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Considerations for the incorporation of measured surfaces in finite element models.
Thompson, Mary Kathryn,Thompson, John M Gerhard Witzstrock Pub. House 2010 Scanning Vol.32 No.4
<P>This work discusses some of the benefits, techniques, challenges, and considerations associated with the incorporation of measured surfaces in finite element (FE) models including how much surface data to measure and import into the model, the shape of the surface geometry to create, the presence and effect of surface layers and impurities, the required mesh density for rough surfaces, the nature of the element formulations and material properties at small length scales, the differences between measurement and FE coordinate systems, the limitations and idealizations of the FE method, issues associated with boundary conditions and their ability to impose or prevent conformal contact, and issues associated with the size of the pinball region and the contact stiffness relative to the nature of the surface. It also describes some current and future research directions that can be used to validate and expand existing techniques and to improve our understanding of surface phenomena. SCANNING 32: 183-198, 2010. © 2010 Wiley Periodicals, Inc.</P>
Kim, Dong-Ho,Jitrapakdee, Sarawut,Thompson, Mary Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.6
UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.
Dual functionality of lamprey VLRB C-terminus (LC) for multimerization and cell surface display
Lee, Jung Seok,Kim, Jaesung,Im, Se Pyeong,Kim, Si Won,Jung, Jae Wook,Lazarte, Jassy Mary S.,Lee, Jeong Ho,Thompson, Kim D.,Jung, Tae Sung Elsevier 2018 Molecular immunology Vol.104 No.-
<P><B>Abstract</B></P> <P>Lamprey, one of the living representatives of jawless vertebrates, uses variable lymphocyte receptors B (VLRB) for antigen recognition, rather than immunoglobulin (Ig) based receptors as used by higher vertebrates. The C-terminus of lamprey VLRB (LC) possess a glycosylphosphatidylinositol (GPI) signal sequence and seven cysteine residues providing dual functionality of the VLRB antibody in the form of a humoral agglutinin and cell membrane receptors. Here, we show that the LC can be either secreted or be membrane anchored as a heterologous fused protein in a multimeric form comprising of eight or ten monomeric units. Using serially truncated LC variants, we showed that the LC, in which the last three amino acid “RKR” were deleted, referred to as LC7, was the most suitable domain for multimeric construction, whereas, the intact LC is more tailored for applications involving membrane anchorage. We show that an antibody specific for viral hemorrhagic septicemia virus (VHSV) (VLR43), displayed on HEK-293F cells using a PiggyBac (PB) transposase system, exhibited a dose-dependent reaction with its antigen, verifying that the LC can be applied in antibody display technology. Therefore, the present report provides valuable insight into the structure of the lamprey VLRB and highlights its potential use as a novel fusion partner for multimerization and membrane anchorage of chimeric proteins.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Lamprey VLRB has dual functionality like as humoral agglutinin and membrane receptors. </LI> <LI> The LC can perform multimerization of fused protein via disulfide-linkages. </LI> <LI> The LC can mediate cell surface localization of fused proteins via GPI anchoring. </LI> <LI> The LC can be used at an antibody display system as membrane tethering domain. </LI> </UL> </P>
Jung, Jae Wook,Lee, Jung Seok,Kim, Young Rim,Im, Se Pyeong,Kim, Si Won,Lazarte, Jassy Mary S.,Kim, Jaesung,Thompson, Kim D.,Suh, Jong Pyo,Jung, Tae Sung Elsevier 2017 Fish & shellfish immunology Vol.65 No.-
<P><B>Abstract</B></P> <P>The T cell receptor (TCR) is the binding site of antigen and is responsible for specifically activating the adaptive immune response. CD3, an essential component of the CD3-TCR complex, is known to be composed of γδ and ε chains in teleost. However, there are few monoclonal antibodies (mAb) available to identify these molecules on T cells, so we aimed to produce a mAb against CD3ε to improve our understanding of T cell immune response in olive flounder (<I>Paralichthys olivaceus)</I>. CD3ε recombinant protein was expressed in yeast, the expression of which was confirmed by SDS-PAGE, MALDI-TOF/TOF MS and Western blot analysis. A CD3ε-specific mAb 4B2 was selected, the specificity of which was examined by confocal microscopy, flow