ARAP, a Novel Adaptor Protein, Is Required for TCR Signaling and Integrin-Mediated Adhesion

A novel adaptor protein was identified by analyzing phosphotyrosine proteomes from membrane rafts of activated T cells. This protein showed sequence similarity to a well-known T cell adaptor protein, adhesion and degranulation-promoting adaptor protein (ADAP); therefore, the novel protein was designated activation-dependent, raft-recruited ADAP-like phosphoprotein (ARAP). Suppression of ARAP impaired the major signaling pathways downstream of the TCR. ARAP associated with the Src homology 2 domain of Src homology 2-containing leukocyte protein of 76 kDa via the phosphorylation of two YDDV motifs in response to TCR stimulation. ARAP also mediated integrin activation but was not involved in actin polymerization. The results of this study indicate that a novel T cell adaptor protein, ARAP, plays a unique role in T cells as a part of both the proximal activation signaling and inside-out signaling pathways that result in integrin activation and T cell adhesion

Increased p190RhoGEF expression in activated B cells correlates with the induction of the plasma cell differentiation

Previously, we demonstrated that the p190 Rho guanine nucleotide exchange factor (p190RhoGEF) was induced following CD40 stimulation of B cells. In this study, we examined whether p190RhoGEF and a downstream effector molecule RhoA are required for B cell differentiation. Expression of p190RhoGEF positively correlated with the expression of surface markers and transcriptional regulators that are characteristic of mature B cells with plasma cell (PC) phenotypes. Moreover, either the overexpression of p190RhoGEF or the expression of a constitutively active RhoA drove cellular differentiation toward PC phenotypes. B cell maturation was abrogated in cells that overexpressed p190RhoGEF and a dominant-negative form of RhoA simultaneously. CD40-mediated maturation events were also abrogated in cells that overexpressed either dominant-negative p190RhoGEF or RhoA. Together, these data provide evidence that p190RhoGEF signaling through RhoA in CD40-activated B cells drives the induction of the PC differentiation. © 2012 by the The Korean Society for Biochemistry and Molecular Biology

Insulin-sensitizing activities of tanshinones, diterpene compounds of the root of Salvia miltiorrhiza Bunge

In this study, the effects of the extract and four tanshinone compounds from the dried root of Salvia miltiorrhiza Bunge (Labiatae) on the tyrosine phosphorylation of the insulin receptor (IR) β-subunit and the downstream signaling were examined in Chinese-hamster ovary cells expressing human insulin receptors (CHO/IR cells) as well as in 3T3-L1 adipocytes. In addition the translocation of the glucose transporter 4 was investigated in 3T3-L1 adipocytes. Total extract of Danshen (1-10 μg/ml) and the four tanshinones (10 μM) did not show any activity, but the total extract and the tanshinone I, IIA and 15, 16-dihydrotanshinone I except cryptotanshinone enhanced the activity of insulin (1 nM) on the tyrosine phosphorylation of the IR as well as the activation of the downstream kinases Akt, ERK1/2, and GSK3β. In the adipocytes the same IR-downstream signaling and the translocation of glucose transporter 4 were demonstrated by the three tanshinones in the presence of insulin. These insulin-sensitizing activities of tanshinones may be useful for developing a new class of specific IR activators as anti-diabetic agents. © 2008 Elsevier GmbH. All rights reserved

Reactive oxygen species play roles on B cell surface receptor CD40-mediated proximal and distal signaling events: Effects of an antioxidant, N-acetyl-L-cysteine treatment

Reactive oxygen species (ROS) have been indicated as important signal mediators for many cell surface receptors. We previously demonstrated that ROS are generated by cross-linking surface receptor CD40 and consequently induce c-Jun N-terminal kinase activation and interleukin-6 secretion in murine B cells. In this study, we investigated further the involvement of ROS in CD40-mediated signaling events in B cells. CD40-mediated proximal events, which include protein serine phosphorylation, protein translocation between membranes and cytosol, as well as receptor complex formation, were inhibited after the pre-incubation of cells with an antioxidant N-acetyl-L-cysteine (NAC). Additionally, B cell responses after long-term ligation of CD40, such as protein expression, nuclear transcription factor κB (NFκB) activation, and cell proliferation, were also affected when cells were treated with NAC. These data suggest that CD40-induced ROS play critical roles in CD40-mediated B cell regulation

