Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-γ2 in B Cells

Genetic studies have revealed that Cbl-b plays a negative role in the antigen receptor–mediated proliferation of lymphocytes. However, we show that Cbl-b–deficient DT40 B cells display reduced phospholipase C (PLC)-γ2 activation and Ca2+ mobilization upon B cell receptor (BCR) stimulation. In addition, the overexpression of Cbl-b in WEHI-231 mouse B cells resulted in the augmentation of BCR-induced Ca2+ mobilization. Cbl-b interacted with PLC-γ2 and helped the association of PLC-γ2 with Bruton's tyrosine kinase (Btk), as well as B cell linker protein (BLNK). Cbl-b was indispensable for Btk-dependent sustained increase in intracellular Ca2+. Both NH2-terminal tyrosine kinase-binding domain and COOH-terminal half region of Cbl-b were essential for its association with PLC-γ2 and the regulation of Ca2+ mobilization. These results demonstrate that Cbl-b positively regulates BCR-mediated Ca2+ signaling, most likely by influencing the Btk/BLNK/PLC-γ2 complex formation

Protein kinase D regulates positive selection of CD4+ thymocytes through phosphorylation of SHP-1

Thymic selection shapes an appropriate T cell antigen receptor (TCR) repertoire during T cell development. Here, we show that a serine/threonine kinase, protein kinase D (PKD), is crucial for thymocyte positive selection. In T cell-specific PKD-deficient (PKD2/PKD3 double-deficient) mice, the generation of CD4 single positive thymocytes is abrogated. This defect is likely caused by attenuated TCR signalling during positive selection and incomplete CD4 lineage specification in PKD-deficient thymocytes; however, TCR-proximal tyrosine phosphorylation is not affected. PKD is activated in CD4+CD8+ double positive (DP) thymocytes on stimulation with positively selecting peptides. By phosphoproteomic analysis, we identify SH2-containing protein tyrosine phosphatase-1 (SHP-1) as a direct substrate of PKD. Substitution of wild-type SHP-1 by phosphorylation-defective mutant (SHP-1S557A) impairs generation of CD4+ thymocytes. These results suggest that the PKD–SHP-1 axis positively regulates TCR signalling to promote CD4+ T cell development

Dok-1 and Dok-2 are negative regulators of lipopolysaccharide-induced signaling

Endotoxin, a bacterial lipopolysaccharide (LPS), causes fatal septic shock via Toll-like receptor (TLR)4 on effector cells of innate immunity like macrophages, where it activates nuclear factor κB (NF-κB) and mitogen-activated protein (MAP) kinases to induce proinflammatory cytokines such as tumor necrosis factor (TNF)-α. Dok-1 and Dok-2 are adaptor proteins that negatively regulate Ras–Erk signaling downstream of protein tyrosine kinases (PTKs). Here, we demonstrate that LPS rapidly induced the tyrosine phosphorylation and adaptor function of these proteins. The stimulation with LPS of macrophages from mice lacking Dok-1 or Dok-2 induced elevated Erk activation, but not the other MAP kinases or NF-κB, resulting in hyperproduction of TNF-α and nitric oxide. Furthermore, the mutant mice showed hyperproduction of TNF-α and hypersensitivity to LPS. However, macrophages from these mutant mice reacted normally to other pathogenic molecules, CpG oligodeoxynucleotides, poly(I:C) ribonucleotides, or Pam3CSK4 lipopeptide, which activated cognate TLRs but induced no tyrosine phosphorylation of Dok-1 or Dok-2. Forced expression of either adaptor, but not a mutant having a Tyr/Phe substitution, in macrophages inhibited LPS-induced Erk activation and TNF-α production. Thus, Dok-1 and Dok-2 are essential negative regulators downstream of TLR4, implying a novel PTK-dependent pathway in innate immunity

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis

The changes in treatment strategies in ABOi living donor liver transplantation for acute liver failure

