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1.
The CD19/CD21 complex functions to prolong B cell antigen receptor signaling from lipid rafts 总被引:9,自引:0,他引:9
The CD19/CD21 complex functions to significantly enhance B cell antigen receptor (BCR) signaling in response to complement-tagged antigens. Recent studies showed that following antigen binding the BCR translocates into plasma membrane lipid rafts that serve as platforms for BCR signaling. Here, we show that the binding of complement-tagged antigens stimulates the translocation of both the BCR and the CD19/CD21 complex into lipid rafts, resulting in prolonged residency in and signaling from the rafts, as compared to BCR cross-linking alone. When coligated to the BCR, the CD19/CD21 complex retards the internalization and degradation of the BCR. The colocalization and stabilization of the BCR and the CD19/CD21 complex in plasma membrane lipid rafts represents a novel mechanism by which a coreceptor enhances BCR signaling. 相似文献
2.
A major function of the B cell is the internalization of antigen through the BCR for processing and presentation to T cells. While there is evidence suggesting that lipid raft signaling may regulate internalization, the molecular machinery coordinating these two processes remains to be defined. Here we present a link between the B cell signaling and internalization machinery and show that Src-family kinase activity is required for inducible clathrin heavy chain phosphorylation, BCR colocalization with clathrin, and regulated internalization. An analysis of different B cell lines shows that BCR uptake occurs only when clathrin is associated with rafts and is tyrosine phosphorylated following BCR crosslinking. We therefore propose that lipid rafts spatially organize signaling cascades with clathrin to regulate BCR internalization. 相似文献
3.
Valensin S Paccani SR Ulivieri C Mercati D Pacini S Patrussi L Hirst T Lupetti P Baldari CT 《European journal of immunology》2002,32(2):435-446
Following ligand binding the TCR segregates to plasma membrane microdomains, termed lipid rafts, characterized by a highly ordered lipid structure favoring partitioning of glycosyl phosphatidyl inositol-linked costimulatory receptors and acylated signaling molecules. Here we show that the inducible association of the TCR and key signaling proteins with lipid rafts is dependent on the actin cytoskeleton through a mechanism involving raft coalescence. Although lipid rafts are required for full activation of the TCR-dependent tyrosine phosphorylation cascade and sustained signaling, triggering of TCR-proximal events, including Fyn activation and a first wave of Vav phosphorylation, is independent of lipid rafts, while a second wave of raft-dependent Vav phosphorylation occurs after raft coalescence, as also supported by the finding that Vav is phosphorylated in response to lipid raft clustering by GM1 aggregation. The constitutive association found between Vav and the CD3zeta chain suggests a model whereby the TCR-associated signaling machinery initiates raft aggregation by promoting F-actin reorganization, which permits full activation of the tyrosine phosphorylation cascade, further reorganization of the actin cytoskeleton and sustained signaling, leading to cell activation. 相似文献
4.
Nadiri A Polyak MJ Jundi M Alturaihi H Reyes-Moreno C Hassan GS Mourad W 《European journal of immunology》2011,41(8):2358-2367
CD40, a member of the TNF receptor family, is expressed on a variety of immune and non-immune cells. Its interaction with its ligand, CD154, plays a pivotal role in humoral and cell-mediated immunity. A low level of CD40 is constitutively associated within membrane lipid rafts and, upon engagement, this level is significantly enhanced. In this study, our objective is to evaluate the process of CD40/lipid raft association in terms of the signals required for its initiation and the resulting biological outcomes. Here, we show the CD40/lipid raft association to be independent of PI-3-kinase, Src family kinases and p38 MAPK pathways. Moreover, CD40 lacking its intracellular domain, which is usually required for CD40-mediated signaling, still localizes to lipid rafts upon engagement, confirming that the CD40/lipid raft association is independent of signaling events. As to the biological outcomes of the CD40/lipid raft association, we show that disrupting lipid raft integrity selectively abolishes CD40-mediated Akt phosphorylation. In addition, replacing the transmembrane domain of CD40 with that of CD45 (a protein excluded from lipid rafts) dramatically reduced CD40-mediated Akt phosphorylation and B7.1 upregulation, while not influencing p38, ERK and JNK activation. Together, these findings clarify the requirements for CD40/lipid raft association and the signals triggered upon CD40 engagement by CD154. 相似文献
5.
L. Gergely J. Czeglédy L. Váczi K. Pálóczi G. Szegedi 《Medical microbiology and immunology》1987,176(3):175-179
Human antibodies against the first and second Epstein-Barr virus encoded nuclear antigens (EBNA-1 and EBNA-2) were analysed by modified ELISA reaction on immunoblots: 5 out of 20 EBNA-1 positive, healthy donor sera reacted with the EBNA-2. Healthy EBV seronegatives contained no antibodies against EBNA-2. The antibodies against this EBNA-2 developed several months after acute EBV infections. Four out of 8 infectious mononucleosis sera failed to react EBNA-2 in the late stage. In the group of EBV seropositive patients with non-Hodgkin lymphoma, 14 out of 18 sera contained EBNA-2 antibodies. 相似文献
6.