Ligation of CD40 receptor in human B lymphocytes triggers the 5-lipoxygenase pathway to produce reactive oxygen species and activate p38 MAPK

Previously, we reported that CD40-induced production of reactive oxygen species (ROS) by NADPH oxidase requires the TNF receptor-associated factor (TRAF) 3, as well as the activities of phosphatidylinositol 3-kinase (PI3K) and Rac1. Here we investigated the possible mechanisms of the production of ROS after CD40 ligation in B cells. We describe an alternative ROS production pathway that is triggered by CD40 ligation, involves 5-lipoxygenase (5-LO), and results in activation of p38 MAPK. Our studies in Raji human B lymphomas revealed that CD40-induced ROS production by 5-LO also requires the activities of PI3K and Rac1. In contrast to the NADPH oxidase pathway, however, TRAF molecules are not required for the CD40-induced ROS production by 5-LO. The association of CD40 with 5-LO is dependent on CD40 ligation in Raji B cells, and co-immunoprecipitation experiments using epitopetagged proteins transiently expressed in human embryonic kidney 293T cells revealed the role of the regulatory subunit of PI3K, p85, in this association. Collectively, these data suggest a separate pathway for the CD40-induced ROS production in B cells and demonstrate that this pathway requires 5-LO via direct association of p85 with both CD40 and 5-LO

Role of two adaptor molecules SLP-76 and LAT in the PI3K signaling pathway in activated T cells

Previously, we identified p85, a subunit of PI3K, as one of the molecules that interacts with the N-terminal region of Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76).We also demonstrated that tyrosine phosphorylation either at the 113 and/ or 128 position is sufficient for the association of SLP-76 with the Src homology 2 domain near the N terminus of p85. The present study further examines the role of the association of these two molecules on the activation of PI3K signaling cascade. Experiments were done to determine the role of SLP-76, either wild-type, tyrosine mutants, or membrane-targeted forms of various SLP-76 constructs, on the membrane localization and phosphorylation of Akt, which is an event downstream of PI3K activation. Reconstitution studies with these various SLP-76 constructs in a Jurkat variant cell line that lacks SLP-76 or linker for activation of T cells (LAT) show that the activation of PI3K pathway following TCR ligation requires both SLP-76 and LATadaptor proteins. The results suggest that SLP-76 associates with p85 after T cell activation and that LAT recruits this complex to the membrane, leading to Akt activation. Copyright © 2011 by The American Association of Immunologists, Inc

Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator

Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) β-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 μg/ml and as an insulin-sensitizer at doses as low as 1 μg/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR β-subunit, phosphorylation of Akt and glycogen synthase kinase-3β), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes. © 2007 Biochemical Society

Over-expression of p190RhoGEF enhances B-cell activation and germinal center formation in T-cell-dependent humoral immune responses

Previously, we reported induced expression of the p190 Rho guanine nucleotide exchange factor (p190RhoGEF, ARHGEF28) following CD40 stimulation of B cells isolated from mouse spleen. We also reported that p190RhoGEF and a downstream effector molecule RhoA are required for B-cell differentiation, especially for the induction of the plasma cell (PC) differentiation. This study investigates the role of p190RhoGEF in B-cell biology in vivo, using p190RhoGEF transgenic (TG) mice that overexpress a wild-type full gene in B cells. Immunization of these mice with T-cell-dependent antigen showed that populations of germinal center B cells and PCs were significantly increased in TG mice. Furthermore, similar results were shown in recombination activating 1 (Rag1) knockout mice that were reconstituted with B cells isolated from TG mice in combination with T cells isolated from littermate control mice. Analyses of isotype class switching and transcription factors involved in a germinal center reaction and PC differentiation also supported the findings from the cellular responses. These results suggest that p190RhoGEF may play a role in the stage of PC differentiation during T-cell-dependent humoral immune responses

