Plasmacytoid dendritic cells (pDCs) play a key role in antiviral immunity, but also contribute to the pathogenesis of certain autoimmune diseases, by producing large amounts of type I IFNs. Although activation of pDCs is triggered by engagement of nucleotide-sensing toll-like receptors (TLR) 7 and 9, type I IFN induction additionally requires IκB kinase (IKK) α–dependent activation of IFN regulatory factor (IRF) 7. However, the signaling pathway mediating IKK-α activation is poorly defined. We show that DOCK2, an atypical Rac activator, is essential for TLR7- and TLR9-mediated IFN-α induction in pDCs. We found that the exposure of pDCs to nucleic acid ligands induces Rac activation through a TLR-independent and DOCK2-dependent mechanism. Although this Rac activation was dispensable for induction of inflammatory cytokines, phosphorylation of IKK-α and nuclear translocation of IRF-7 were impaired in Dock2-deficient pDCs, resulting in selective loss of IFN-α induction. Similar results were obtained when a dominant-negative Rac mutant was expressed in wild-type pDCs. Thus, the DOCK2–Rac signaling pathway acts in parallel with TLR engagement to control IKK-α activation for type I IFN induction. Owing to its hematopoietic cell-specific expression, DOCK2 may serve as a therapeutic target for type I IFN–related autoimmune diseases.
The lytic polysaccharide monooxygenases (LPMOs) have received considerable attention subsequent to their discovery because of their ability to boost the enzymatic conversion of recalcitrant polysaccharides. In the present study, we describe the enzymatic properties of SgLPMO10F, a small (15 kDa) auxilliary activity (AA) family 10 LPMO from Streptomyces griseus belonging to a clade of the phylogenetic tree without any characterized representative. The protein was expressed using a Brevibacillus-based expression system that had not been used previously for LPMO expression and that also ensures correct processing of the N-terminus crucial for LPMO activity. The enzyme was active towards both a-and b-chitin and showed stronger binding and a greater release of soluble oxidized products for the latter allomorph. In chitinase synergy assays, however, SgLPMO10F worked slightly better for a-chitin, increasing chitin solubilization yields by up to 30-fold and 20-fold for a-and b-chitin, respectively. Synergy experiments with various chitinases showed that the addition of SgLPMO10F leads to a substantial increase in the (GlcNAc) 2 :GlcNAc product ratio, in reactions with a-chitin only. This underpins the structural differences between the substrates and also shows that, on a-chitin, SgLPMO10F affects the binding mode and/or degree of processivity of the chitinases tested. Variation in the only exposed aromatic residue in the substrate-binding surface of LPMO10s has previously been linked to preferential binding for a-chitin (exposed Trp) or b-chitin (exposed Tyr). Mutation of this residue, Tyr56, in SgLPMO10F to Trp had no detectable effect on substrate-binding preferences but, in synergy experiments, the mutant appeared to be more efficient on a-chitin.
We have isolated a cDNA clone for a novel glutathionedependent dehydroascorbate reductase from a rat liver cDNA library in gt11 by immunoscreening. The authenticity of the clone was confirmed as follows: first, the antibody that had been purified through affinity for the protein expressed by the cloned gt11 phage recognized only the enzyme in a crude extract from rat liver; and second, two internal amino acid sequences of purified enzyme were identified in the protein sequence predicted from the cDNA. The predicted protein consists of 213 amino acids with a molecular weight of 24,929, which is smaller by ϳ3,000 than the value obtained by matrix-assisted laser desorption/ionization time-offlight mass spectrometry. This discrepancy of the molecular weight was explained by post-translational modification because the recombinant protein expressed by a mammalian system (Chinese hamster ovary cells) was of the same size as rat liver enzyme but larger than the protein expressed by a bacterial system (Escherichia coli). Chinese hamster ovary cells, originally devoid of glutathione-dependent dehydroascorbate reductase activity, was made to elicit the enzyme activity (1.5 nmol/ min/mg of cytosolic protein) by expression of the recombinant protein. Additionally, the cells expressing the enzyme were found to accumulate 1.7 times as much ascorbate as the parental cells after incubation with dehydroascorbate. This result points to the importance of the dehydroascorbic acid reductase in maintaining a high concentration of ascorbate in the cell.L-Ascorbic acid (AA) 1 acts as an important cofactor in various enzymatic reactions and also as an effective antioxidant in scavenging reactive oxygen species in vivo. These physiological functions of AA are associated with its univalent or divalent oxidation. The univalent oxidation of AA leads to the formation of monodehydroascorbate that is converted to the divalent oxidation product dehydroascorbic acid (DHA) through spontaneous disproportionation or further oxidation. Because DHA is unstable at physiological pH and temperature (1), regeneration of AA from DHA could be a beneficial process even for many organisms that can synthesize AA themselves. Especially for humans and primates that cannot synthesize it, dietary intake of AA is the only way to supply this vitamin; therefore, a system for regeneration of AA from its oxidized forms would be important for the cell to maintain a normal cellular level of AA.Many reactions potentially contributing to the regeneration of AA in animal cells have been reported. Monodehydroascorbate is reduced to AA by an NADH-dependent enzymatic reaction occurring on subcellular membranes of mitochondria (2) and microsomes (3, 4). As for the conversion of DHA to AA, nonenzymatic reduction by GSH has been suggested for a long time (5). However, because of the slowness of the reaction, much attention has been directed to enzyme-catalyzed reduction of DHA in recent years. Wells et al. (6) reported that porcine liver thioltransferase (glutaredoxin) and bovin...