Improved immunofluorescence antigens for detection of immunoglobulin M antibodies to Epstein-Barr viral capsid antigen and antibodies to Epstein-Barr virus nuclear antigen. 总被引:4,自引:3,他引:1
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Epstein-Barr viral capsid antigen and nuclear antigen produced by modified procedures were evaluated for use in measuring viral capsid antigen immunoglobulin M and Epstein-Barr virus nuclear antigen antibody responses in sera from patients with suspected Epstein-Barr virus infections. Viral capsid antigen production was stimulated with a phorbol ester, and the Epstein-Barr virus nuclear antigen cells were fixed in suspension to eliminate loss of antigen during the drying process. Both preparations proved to be sensitive and reliable. 相似文献
7.
Su TT Guo B Kawakami Y Sommer K Chae K Humphries LA Kato RM Kang S Patrone L Wall R Teitell M Leitges M Kawakami T Rawlings DJ 《Nature immunology》2002,3(8):780-786
NF-kappa B signaling is required for the maintenance of normal B lymphocytes, whereas dysregulated NF-kappa B activation contributes to B cell lymphomas. The events that regulate NF-kappa B signaling in B lymphocytes are poorly defined. Here, we demonstrate that PKC-beta is specifically required for B cell receptor (BCR)-mediated NF-kappa B activation. B cells from protein kinase C-beta (PKC-beta)-deficient mice failed to recruit the I kappa B kinase (IKK) complex into lipid rafts, activate IKK, degrade I kappa B or up-regulate NF-kappa B-dependent survival signals. Inhibition of PKC-beta promoted cell death in B lymphomas characterized by exaggerated NF-kappa B activity. Together, these data define an essential role for PKC-beta in BCR survival signaling and highlight PKC-beta as a key therapeutic target for B-lineage malignancies. 相似文献
8.
The plasma membranes of eukaryotic cells are not uniform and possess distinct cholesterol- and sphingolipid-rich raft microdomains that are enriched in proteins known to be essential for cellular function. Lipid raft microdomains are important for T cell receptor (TCR)-mediated activation of T cells. However, the importance of lipid rafts on antigen presenting cells (APCs) and their role in major histocompatibility (MHC) class II-restricted antigen presentation has not been examined. MHC class II molecules were found to be constitutively present in plasma membrane lipid rafts in B cells. Disruption of these microdomains dramatically inhibited antigen presentation at limiting concentrations of antigen. The inhibitory effect of raft disruption on antigen presentation could be overcome by loading the APCs with exceptionally high doses of antigen, showing that raft association concentrates MHC class II molecules into microdomains that allow efficient antigen presentation at low ligand densities. 相似文献
9.
J. Sánchez-Wandelmer A. Dávalos G. de la Peña S. Cano M. Giera A. Canfrán-Duque F. Bracher A. Martín-Hidalgo C. Fernández-Hernando M.A. Lasunción R. Busto 《Neuroscience》2010
Haloperidol exerts its therapeutic effects basically by acting on dopamine receptors. We previously reported that haloperidol inhibits cholesterol biosynthesis in cultured cells. In the present work we investigated its effects on lipid-raft composition and functionality. In both neuroblastoma SH-SY5Y and promyelocytic HL-60 human cell lines, haloperidol inhibited cholesterol biosynthesis resulting in a decrease of the cell cholesterol content and the accumulation of different sterol intermediates (7-dehydrocholesterol, zymostenol and cholesta-8,14-dien-3β-ol) depending on the dose of the drug. As a consequence, the cholesterol content in lipid rafts was greatly reduced, and several pre-cholesterol sterols, particularly cholesta-8,14-dien-3β-ol, were incorporated into the cell membrane. This was accompanied by the disruption of lipid rafts, with redistribution of flotillin-1 and Fyn and the impairment of insulin-Akt signaling. Supplementing the medium with free cholesterol abrogated the effects of haloperidol on lipid-raft composition and functionality. LDL (low-density lipoprotein), a physiological vehicle of cholesterol in plasma, was much less effective in preventing the effects of haloperidol, which is attributed to the drug's inhibition of intracellular vesicular trafficking. These effects on cellular cholesterol homeostasis that ultimately result in the alteration of lipid-raft-dependent insulin signaling action may underlie some of the metabolic effects of this widely used antipsychotic. 相似文献
10.
