Th2 Suppressor Cells Are More Susceptible to Sphingosine Than Th1 Cells in Murine Contact Photosensitivity

Murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) is a cutaneous delayed-type hypersensitivity reaction in which both positive and negative regulatory pathways exist. The latter pathway is mediated by antigen-specific, CD4+ suppressor T cells (CPS-Ts) that are Th2 cells. We examined the effects of sphingosine and synthetic cell-permeable analogs of ceramide on the cellular kinetics of CPS-Ts and immune lymph node cells from TCSA-photosensitized mice (CPS-LNC), along with other murine T-cell populations. The addition of sphingosine at 10 or 3 μM to in vitro cultures suppressed DNA synthesis of CPS-Ts and Th2 clones, including D10 cells and 24-2 cells, but not that of CPS-LNC or Th1 clones, including 23-1-8 and 28-4 cells. This suggested that sphingosine exerts its inhibitory effects preferentially on the proliferation of Th2 cells. Although suppressing DNA synthesis, sphingosine augmented the production and mRNA expression of interleukin-4 (IL-4) and enhanced the expression of the IL-4 receptor in CPS-Ts. In addition, the ability of sphingosine to induce signal transduction of CPS-Ts was confirmed by elevation of the intracellular free Ca++ concentration. Because CPS-Ts exposed to sphingosine exhibited a lower G2M/G1 ratio than control, these seemingly ambivalent phenomena may be caused by retardation of the G1 to S phase progression, a cell-cycle dysregulation known to augment cytokine production. In contrast to sphingosine, cell-permeable ceramide did not affect the proliferation of these cells when stimulated with mitogen/antigen and did not augment IL-4 production by CPS-Ts. Our study suggests that sphingosine modifies the Th1/Th2 balance by preferentially affecting the cellular kinetics of Th2

Treatment of T Lymphocytes with 8-Methoxypsoralen Plus Ultraviolet A Induces Transient but Biologically Active Th1-Skewing Cytokine Production

8-Methoxypsoralen plus ultraviolet A light is suggested to shift T lymphocytes from Th2 to Th1 cells. To clarify this issue, we examined the effects of 8-methoxypsoralen/ultraviolet A on the expression/production of cytokines in peripheral blood mononuclear cells from normal subjects and a Sézary syndrome patient. 8-Methoxypsoralen/ultraviolet A augmented the expression of mRNAs for interferon-γ and interleukin-2 and reduced those for interleukin-4 and interleukin-10. It seems that this enhancement of Th1 cytokines is caused by increment of cytokine production by Th1 cells but not by conversion of Th2 cells to produce Th1 cytokines. The number of interferon-γ-secreting lymphocytes was markedly increased in 8-methoxypsoralen/ultraviolet A-treated peripheral blood mononuclear cells 20 h after treatment, whereas that of Th2 cytokine-producing cells was decreased. Accordingly, the amount of interferon-γ was elevated in culture supernatants from 8-methoxypsoralen-phototreated peripheral blood mononuclear cells, whereas interleukin-4 was significantly reduced. This enhanced production of interferon-γ, however, was found only until 3 d after 8-methoxypsoralen phototreatment and was declined by 5 d after treatment. Finally, 8-methoxypsoralen/ultraviolet A treatment of T cells regulated their ability to induce keratinocyte CD54 expression. Our results show that 8-methoxypsoralen/ultraviolet A has a transient but biologically active Th1-skewing action in human T cells, suggesting that 8-methoxypsoralen/ultraviolet A exerts a beneficial therapeutic effect on Th2-mediated or Th2-malignant diseases

Rad9 modulates the P21WAF1 pathway by direct association with p53

<p>Abstract</p> <p>Background</p> <p>Previous studies suggest that human <it>RAD9 </it>(hRad9), encoding a DNA damage checkpoint molecule, which is frequently amplified in epithelial tumor cells of breast, lung, head and neck cancer, participates in regulation of the tumor suppressor p53-dependent transactivation of pro-survival <it>P21</it>^{<it>WAF1</it>}. This study examined the exact mechanism of the hRad9 function, especially through the phosphorylation of the C-terminus, in the transcription regulation of <it>P21</it>^{<it>WAF1</it>}.</p> <p>Results</p> <p>The transfection of phosphorylation-defective <it>hRAD9 </it>mutants of C-terminus resulted in reduction of the p53-dependent <it>P21</it>^{<it>WAF1 </it>}transactivation; the knockdown of total hRad9 elicited an increased <it>P21</it>^{<it>WAF1 </it>}mRNA expression. Immunoprecipitation and a ChIP assay showed that hRad9 and p53 formed a complex and both were associated with two p53-consensus DNA-binding sequences in the 5' region of <it>P21</it>^{<it>WAF1 </it>}gene. The association was reduced in the experiment of phosphorylation-defective <it>hRAD9 </it>mutants.</p> <p>Conclusion</p> <p>The present study indicates the direct involvement of hRad9 in the p53-dependent <it>P21</it>^{<it>WAF1 </it>}transcriptional mechanism, presumably via the phosphorylation sites, and alterations of the hRad9 pathway might therefore contribute to the perturbation of checkpoint activation in cancer cells.</p

Structural basis of Sec-independent membrane protein insertion by YidC

[プレスリリース]バイオサイエンス研究科膜分子複合機能学研究室の塚崎智也准教授らの研究グループが、タンパク質を細胞膜に組み込むメカニズムを解明しました（2014/04/17）Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively1, 2. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG3, 4, 5. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane