Bilayer-mediated clustering and functional interaction of MscL channels

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.     

GITR Intrinsically Sustains Early Type 1 and Late Follicular Helper CD4 T Cell Accumulation to Control a Chronic Viral Infection

CD4 T cells are critical for control of persistent infections; however, the key signals that regulate CD4 T help during chronic infection remain incompletely defined. While several studies have addressed the role of inhibitory receptors and soluble factors such as PD-1 and IL-10, significantly less work has addressed the role of T cell co-stimulatory molecules during chronic viral infection. Here we show that during a persistent infection with lymphocytic choriomeningitis virus (LCMV) clone 13, mice lacking the glucocorticoid-induced tumor necrosis factor receptor related protein (GITR) exhibit defective CD8 T cell accumulation, increased T cell exhaustion and impaired viral control. Differences in CD8 T cells and viral control between GITR^+/+ and GITR^-/- mice were lost when CD4 T cells were depleted. Moreover, mixed bone marrow chimeric mice, as well as transfer of LCMV epitope-specific CD4 or CD8 T cells, demonstrated that these effects of GITR are largely CD4 T cell-intrinsic. GITR is dispensable for initial CD4 T cell proliferation and differentiation, but supports the post-priming accumulation of IFNγ⁺IL-2⁺ Th1 cells, facilitating CD8 T cell expansion and early viral control. GITR-dependent phosphorylation of the p65 subunit of NF-κB as well as phosphorylation of the downstream mTORC1 target, S6 ribosomal protein, were detected at day three post-infection (p.i.), and defects in CD4 T cell accumulation in GITR-deficient T cells were apparent starting at day five p.i. Consistently, we pinpoint IL-2-dependent CD4 T cell help for CD8 T cells to between days four and eight p.i. GITR also increases the ratio of T follicular helper to T follicular regulatory cells and thereby enhances LCMV-specific IgG production. Together, these findings identify a CD4 T cell-intrinsic role for GITR in sustaining early CD8 and late humoral responses to collectively promote control of chronic LCMV clone 13 infection.     

The mass-spectrometric identification of hypoxanthine and xanthine (`oxypurines') in skeletal muscle from two patients with congenital xanthine oxidase deficiency (xanthinuria)

1. The presence of hypoxanthine and xanthine in the skeletal muscle of two patients with congenital xanthine oxidase deficiency (xanthinuria) was demonstrated by high-resolution mass spectrometry. 2. Evidence was obtained for the presence of a trace of hypoxanthine only in normal muscle. 3. Dry pulverized tissue was introduced directly into the mass spectrometer and preliminary chemical processing of the tissue was therefore unnecessary. 4. The criteria for the mass-spectrometric identification of hypoxanthine and xanthine in the tissue and the significance of the observations are discussed.     

Quantitative gas-liquid chromatographic analysis of rodent milk triglycerides

A comparison has been made of the milk and adipose tissue triglycerides of rabbits and guinea pigs provided with one diet and of rats and mice provided with another. Both intact triglycerides and component fatty acids were analyzed by gas-liquid chromatography. Good correlation of the data obtained by the two techniques was obtained by calculating the average chain length of the fatty acid moieties. Little difference was found in the triglyceride composition of the adipose tissue of the different species. However, wide variation in the triglyceride composition of the milk was found between the species: the average fatty acid chain length in milk was 11.7 for rabbits, 14.2 for rats, 15.3 for mice, and 17.2 for guinea pigs. The corresponding values for adipose tissue were in the range 16.9-17.4 in all animals. The significance of enzymes that synthesize short-chain fatty acids in mammary gland is discussed.