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Production of reactive oxygen species (ROS) and consequent glutathione oxidation are associated with various physiological processes and diseases, including cell differentiation, senescence, and inflammation. GFP-based redox sensors provide a straight-forward approach to monitor ROS levels and glutathione oxidation within a living cell at the subcellular resolution. We utilized palmitoylated versions of cytosolic glutathione and hydrogen peroxide sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively) and demonstrated a unique redox environment near biological membranes. In HeLa cells, cytosolic glutathione was practically completely reduced (EGSH/GSSG = â 333Â mV) and hydrogen peroxide level was under the detectable range. In contrast, the cytoplasmic milieu near membranes of intracellular vesicles exhibited significant glutathione oxidation (EGSH/GSSG > â 256Â mV) and relatively high H2O2 production, which was not observed for the plasma membrane. These vesicles colocalized with internalized EGFR, suggesting that H2O2 production and glutathione oxidation are characteristics of cytoplasmic surfaces of the endocytosed vesicles. The results visually illustrate local redox heterogeneity within the cytosol for the first time.

Glutathione is a small thiol-containing peptide that plays a central role in maintaining cellular redox homeostasis. Glutathione serves as a physiologic redox buffer by providing thiol electrons for catabolizing harmful oxidants and reversing oxidative effects on biomolecules. Recent evidence suggests that the balance of reduced and oxidized glutathione (GSH/GSSG) defines the redox states of Cys residues in proteins and fine-tunes their stabilities and functions. To elucidate the redox balance of cellular glutathione at subcellular resolution, a number of redox-sensitive green fluorescent protein (roGFP) variants have been developed. In this study, we constructed and functionally validated organelle- and cytoskeleton-targeted roGFP and elucidated the redox status of the cytosolic glutathione at a subcellular resolution. These new redox sensors firmly established a highly reduced redox equilibrium of cytosolic glutathione, wherein significant deviation was observed among cells. By targeting the sensor to the cytosolic and lumen sides of the Golgi membrane, we identified a prominent redox gradient across the biological membrane at the Golgi body. The results demonstrated that organelle- and cytoskeleton-targeted sensors enable the assessment of glutathione oxidation near the cytosolic surfaces of different organelle membranes.

Two different sizes of siRNAs, of which one type was 21-nucleotide (nt) siRNA containing 2-nt dangling ends and the other type was 27-nt siRNA with blunt ends, were conjugated with a nuclear export signal peptide of HIV-1 Rev at the 5′-sense end. Processing by Dicer enzyme, cell membrane permeability, and RNAi efficiency of the peptide-conjugated siRNAs were examined. Dicer cleaved the peptide-conjugated 27-nt siRNA leading to the release of 21-nt siRNA, whereas the peptide-conjugated 21-nt siRNA was not cleaved. High membrane permeability and cytoplasmic localization was found in the conjugates. Moreover, the peptide-conjugated 27-nt siRNA showed increased potency of RNAi in comparison with the nonmodified 21-nt and 27-nt siRNAs, whereas the peptide-conjugated 21-nt siRNA showed decreased RNAi efficacy. This potent RNAi efficacy is probably owing to acceleration of RISC through recognition by Dicer, as well as to the improvement of cell membrane permeability and intracellular accumulation.

Abstract.: We established a novel allergic dermatitis model in mouse ear lobes in which antigen-nonspecific inflammation was induced by painting 12-O-tetradecanoylphorbol 13-acetate (TPA) between sensitization and challenge with picryl chloride (PiCl). This model has an advantage for analyzing atopic dermatitis-like inflammation within a short period. Analysis of the time course changes in the PiCl-induced swelling showed that the allergic inflammation was shifted from a delayed-type response to a biphasic response consisting of a weak immediate-phase response and a late-phase response by painting with TPA. The application of TPA increased the PiCl-induced infiltration of eosinophils and mast cells at the inflammatory site and shifted the cytokine milieu from Th1 to Th2. The expression of the Th2-inducing cytokine thymic stromal lymphopoietin (TSLP) mRNA was also increased by TPA. These findings suggested that the induction of antigen-nonspecific inflammation by TPA before the antigen challenge enhanced the Th2 response and modified the PiCl-induced delayed type-hypersensitivity. Using this model, we clarified that histamine plays significant roles in the early-phase swelling via H1 receptors and the late-phase swelling via H3/H4 receptors. Thus, we suggested the usefulness of the combined treatment with an H1 antagonist and an H4 antagonist for the suppression of atopic dermatitis. Keywords:: allergic dermatitis, histamine, picryl chloride, 12-O-tetradecanoylphorbol 13-acetate, thymic stromal lymphopoietin, allergic inflammation

Novel anti-HIV lectin family which shows a strict binding specificity for high mannose glycans has been found in lower organisms. The bacterial orthologue has been identified in the genome of Pseudomonas fluorescens Pf0-1 and the gene coding a putative lectin was cloned, expressed in Escherichia coli and purified by one step gel filtration. Glycan array screening of the recombinant lectin, termed PFL, has revealed that PFL preferentially recognizes high mannose glycans with α1-3 Man that was highly exposed at the D2 position. In contrast, masking of this α1-3 Man with α1-2 Man dramatically impaired lectin-carbohydrate interactions. Reducing terminal disaccharide, GlcNAc-GlcNAc of high mannose glycans was also essential for PFL-binding. PFL showed a potent anti-influenza virus activity by inhibiting the virus entry into cells at doses of low nanomolar concentration. At micromolar concentration or higher, PFL showed a cytotoxicity accompanying loss of the cell adhesion against human gastric cancer MKN28 cells. The cell surface molecule to which PFL bound was co-precipitated with biotin-labeled PFL and identified as integrin α2 by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Intriguingly, upon treatment with exogenous PFL, integrin α2 on the cell surface underwent rapid internalization to the cytoplasm and accumulated to perinuclear region, together with the bound PFL. The resulting loss of cell adherence would trigger a signaling pathway that induced anoikis-like cell death. These events were effectively inhibited by pretreatment of PFL with mannnan, indicating the involvement of high mannose glycans on PFL-induced cell death that was triggered by PFL-integrin α2 interactions.

Background: Pseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated.Methods: Cell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated.Results: The strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth.Conclusion: PFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs. [ABSTRACT FROM AUTHOR]