysis, low physical activity and high sedentary time were associated with an increased MetS-score (all P<0.005); however, after mutual adjustments for movement behaviors, physical activity and sleep duration, but not sedentary time, were associated with the MetS-score (all P<0.03). Further adjusting for fat mass index while removing waist circumference from the MetS-score rendered the associations no longer statistically significant (all P>0.17). Children in the most favorable tertiles of changes in moderate-to-vigorous physical activity, sleep duration and sedentary time during the 200-day follow-up period had an improved MetS-score relative to children in the opposite tertiles (P = 0.005).

Conclusion

The present findings indicate that physical activity, sedentary time and sleep duration should all be targeted to improve cardio-metabolic risk markers in childhood; this is possibly mediated by adiposity.     

Lipid raft localization of GABA A receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells

The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na(+), K(+)-ATPase, was found in discrete rafts of lower density, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy "hot spots" at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-beta-cyclodextrin. In contrast, both the GABA(A) receptor and Na(+), K(+)-ATPase were largely soluble in ice cold Triton X-100. This indicates that Brij 98 extraction defines an unusual type of cholesterol-independent lipid rafts that harbour membrane proteins also associated with underlying scaffolding/cytoskeletal proteins such as gephyrin (GABA(A) receptor) and ankyrin G (Na(+), K(+)-ATPase). By providing an ordered membrane microenvironment, lipid rafts may contribute to the clustering of the GABA(A) receptor and the Na(+), K(+)-ATPase at distinct functional locations on the cell surface.     

Cholera toxin entry into pig enterocytes occurs via a lipid raft- and clathrin-dependent mechanism

The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.