HLA-G protein is the functional homolog of Qa-2, the product of the mouse preimplantation embryo development (Ped) gene. Embryos expressing Qa-2 on the cell surface exhibit a faster rate of preimplantation cleavage and preferential survival in utero compared with Qa-2-negative embryos. Qa-2 is glycosylphosphatidylinositol (GPI) linked in the cell membrane. As a result, Qa-2 proteins cluster in cholesterol- and sphingolipid-rich lipid raft microdomains in the cell membrane and can signal via raft-associated intracellular signaling molecules. Using T cells as a model system, cross-linking of Qa-2 on the cell membrane has been shown to induce proliferation of resting cells. HLA-G, like Qa-2, lacks a cytoplasmic domain capable of transducing signals from the cell surface to the nucleus, but unlike Qa-2, HLA-G has a short six-amino acid cytoplasmic tail rather than a GPI anchor. To test whether HLA-G, like Qa-2, is located in lipid rafts and can act as a signaling molecule, we used an HLA-G transgenic mouse system. T cells were isolated and tested for HLA-G expression by immunofluorescence and for localization of HLA-G in lipid rafts by immunofluorescence and Western blotting. Next, the T cells were cross-linked with anti-HLA-G antibody to test for induction of proliferation. Our novel results show that HLA-G, like GPI-linked Qa-2, is present in lipid rafts in the cell membrane and can act as a signaling molecule to induce proliferation of resting T cells. 相似文献
11.
Subclass reactivity to Epstein-Barr virus capsid antigen in primary and reactivated EBV infections 总被引:8,自引:0,他引:8
A new method for analysis of virus-specific Immunoglobulin G (IgG) subclasses was developed using indirect immunofluorescence. Three hundred thirty-three serum samples from patients with different types of Epstein-Barr virus (EBV)-associated diseases and healthy controls were examined for subclass distribution to the virus capsid antigen (EBV VCA). EBV-VCA-expressing cell preparations were incubated with patient serum followed by monoclonal antibodies to human IgG1 through IgG4 and labelled anti-mouse IgG. Virus-specific IgG1 was found to be the dominant antibody. The titers for IgG1 and total Ig to EBV VCA correlated well. EBV VCA-specific IgG2 was not found. EBV VCA-specific IgG3 in a titer of greater than or equal to 10 was found in 33% of healthy seropositive donors, in 97% of patients with suspected reactivated EBV infection, and in 100% of symptomatic patients with suspected reactivated EBV infection. EBV VCA specific IgG3 occurred in 90% of placebo-treated compared to 30% in long-term acyclovir-treated bone marrow transplant recipients, indicating more frequent reactivations in the former group. IgG4 to VCA was infrequently found in seropositive persons. In serum samples from patients with nasopharyngeal carcinoma and high EBV VCA Ig and IgA titers, IgG4 to VCA was always present. Analysis of EBV VCA specific IgG subclasses seems to be valuable for the diagnosis of reactivated EBV infection. 相似文献
12.
Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts 总被引:8,自引:0,他引:8
The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly characterized. Our previous study has shown that HCV RNA synthesis occurs on a lipid raft membrane structure [J. Virol. 77 (2003) 77 4160]. In this study, we further characterized these replication complexes (RCs) in Huh-7 cells that support active RNA replication of a subgenomic HCV replicon. Biochemical analysis showed that these membrane structures were resistant to Nonidet P-40 or Triton X-100 (TX-100) at 4 degrees C while solubilized by beta-octylglucoside at 4 degrees C or Triton TX-100 at 37 degrees C, characteristic of lipid rafts. Cholesterol sequestration assay further demonstrated the association between HCV nonstructural (NS) proteins and cholesterol-rich lipid rafts. The RCs contained both minus- and plus-strand HCV RNA, with the plus-stranded RNA being approximately 10-fold more abundant than the minus-strand. Furthermore, the HCV RNA and NS proteins were resistant to RNase and protease digestion, respectively, but became sensitive after treatment with the raft-disrupting agents. These results suggested that the HCV RCs are protected within lipid rafts. Detergent-resistant membrane (DRM) fractions containing NS proteins and viral RNA were capable of HCV RNA synthesis using the endogenous HCV RNA template. NS proteins were distributed in both the ER and the Golgi, but the majority of the active RCs were detected in the Golgi-derived membrane. Depletion of cellular cholesterol selectively reduced HCV RNA replication. These findings provide further insights into the mechanism of HCV replication in vivo. 相似文献
13.