We have isolated from bovine brain a protein with a high capacity to inhibit the copper ion-catalyzed oxidation of L-ascorbate and identified it as S100b protein, an EF-hand calcium-binding protein, by sequencing its proteolytic peptides. Copper binding studies showed that this protein has four copper-binding sites per dimeric protein molecule with a dissociation constant of 0.46 M and that in the presence of L-ascorbate, copper ions bind to a total of six binding sites with a great increase in affinity. Furthermore, we examined whether S100b protein can prevent copper-induced cell damage. Bovine S100b protein was found to suppress dose-dependently the hemolysis of mouse erythrocytes induced by CuCl 2 . We transformed Escherichia coli cells with pGEX-5X-3 vector containing a cDNA for rat S100b protein, so that this protein could be expressed as a fusion protein with glutathione S-transferase. The transformed cells were demonstrated to be markedly resistant to a treatment with CuCl 2 plus H 2 O 2 as compared with the control cells expressing glutathione S-transferase alone. These results indicate that S100b protein does suppress oxidative cell damage by sequestering copper ions.
The pyramidal indentation-induced surface deformation of brittle ceramics is examined on the basis of extensive test results for indentation load (P)-depth (h) curves during loading/unloading cycle. A mechanically stiff test system is essential for obtaining P-h curves acceptable and reliable for subsequent analyses. Both the loading and unloading P-h curves are expressed by quadratic functions within experimental variations for all the indenters used (Vickers, Berkovich, and Knoop). The loading curve is then related to the Meyer hardness and the unloading curve to Young's modulus by the use of semiempirical equations which enable one to estimate these moduli from the observed loading/unloading parameters. An elastoplastic constitutive equation for indentation surface deformation is theoretically derived. This equation not only predicts well the experimental observations but also gains an important physical insight into the Meyer hardness. The Meyer hardness of brittle materials is not a measure for plasticity, but an elastic/plastic parameter which significantly depends on the geometry of indenter. The concept and experimental determination of “true” hardness as a characteristic material measure for plasticity are proposed.
The Single Particle Irradiation system to Cell (SPICE) facility at the National Institute of Radiological Sciences (NIRS) is a focused vertical microbeam system designed to irradiate the nuclei of adhesive mammalian cells with a defined number of 3.4 MeV protons. The approximately 2-μm diameter proton beam is focused with a magnetic quadrupole triplet lens and traverses the cells contained in dishes from bottom to top. All procedures for irradiation, such as cell image capturing, cell recognition and position calculation, are automated. The most distinctive characteristic of the system is its stability and high throughput; i.e. 3000 cells in a 5 mm × 5 mm area in a single dish can be routinely irradiated by the 2-μm beam within 15 min (the maximum irradiation speed is 400 cells/min). The number of protons can be set as low as one, at a precision measured by CR-39 detectors to be 99.0%. A variety of targeting modes such as fractional population targeting mode, multi-position targeting mode for nucleus irradiation and cytoplasm targeting mode are available. As an example of multi-position targeting irradiation of mammalian cells, five fluorescent spots in a cell nucleus were demonstrated using the γ-H2AX immune-staining technique. The SPICE performance modes described in this paper are in routine use. SPICE is a joint-use research facility of NIRS and its beam times are distributed for collaborative research.
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