한의학 진단기기의 법적 논쟁 : 규제법과 촉진정책 사이에 한의학의 정체성

학위논문(석사) - 한국과학기술원 : 과학기술정책대학원, 2016.8 ,[iii, 39 p. :]한국은 이원화된 특수한 의료체계를 보유한 나라이며, 한의학과 서양의학을 의료법 내에서 동등하게 인정하고 있다. 그러나 완벽히 분리될 수 없는 의학의 특성으로 인해 한의학과 서양의학의 갈등은 두 의학의 발전과 함께 존재해왔다. 본 연구에서는 최근 대두된 의료기기 사용 분쟁을 통해 정책과 법률이 의료기술의 발전에 중요한 역할을 한다는 것을 강조하고자 하였다. 정책과 법률은 과학기술 발전의 원동력이 되기도 하지만, 변화하는 과학기술의 발전 상황과 의료환경을 을 적절히 반영하지 못하면 오히려 의료기술의 발전을 저해하는 요인으로 작용할 수도 있다. 본 논문에서는 먼저 육성 정책에 따른 한의학의 발전 과정을 짚어보고, 발전에 따른 사회구성원의 한의학에 대한 인식 변화 및 한의학의 과학화를 위한 행동의 변화를 살펴본 후, 변화된 사회적 인식과 행동을 법체계가 적절히 수렴하지 못할 때 발생하는 사회적 갈등을 보여주었다. 특히 과학기술의 산물인 의료기기와 관련하여 한의사의 의료기기 사용 판례 분석을 통해 소송 과정에서 한의학이 과학기술과 더불어 발전해야 한다는 변화된 인식이 보여지지만, 최종 판결에서는 과거에 만들어진 판결 기준에 주로 의존하여 변화하는 사회적 인식을 반영하지 못하고 한의사의 의료기기를 제한하는 경우가 대부분이었다. 이는 한의학 규제법과 촉진 정책 사이에 일관성이 결여되어 나타나는 것으로, 그 결과 사회적인 혼란이 초래되고, 한의학계와 서양의학계의 갈등이 심화되며, 한의학의 과학적 발전이 저해되고 있음을 알 수 있었다.한국과학기술원 :과학기술정책대학원

Role of TNF Receptor-Associated Factor 3 in the CD40 Signaling by Production of Reactive Oxygen Species through Association with p40 phox, a Cytosolic Subunit of Nicotinamide Adenine Dinucleotide Phosphate Oxidase

To extend our previous report, which showed the production of the reactive oxygen species (ROS) after the CD40 ligation in the B cells, we further examined the possible mechanisms for ROS production and the involvement of CD40-induced ROS in p38 activation. Our research shows that the stimulation of WEHI 231 B lymphomas with anti-CD40 induced ROS production and p38 activation. An antioxidant N-acetyl-L-cysteine or an inhibitor for NADPH oxidase blocked both of these, but the inhibitors for 5-lipoxygenase did not. We also show that the treatment of cells with inhibitors for the phosphatidylinositol 3-kinase (PI3-K) interfered with the CD40-induced ROS production and p38 activation. In addition, when overexpressed with a dominant negative form of either Rac1 (N17Rac1) or the TNFR-associated factor (TRAF) 3, the WEHI 231 B cells did not show a full response to the CD40 stimulation to produce ROS. Molecular association studies further revealed that the TRAF3 association with p40 phox, a cytosolic subunit of NADPH oxidase and p85 (a subunit of PI3-K), may possibly be responsible for the production of ROS by CD40 stimulation in WEHI 231 B cells. Collectively, these data suggest that the CD40-induced ROS production by NADPH oxidase in WEHI 231 requires the role of TRAF3, as well as activities of PI3-K and Rac1