cytometry and RT-PCR, and the mAb was subsequently used to examine the CD3ε lymphocyte population in several different immune organs, with relatively high percentages of these cells seen in trunk-kidney and spleen, while lower percentages were seen in the liver and peripheral blood of olive flounder. During a viral hemorrhagic septicemia virus (VHSV) infection in olive flounder, the number of CD3ε lymphocytes was seen to gradually increase in the liver, spleen and trunk-kidney of infected fish until 7 days post infection (dpi). In peripheral blood, on the other hand, the increase in CD3ε lymphocyte numbers peaked by 3 dpi. These results suggest that CD3ε lymphocytes might be involved in the immune response against VHSV.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Recombinant CD3ε was produced using yeast expression system. </LI> <LI> Monoclonal antibody 4B2 specifically detects the CD3ε lymphocyte in olive flounder. </LI> <LI> CD3ε lymphocytes might be involved in immune response related to VHSV. </LI> </UL> </P>
Lee, Jung Seok,Kim, Jaesung,Im, Se Pyeong,Kim, Si Won,Jung, Jae Wook,Lazarte, Jassy Mary S.,Lee, Jeong-Ho,Thompson, Kim D.,Jung, Tae Sung Elsevier 2018 Journal of immunological methods Vol.462 No.-
<P><B>Abstract</B></P> <P>Monomeric variable lymphocyte receptor B (VLRB) is one of the smallest binding scaffold (20–25 kDa) from jawless vertebrates, hagfish and lamprey. This relatively new class of binding scaffold has various advantages: i) it has a single peptide composition, amenable to molecular engineering for enhancing its stability and affinity; ii) it has a small size, contributing better tissue penetration and easier production using microorganism expression system. Monomeric arVLRB142, which can specifically bind to the glycoprotein of viral hemorrhagic septicemia virus (VHSV), was expressed in <I>Pichia pastoris</I>. High quantity recombinant monomeric arVLRB142 (rVLR142<SUP>mono</SUP>) was purified from 100 ml of culture with a resulting yield of 2.6 ±1.3 mg of target protein. Functional studies revealed that the purified rVLR142<SUP>mono</SUP> can specifically recognize low levels of the target antigen (recombinant glycoprotein) (i.e. as low as 0.1 nM), but also the native glycoprotein of VHSV. The expressed rVLR142<SUP>mono</SUP> exhibited high levels of stability and it retained it binding capacity over broad temperature (4 °C ~ 60 °C) and pH ranges (pH 1.5–12.5). We developed an effective expression system for mass production of monomeric VLRB based on <I>P. pastoris</I>. The recombinant protein that was obtained offers promising binding avidity and biophysical stability and its potential use in various biotechnological applications.</P>
Jung, Jae Wook,Lee, Jung Seok,Kim, Jaesung,Im, Se Pyeong,Kim, Si Won,Lazarte, Jassy Mary S.,Kim, Young Rim,Chun, Jin Hong,Ha, Min Woo,Kim, Hyeong Su,Thompson, Kim D.,Jung, Tae Sung American Association of Immunologists 2020 Journal of Immunology Vol.204 No.3
<P><B>Key Points</B></P><P><OL><LI>NNV-specific VLRBs were developed using a library screening system.</LI><LI>The binding ability of this Ab was enhanced through modular engineering.</LI><LI>VLRB could be an alternative neutralizing Ab against Betanodavirus infection.</LI></OL></P><P>The variable lymphocyte receptor (VLR) mediates the humoral immune response in jawless vertebrates, including lamprey (<I>Petromyzon marinus</I>) and hagfish (<I>Eptatretus burgeri</I>). Hagfish VLRBs are composed of leucine-rich repeat (LRR) modules, conjugated with a superhydrophobic C-terminal tail, which contributes to low levels of expression in recombinant protein technology. In this study, we screened Ag-specific VLRBs from hagfish immunized with nervous necrosis virus (NNV). The artificially multimerized form of VLRB was constructed using a mammalian expression system. To enhance the level of expression of the Ag-specific VLRB, mutagenesis of the VLRB was achieved in vitro through domain swapping of the LRR C-terminal cap and variable LRR module. The mutant VLRB obtained, with high expression and secretion levels, was able to specifically recognize purified and progeny NNV, and the Ag binding ability of this mutant was increased by at least 250-fold to that of the nonmutant VLRB. Furthermore, preincubation of the Ag-specific VLRB with NNV reduced the infectivity of NNV in E11 cells in vitro, and in vivo experiment. Our results suggest that the newly developed Ag-specific VLRB has the potential to be used as diagnostic and therapeutic reagents for NNV infections in fish.</P>