A hybrid cell line (Cl-5l) producing an anti-capsid antibody was obtained by fusion of mouse myeloma cells with spleen cells from mice immunized with purified P3HR-1 Epstein-Barr virus (EBV). Immunofluorescence showed that the Cl-5l antibody reacted with the cytoplasm and the nucleus of P3HR-1 and B95-8 cells, but not with Raji, BJAB, Molt-4, and superinfected Raji cells in the presence of cytosine arabinoside (Ara-C). The viable P3HR-1 and B95-8 cells were not stained nor was the viral infectivity neutralized. The Cl-5l antibody immunoprecipitated 123,000 and 120,000 dalton polypeptides of P3HR-1 and B95-8 cells, respectively, and both were sensitive to phosphonoacetic acid. Specific reactions were not evident with extracts of Raji cells and superinfected Raji cells in the presence of Ara-C. An analysis of the purified virus particles showed that this antibody recognized a capsid component of EBV. 相似文献
14.
15.
EB病毒膜蛋白在CHO细胞中的表达 总被引:1,自引:0,他引:1
目的研制EB(Epstein-Bar)病毒基因工程疫苗。方法构建了含有Epstein-Bar病毒(EBV)膜蛋白(MA)基因的重组表达质粒pCMV/MA,转染CHO细胞,研究表达产物的生物学性状,及培养液中不同血清含量、冻存、G418对CHO细胞分泌MA的影响。结果获得两个稳定表达MA的细胞株。免疫印迹检测表达产物的分子量约为340kD和220kD,间接免疫荧光和免疫斑点确定表达产物能与抗MA的单克隆抗体特异性结合,用薄层扫描和Lowry法计算MA的表达量为每天19μg/ml。经纯化的MA免疫小鼠,在血清中检测到抗MA的特异性抗体。结论为开发有效的表达系统用于EB病毒基因工程疫苗的生产提供有利的实验基础。 相似文献
16.
Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor-mediated lipid raft dynamics 总被引:4,自引:0,他引:4
Ligation of the B cell antigen receptor (BCR) with antigen induces lipid raft coalescence, a process that occurs after crosslinking of a variety of signaling receptors and is thought to potentiate cellular activation. To investigate lipid raft dynamics during BCR signaling, we quantitatively analyzed the B cell lipid raft proteome. BCR engagement induced dissociation of the adaptor protein ezrin from lipid rafts as well as threonine dephosphorylation of ezrin and its concomitant detachment from actin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Expression of constitutively active ezrin chimeras inhibited the BCR-induced coalescence of lipid rafts. Our data demonstrate that the release of ezrin from lipid rafts acts as a critical trigger that regulates lipid raft dynamics during BCR signaling. 相似文献
17.
The significance of lipid rafts in lipopolysaccharide (LPS) signaling in macrophages was studied through isolation of them by gradient centrifugation and subsequent visualization of signal molecules using antibodies. LPS signaling is initiated by binding to Toll-like receptor-4 (TLR4) and the co-receptor CD-14, leading to activation of downstream targets, such as MAP kinases. In this study, we show that LPS causes translocation of CD-14 and MAP kinases (ERK-2 and p38) to lipid rafts in the macrophage cell line RAW 264.7. The adaptor proteins MyD88 and Gab-2, on the other hand, were not detected in the lipid raft fractions. These results indicate that lipid rafts play a role in LPS-induced signaling in macrophages. 相似文献
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19.
为了建立纯化Epstein-Barr(EB)病毒壳抗原(EBV-VCA)的方法,用于酶联免疫吸附试验(ELISA)检测人因清中的相关抗体,我们用重组昆虫病毒在感染的sf9细胞中表达EBV-VCA。感染的细胞经裂解后,层析纯化表达产物。用经的VCA作为抗原包虫被ELISA板或硝基纤维膜,检查血清中VCA/IgA和VCA/gG抗体,为EBV感染的的检测和NPC的诊断发展了一个敏感、特异和简便的方法。 相似文献
20.
The pathway by which rotavirus is released from the cell is poorly understood but recent work has shown that, prior to cell lysis, rotavirus is released almost exclusively from the apical surface of the infected cell. By virtue of their unique biochemical and physical properties, viruses have exploited lipid rafts for host cell entry and/or assembly. Here we characterized the association of rhesus rotavirus (RRV) with lipid rafts during the rotavirus replication cycle. We found that newly synthesized infectious virus associates with rafts in vitro and in vivo. RRV proteins cosegregated with rafts on density gradients. Viral infectivity and genomic dsRNA also cosegregated with the raft fractions. Confocal microscopic analysis of raft and RRV virion proteins demonstrated colocalization within the cell. In addition, cholesterol depletion interfered with the association of RRV particles with rafts and reduced the release of infectious particles from the cell. Furthermore, murine rotavirus associates with lipid rafts in intestinal epithelial cells during a natural infection in vivo. Our results confirm the association of rotavirus infectious particles with rafts during replication in vitro and in vivo and strongly support the conclusion that this virus uses these microdomains for transport to the cell surface during replication. 相似文献