Our previous studies have revealed that the signaling protein BCL10 plays a major role in adaptive immunity by mediating NF-B activation in the LPS/TLR4 pathway. In this study, we show that IRAK-1 acts as the essential upstream adaptor that recruits BCL10 to the TLR4 signaling complex and mediates signaling to NF-B through the BCL10-MALT1-TRAF6-TAK1 cascade. Following dissociation from IRAK-1, BCL10 is translocated into the cytosol along with TRAF6 and TAK1, in a process bridged by a direct BCL10-Pellino2 interaction. RNA interference against MALT1 markedly reduced the level of NF-B activation stimulated by lipopolysaccharide (LPS) in macrophages, which suggests that MALT1 plays a major role in the LPS/TLR4 pathway. MALT1 interacted with BCL10 and TRAF6 to facilitate TRAF6 self-ubiquitination in the cytosol, which was strictly dependent on the dissociation of BCL10 from IRAK-1. We show that BCL10 oligomerization is a prerequisite for BCL10 function in LPS signaling to NF-B and that IRAK-1 dimerization is an important event in this process.BCL10 and MALT1 are intracellular NF-B activators that are involved in MALT 4 lymphoma translocations (1, 2). These translocations are associated with MALT tumorigenesis via constitutive NF-B activation, which provides both anti-apoptotic and proliferative signals by up-regulating the transcription of specific targets (3). BCL10 contains the caspase recruitment domain (CARD), which is involved in protein oligomerization and protein-protein associations (4), as well as the MALT1-binding sequence containing 16 amino acids.BCL10 proteins can form homo-oligomers through CARD-CARD interaction under suitable conditions. In TCR signaling, BCL10 binds to its downstream adaptor MALT1 and co-operatively activates NF-B (4 -6). Recent studies have shown that BCL10 and MALT1 mediate NF-B activation by facilitating Lys-63 polyubiquitination of NEMO, which is an essential regulatory subunit of the IKK complex (7). In this process, tumor necrosis factor receptor-associated factor 6 (TRAF6), which is a polyubiquitin ligase, fills the gap between BCL10-MALT1 and NEMO in TCR signaling (8). MALT1 binds to TRAF6 through two putative C-terminal TRAF6-binding motifs. In vitro experiments using purified MALT1, TRAF6, TGF-␤-activated kinase (TAK1), and ubiquitination enzymes, which include Ubc13/ Uev1A, allowed reconstitution of the pathway from BCL10 to IKK activation. As reported previously, BCL10 and MALT1 form oligomers that bind to TRAF6, induce TRAF6 oligomerization, and activate the ligase activity of TRAF6 to promote self-ubiquitination and polyubiquitination of NEMO. This cascade model is predicted to be responsible for TCR signaling and NF-B activation in T lymphocytes (8).Toll-like receptors (TLRs) play essential roles in innate immune recognition in mammalian species. Upon engagement of their respective ligands, TLRs stimulate the transcription of effector genes through activation of common transcription factors, which include nuclear factor-B (NF-B), activating protein 1, and activating tr...

To develop early diagnostic reagents, effective vaccines, and even drugs against SARS-associated coronavirus (SARS-CoV), the human single fold single-chain antibody fragments, (scFv) libraries I+J (Tomlinson I+J) were used to identify novel scFvs, which can specifically bind to SARS-CoV. Interestingly, two scFvs (B5 and B9) exhibited higher binding specificity to SARS-CoV with the OD(450) value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of V(H) gene (351bp) and V(L) gene (327bp), so the two scFvs were uniformly named as SA59B and chosen for further analysis. SA59B scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The soluble 30kDa SA59B scFv-antibody was verified in SDS-PAGE and Western-blot. The purified SA59B scFv-antibody was labeled with HRP by the glutaraldehyde method, and the concentration of HRP and SA59B scFv-antibody in the SA59B-HRP solution reached 2.4 and 2.28mg/ml, respectively. Then, the binding ability of SA59B-HRP to SARS-CoV was evaluated by ELISA with S/N of 11.6, indicating higher binding specificity between them. Finally, both the SA59B sequence specificity and its application for diagnosis, prophylaxis or therapy of SARS were discussed.

Various sensor-based immunoassay methods have been extensively developed for the detection of interleukin-6 (IL6), but most often exhibit low detection signals and low detection sensitivity, and are unsuitable for routine use. The aim of this work is to develop a simple and sensitive conductometric immunoassay for IL6 in human serum by using an organic/inorganic hybrid membrane-functionalized interface. Initially, thionine-bound 3,4,9,10-perylenetetracarboxylic acid was doped into colloidal alumina, then nanogold particles were immobilized onto the thionine surface, and then horseradish peroxidase-labeled anti-IL6 antibodies were conjugated on the nanogold surface. The organic/inorganic hybrid membrane provides a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The performance and factors influencing the performance of the immunosensor were evaluated. The detection is based on the change in local conductivity before and after the antigen-antibody interaction in 0.02 M phosphate buffer solution (pH 6.8) containing 50 microM H(2)O(2), 0.01 M KI and 0.15 M NaC1. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 25 to 400 pg/ml towards IL6 with a relatively low detection limit of 5 pg/ml (S/N = 3). The stability, reproducibility and precision of the immunosensor were acceptable. 37 serum specimens were assayed by the developed immunosensor and standard enzyme-linked immunosorbent assay, respectively, and the results obtained were almost consistent. More importantly, the detection methodology provides a promising approach for other proteins or biosecurity.

Previous studies based on cell culture and xenograft animal models suggest that Smad3 has tumor suppressor function for breast cancer during early stages of tumorigenesis. In this report, we show that DMBA (7,12-dimethylbenz[a]anthracene), a chemical carcinogen, induces mammary tumor formation at a significantly higher frequency in the Smad3 heterozygous mice than in the Smad3 wild type mice. This is the first genetic evidence showing that Smad3 inhibits mammary tumor formation in a mouse model. Our findings support the notion that Smad3 has important tumor suppressor function for breast cancer.