IFN-γ/IL-17 double knockout mice did not develop arthritis.

<p>(a) Comparison of arthritis score during 10 weeks after 1^st immunization. (b) Serum was collected at 5^th week after CIA induction, the level of anti CII IgG, IgG1, IgG2 was determined by ELISA. (c) Joint histology of CIA (IFN-γ KO, n = 14) and CIA (IFN-γIL-17 DKO, n = 14) at 10^th week after 1^st immunization. Joint tissue sections were stained with Hematoxylin and Eosin, Toluidine Blue, Safranin O. (d) Immunohistochemistry. RANKL or RANK positive cells were not seen in CIA (IFN-γ/IL-17 DKO) joint section. Original magnification,200x. Mann-Whitney U test (b) used. Values are presented as the mean ± standard deviation. *, p<0.05, **, p<0.005 ***, p<0.001.</p

The proportion of naïve T cells increased in IFN-γ/IL-17 double knockout mice compared to that of IFN-γ KO mice.

<p>Spleen, draining lymph nodes and mesenteric lymph node were removed at 5^th week after 1^st immunization, and prepared for single cell suspension. They were stained with PerCP - anti CD4 ab, APC - anti CD44 ab, and FITC - anti CD62L ab for flowcytometry. (a) Data represents one of three experiments. (b) CD4⁺CD44^highCD62L^low (memoryCD4⁺T cells) T cell population was decreased in CIA (IFN-γIL-17 DKO) group while CD4⁺CD44^lowCD62L^high (naïve CD4⁺ T cells) population was increased. (c) IL-2, IL-15 and IL-21 were strongly expressed in the joint synovium of CIA (IFN-γ KO). Original magnification, 200x. Mann-Whitney U test used (b). Values are presented as the mean ± standard deviation of four independent experiments. *, p<0.05, **, p<0.005 ***, p<0.001.</p

Suppression of IL-17 by IFN- γ is associated with IDO.

<p>(a) CD4⁺ T cells isolated from wild type (WT) C57BL/6 (B6) mice and IFN-γ knock out (KO) B6 mice. The cells were cultured in Th17-polarizing conditions (anti CD3 mAb, anti CD28 mAb, anti IFN-γAb, anti IL-4 Ab, TGF-β, IL-6). Non-CD4 cells were cultured with media only or 100 ng/ml of recombinant IFN-γ. After 72hours, cells were washed and only APCs were irradiated at 3000 rad. After Th17 cells and APC/APC_IFN-γ were co-cultured (1∶1) for 16 hrs, the culture supernatant was measured for IL-17 ELISA (right). (b) Splenocytes derived from IFN-γ KO mice were cultured solely or with combined condition with 10 ng/ml of IL-23, 50 ng/ml of IFN-γ, 100 µM 1-MT for 72 hrs. IFN-γ and 1-MT was pretreated. (c) IL-17 mRNA expression of Th17 polarized cells derived from B6 and IDO KO mice splenocyte. (d) Splenocytes derived from IL-1Ra KO mice were cultured solely or with combined condition with 10 ng/ml of IL-23, 50 ng/ml of IFN-γ, 100 µM 1-MT for 72 hrs. IFN-γ and 1-MT was pretreated. ANOVA with post hoc analysis (d,f) was used. Values are presented as the mean ± standard deviation of three independent experiments. *, p<0.05, **, p<0.005 ***, p<0.001.</p

Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i>²‑Guanyl DNA Adducts

DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i>²-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i>²-CH₂(9-anthracenyl)­G (<i>N</i>²-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i>_cat/<i>K</i>_m for dCTP insertion opposite G and <i>N</i>²-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i>_cat/<i>K</i>_m for next-base extension from C paired with <i>N</i>²-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i>²-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens

Flounder tissues fixed in neutral buffered formalin were probed with a SH2 domain-specific cRNA probe

The left panels in each tissue hybridized with the probe show blue color, which indicates the presence of the mRNA. Right panels in each tissue showing no blue region are the same tissue probed with a sense probe as a negative control.<p><b>Copyright information:</b></p><p>Taken from "Molecular cloning and expression analysis of the STAT1 gene from olive flounder, "</p><p>http://www.biomedcentral.com/1471-2172/9/31</p><p>BMC Immunology 2008;9():31-31.</p><p>Published online 26 Jun 2008</p><p>PMCID:PMC2443792.</p><p></p

