S-glutathionylation activates STIM1 and alters mitochondrial homeostasis

Oxidant stress influences many cellular processes, including cell growth, differentiation, and cell death. A well-recognized link between these processes and oxidant stress is via alterations in Ca²⁺ signaling. However, precisely how oxidants influence Ca²⁺ signaling remains unclear. Oxidant stress led to a phenotypic shift in Ca²⁺ mobilization from an oscillatory to a sustained elevated pattern via calcium release–activated calcium (CRAC)–mediated capacitive Ca²⁺ entry, and stromal interaction molecule 1 (STIM1)– and Orai1-deficient cells are resistant to oxidant stress. Functionally, oxidant-induced Ca²⁺ entry alters mitochondrial Ca²⁺ handling and bioenergetics and triggers cell death. STIM1 is S-glutathionylated at cysteine 56 in response to oxidant stress and evokes constitutive Ca²⁺ entry independent of intracellular Ca²⁺ stores. These experiments reveal that cysteine 56 is a sensor for oxidant-dependent activation of STIM1 and demonstrate a molecular link between oxidant stress and Ca²⁺ signaling via the CRAC channel.     

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV_A, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV_A activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV_A activation. Computational studies suggested that unique ionic interactions exist between lipid IV_A and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IV_A interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys³⁶⁷ and Arg⁴³⁴) at the dimerization interface. When replaced with their negatively charged human counterparts Glu³⁶⁹ and Gln⁴³⁶, mouse TLR4 was no longer responsive to lipid IV_A. In contrast, human TLR4 gained lipid IV_A responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV_A species specificity. Thus, using lipid IV_A as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.     

Antihyperglycemic effect of polysaccharide from fermented broth of Pleurotus citrinopileatus

Pleurotus citrinopileatus is an edible mushroom, which has recently become very popular, with a consequent increase in industrial production. Water-soluble polysaccharides (WSPS), extracted from edible mushrooms, have been found to have antitumor and immunoenhancing effects. In this study, we investigate the effects of WSPS extracted from submerged fermented medium of P. citrinopileatus on hyperglycemia and damaged pancreatic cells in rats with streptozotocin (STZ)-induced diabetes. The diabetic rats fed with water-soluble polysaccharide of P. citrinopileatus (SPPC) lost less body weight than those fed SPPC-free regular diet. Serum total cholesterol and triglyceride levels in the diabetic rats fed with SPPC at a dose of 0.4 g/kg bw daily was lower than in the groups fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The fasting blood glucose levels of diabetic rats fed with SPPC were 44% lower than the negative controls. The degree of damage to the islets of Langerhans of the rats fed with the highest dosage of SPPC was significantly lower than those fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The results showed that STZ-induced diabetic rats fed with SPPC might help alleviate the elevation of the level of that in fasting blood glucose.     

Isolation and characterization of 15 microsatellite loci in four endangered Amentotaxus species (Taxaceae)

? Premise of the study: Fifteen microsatellite loci were developed in an endangered species, Amentotaxus formosana, and were tested in an additional three species, A. argotaenia, A. yunnanensis, and A. poilanei, to evaluate the population structure for conservation efforts and reconstruct the phylogeographic patterns of this ancient lineage. ? Methods and Results: Polymorphic primer sets were developed from A. formosana; the number of alleles ranged from two to 10, with an observed heterozygosity ranging from 0 to 0.60. All of the loci were found to be interspecifically amplifiable. ? Conclusions: These polymorphic and transferable loci will be potentially useful for future studies that will focus on identifying distinct genetic units within species and establishing the phylogeographic patterns and the process of speciation among closely related species.     

In vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles as permeating vectors for the blood-brain barrier

A series of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting has been employed for the formulation of aqueous nanoparticulate systems, which were in turn investigated for their effects on a modeled blood-brain-barrier system of mouse-brain endothelial cells. Barrier function measurements employing electric cell-substrate impedance sensing and analyses of tight junction-specific protein profiles have indicated that the alkylglyceryl-modified chitosan nanoparticles impact upon the integrity of the model blood-brain barrier, whereas confocal microscopy experiments have demonstrated the efficient cellular uptake and the perinuclear localization of these nanoparticles. The application of nanoparticles to the model blood-brain barrier effected an increase in its permeability, as demonstrated by following the transport of the tracer molecule fluorescein isothiocyanate.