Introduction. Living donor liver transplantation (LDLT) using ABO-incompatible (ABOi) graft for acute liver failure (ALF) is a developing treatment modality. Methods. We reviewed the changes in our treatment strategies in applying ABOi LDLT for FH over our fourteen years of experience. Results. Five patients with ALF received LDLT in adults using ABOi grafts, with different but gradually renewed protocols. The etiologies for acute liver failure included autoimmune hepatitis (n=3) and unknown (n=2). The desensitization protocol for ABOi barrier included Case #1 ; local infusion (portal vein)+plasma exchange (PE), Case #2 ; local infusion (hepatic artery)+rituximab+PE, Case #3 and #4 ; rituximab+PE, and Case #5 ; rituximab+PE under high-flow continuous hemodiafiltration. Local infusion was abandoned since Case #3, because Case #1 had portal vein thrombosis resulting in graft necrosis and Case #2 had hepatic artery dissection. The patients (Case #2 and #3), who received rituximab within 7 days before LDLT, experienced antibody-mediated rejection. Thus, the most recent protocol for ABOi-LDLT is that rituximab is given 2 weeks before LDLT, followed by high-flow continuous hemodiafiltration to obstacle hepatic encephalopathy until LDLT. The four patients except Case #1 are doing well with good graft function over 3.8±3.7 years. Conclusion. Rituximab-based ABOi-LDLT, most-recently under high-flow hemodiafiltration for treating encephalopathy, is a feasible option for applying LDLT for ALF

Role of Dok-1 and Dok-2 in Myeloid Homeostasis and Suppression of Leukemia

Dok-1 and Dok-2 are closely related rasGAP-associated docking proteins expressed preferentially in hematopoietic cells. Although they are phosphorylated upon activation of many protein tyrosine kinases (PTKs), including those coupled with cytokine receptors and oncogenic PTKs like Bcr-Abl, their physiological roles are largely unidentified. Here, we generated mice lacking Dok-1 and/or Dok-2, which included the double-deficient mice succumbed to myeloproliferative disease resembling human chronic myelogenous leukemia (CML) and chronic myelomonocytic leukemia. The double-deficient mice displayed medullary and extramedullary hyperplasia of granulocyte/macrophage progenitors with leukemic potential, and their myeloid cells showed hyperproliferation and hypo-apoptosis upon treatment and deprivation of cytokines, respectively. Consistently, the mutant myeloid cells showed enhanced Erk and Akt activation upon cytokine stimulation. Moreover, loss of Dok-1 and/or Dok-2 induced blastic transformation of chronic phase CML-like disease in mice carrying the bcr-abl gene, a cause of CML. These findings demonstrate that Dok-1 and Dok-2 are key negative regulators of cytokine responses and are essential for myeloid homeostasis and suppression of leukemia

RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-kappa B pathway

The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNF alpha mRNA decay via a 3'UTR constitutive decay element (CDE). Here we applied PAR-CLIP to human RC3H1 to identify similar to 3, 800 mRNA targets with 416, 000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage-induced mRNAs, indicating a role of this RNA-binding protein in the post-transcriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of the NF-kappa B pathway regulators such as I kappa B alpha and A20. RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 30UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with I kappa B kinase and NF-kappa B activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-kappa B pathway

Angular distribution control of extreme ultraviolet radiation from laser-produced plasma by manipulating the nanostructure of low-density SnO 2 targets

金沢大学先端科学・社会共創推進機構We have found that the divergence of a relatively monochromatic extreme ultraviolet (EUV) emission from a laser-produced plasma can be manipulated by changing the target morphology which is a porous low-density tin oxide (Sn O2) structure. The fundamental light of a Nd-YAG laser was irradiated on the target with laser intensity of ∼ 1011 W cm2 and pulse duration of 10 ns. The nanostructure and density of the targets were tuned by a combination of colloidal polymer template and sol-gel processes [Gu, Nagai, Norimatsu, Fujioka, Nishimura, Nishihara, Miyanaga, and Izawa, Chem. Mater. 17, 1115 (2005)], which has a merit in large-scale preparation. When the target has an open cell nanostructure, the EUV emission directed predominantly along target normal, while a closed cell target exhibited divergent emission. The angular distribution may be affected by the orientation of the microstructured initial target, and this phenomenon can be applied to wavefront control of EUV emission. © 2006 American Institute of Physics.Embargo Period 12 month