EG, egg; LV, larva; 7D, 7 day post-hatch; 14D, 14 day post-hatch; 27D, 27 day post-hatch; 34D, 34 day post-hatch

(B) Expression of STAT1 mRNA in various tissues of the flounder. MS, muscle; LV, liver; IT, intestine; ST, stomach; KD, head kidney; SK, skin; FN, fin; SP, spleen; GI, gill; EY, eye; HR, heart. Data are averages from three replications with standard deviations.<p><b>Copyright information:</b></p><p>Taken from "Molecular cloning and expression analysis of the STAT1 gene from olive flounder, "</p><p>http://www.biomedcentral.com/1471-2172/9/31</p><p>BMC Immunology 2008;9():31-31.</p><p>Published online 26 Jun 2008</p><p>PMCID:PMC2443792.</p><p></p

H₂O₂ induces SHP-2-caveolin-1 complex formation.

<p>(A) Complex formation between caveolin-1 and SHP-2 in CRT-MG cells as investigated by <i>in situ</i> proximity ligation assay (PLA). CRT-MG cells were incubated for 10 min with 5 mM H₂O₂, fixed, and then incubated overnight with antibodies against caveolin-1 and SHP-2. After washing, the PLA probe PLUS and PLA Probemaker probe were incubated for 3 h at 37°C and diluted ligases at 1∶40 were incubated for 2 h at 37°C. Circular oligonucleotides were amplified using polymerase for 100 min at 37°C. Nuclei were stained with DAPI. The experiments were repeated 4 times with similar results. Bars = 20 µm. (B) pCMV-SPORT6, Caveolin-1 WT or caveolin-1 Y14A DNA were transiently transfected into HEK 293T cells. Next, 24 h after transfection, cells were incubated with 5 mM H₂O₂ for 10 min. WCLs (700 µg) were subjected for immunoprecipitation using the anti-caveolin-1 and anti-SHP-2 antibodies and analyzed by immunoblotting. Input (5%) is shown. Data are representative of at least 3 experiments.</p

A phylogenetic tree of the aligned sequences was constructed using the neighbor-joining algorithm in MEGA (version 3

0). The confidence for each node was determined by bootstrap analysis (1000 repetitions).<p><b>Copyright information:</b></p><p>Taken from "Molecular cloning and expression analysis of the STAT1 gene from olive flounder, "</p><p>http://www.biomedcentral.com/1471-2172/9/31</p><p>BMC Immunology 2008;9():31-31.</p><p>Published online 26 Jun 2008</p><p>PMCID:PMC2443792.</p><p></p

Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i>²‑Guanyl DNA Adducts

DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i>²-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i>²-CH₂(9-anthracenyl)­G (<i>N</i>²-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i>_cat/<i>K</i>_m for dCTP insertion opposite G and <i>N</i>²-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i>_cat/<i>K</i>_m for next-base extension from C paired with <i>N</i>²-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i>²-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens

H₂O₂-mediated association of CSK and caveolin-1 is dependent on the phosphorylation of caveolin-1 at Tyr 14.

<p>(A) CRT-MG cells were treated with or without H₂O₂ for 10 min, and whole-cell lysates (WCLs) were extracted. A total of 700 µg of WCL was immunoprecipitated with an anti-caveolin-1 antibody. Immunoprecipitates were analyzed by immunoblotting with anti-CSK and anti-caveolin-1 antibodies. (B) Human embryonic kidney cell line HEK 293T cells were transfected with empty vector (Mock), wild-type (WT) and mutant caveolin-1 (Y14A). After 24 h, cells were treated with 5 mM H₂O₂ for 10 min or left untreated and lysed in RIPA buffer. WCLs (700 µg) were precleared and immunoprecipitated with anti-CSK and anti-caveolin-1 antibodies and then analyzed by immunoblotting. Input (5%) is shown (Cav-1, caveolin-1 and pY14-caveolin-1). Data are representative of at least 3 experiments.</p