Characterizing relationships between cell structures and functions requires mesoscale mapping of intact cells showing subcellular rearrangements following stimulation; however, current approaches are limited in this regard. Here, we report a unique application of soft x-ray tomography to generate three-dimensional reconstructions of whole pancreatic β cells at different time points following glucose-stimulated insulin secretion. Reconstructions following stimulation showed distinct insulin vesicle distribution patterns reflective of altered vesicle pool sizes as they travel through the secretory pathway. Our results show that glucose stimulation caused rapid changes in biochemical composition and/or density of insulin packing, increased mitochondrial volume, and closer proximity of insulin vesicles to mitochondria. Costimulation with exendin-4 (a glucagon-like peptide-1 receptor agonist) prolonged these effects and increased insulin packaging efficiency and vesicle maturation. This study provides unique perspectives on the coordinated structural reorganization and interactions of organelles that dictate cell responses.

No abstract

Many studies have shown that reactive oxygen species including oxygen free radicals are causative factors in the etiology of degenerative diseases, including some hepatopathies. 1)Carbon tetrachloride (CCl 4 ) is most frequently used as a chemical inducer of experimental liver cirrhosis.2) It has been suggested that hepatic necrosis caused by carbon tetrachloride involves bioactivation by the microsomal cytochrome P450-dependent monooxygenase system, resulting in the formation of trichloromethyl free radical and reactive oxygen species that initiate lipid peroxidation and protein oxidation.3) Free radicals in both in vitro and in vivo models have also been shown to modify and damage proteins, carbohydrates, and DNA. 4) Therefore, under such disseminated oxidative stress, biomembrane and bioactive molecules are disturbed or inactivated. Furthermore, hepatic microsomes, mitochondria, and the nuclei of hepatocytes are also impaired by lipid peroxide, with hepatocytes ultimately being destroyed. 5)According to in vitro and in vivo studies, several classical antioxidants have been shown to protect hepatocytes against lipid peroxidation or inflammation, therefore preventing the occurrence of hepatic necrosis.6,7) a-Tocopherol is well known for its antioxidant properties in biomembranes where it acts to prevent lipid peroxidation.8) It also protects against carbon tetrachloride-induced hepatotoxicity.9,10) The a-tocopherol analogue, PMC (2,2,5,7,8-pentamethyl-6-hydroxychromane), in which the phytyl chain is replaced by a methyl group, is more hydrophilic than other a-tocopherol derivatives, and has potent radical scavenging activity and potent inhibition of nuclear factor-kB activity.11,12) The antiperoxidative potency of PMC was approximately 18-times than that of a-tocopherol.12) Recently, we reported that PMC is a potentially effective antioxidant and antiplatelet agent through the inhibition of cyclooxygenase.13) Therefore, we suggested that PMC may be effective against diseases in which reactive oxygen species play a role as potent causative factors.In the present study, we examined and compared the relative inhibitory activities of PMC with silymarin in chronic carbon tetrachloride-induced liver injury in mice. We also evaluated the role of oxidative stress in this model of liver injury. MATERIALS AND METHODSMaterials PMC was obtained from Wako Pure Chemical Ind. (Osaka, Japan). Carbon tetrachloride, silymarin, hydrogen peroxide, sodium azide, 5,5Ј-dithiobis (2-nitro benzoic acid) (DTNB), 2-thiobarbituric acid and other reagents in the study were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). Diagnostic kits for assaying alanine aminotransferase (GPT) and aspartate aminotransferase (GOT) were also purchased from Sigma.Animals Male ICR mice, 5 weeks old, weighing 20-25 g used in this study were obtained from the Laboratory Animal Center of National Taiwan University. All the animal experiments and care were performed according to the Guide for the Care and Use of Laboratory Animals (National Academy Pre...

Objective Germinal center kinase–like kinase (GLK; also called MAPKKKK‐3) activates protein kinase Cθ (PKCθ) during T cell activation and controls autoimmunity in lupus patients. Intracellular kinases are involved in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the role of GLK in RA. Methods The severity of collagen‐induced arthritis (CIA) was studied in GLK‐deficient mice. Expression levels of GLK from RA patients were determined by Western blotting, flow cytometry, real‐time polymerase chain reaction, and immunohistochemical staining. Localization of GLK in T cells was identified by confocal microscopy. RA disease activity was assessed using the Disease Activity Score in 28 joints. Results GLK‐deficient mice displayed impaired CIA development and decreased inflammatory cytokine levels. Local T cell infiltration and collagen restimulation responses were impaired by GLK deficiency. RA patients showed significantly higher GLK protein and messenger RNA levels in peripheral blood T cells than did healthy controls. GLK‐overexpressing T cells in synovial fluid and synovial tissue samples from RA patients were increased compared with those from osteoarthritis patients. Confocal microscopy and flow cytometry showed that GLK colocalized and coexisted with phosphorylated PKCθ in T cells from RA patients. Frequencies of GLK‐expressing T cells were significantly correlated with RA disease activity. Conclusion GLK overexpression in T cells contributes to the pathogenesis of RA, indicating that GLK is a novel biomarker for autoimmune disease severity and a potential therapeutic target for RA.