• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1995.04a
• /
• pp.82-82
• /
• 1995

4-lBB molecule is expressed on the surface of activated CD4$\^$+/ and CD8$\^$+/ T cells. We generated a panel of anti-4-1 B5 murine mAbs using a fusion protein consisting of the extracellular domain of human 4-1 BB fused to Glutathione S-transferase. The binding activity against cell surface 4-1 BB molecule was assessed by flow cytometry analysis. These studies showed that several anti-4-1 BB mAbs bound to 10-30% of CD4$\^$+/ and CD8$\^$+/T cells in PHA or Con A stimulated PBLs, although these mAbs interacted with only, l-2% of CD4$\^$+/ and CD8$\^$+/ T cells in normal PBLs, indicating the specificity of mAbs to the 4-l BB molecule on activated CD4$\^$+/ and CD8$\^$+/ T cells. Next, we examined the effect of an anti-4-l BB mAb (4B4-1-1) on allogeneic mixed lymphocyte reactions (MLRs). The data indicated that the antibody significantly inhibited the proliferative response at higher concentrations. When tested with several T cell mitogens, the antibody had no stimulatory or inhibitory effects on the mitogen-mediated T cell proliferation. These data suggest that 4-1 BB molecule may play a role in the regulation of antigen-mediated immune response.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1995.04a
• /
• pp.81-81
• /
• 1995

It is well known that Src Homology 2(SH2) domain in many intracellular signal transduction proteins is very important. The domain has about 100 amino acid residues and bind phosphotyrosine-containing peptide with high affinity and specificity. Lck SH2 domain is a Src-like, lymphocyte-specific tyrosine kinase. An 11-residue phosphopeptide derived from the hamster polvoma middle-T antigen, EPQp YEEIPIYL, binds with an 1 nM dissociation constant to Lck SH2 domain. And it is known that the phosphotyrosine and isoleucine residues of the peptide are tightly bound by two well-defined pockets on Lck SH2 domain's surface. To investigate the conformational changes during complexation of SH2 domain with phosphopeptide we have performed the molecular dynamics simulation for Lck SH2 domain with peptide and peptide-free form at look in aqueous solution. More than 3000 water molecules were incorporated to solvate Lck SH2 domain and peptide. Periodic boundary condition has been applied in molecular dynamics simulation. Data analysis with the results of that simulation shows that the phosphopeptide makes primary interaction with the Lck SH2 domain at six central residues, The comparison of the complexed and uncomplexed SH2 domain structures in solution has revealed only relatively small change. But the hydrophilic and hydrophobic pockets in the protein surface show the conformational changes in spite of the small structural difference between the complex and peptide-free forms.

• Proceedings of the Korean Institute of Surface Engineering Conference
• /
• 2012.11a
• /
• pp.4-4
• /
• 2012

Proteins enable biology to be viable through molecular interactions (such as in antigen-antibody, ligand-receptor, and drug-target) with

• Korean Journal of Food Science and Technology
• /
• v.31 no.1
• /
• pp.1-6
• /
• 1999

A piezoelectric (PZ) biosensor system detecting Salmonella spp. was developed. The system consisted of an oscillator, a frequency counter and an antibody-immobilized quartz crystal. An anti-Salmonella antibody was immobilized on one gold. surface of the quartz crystal with protein A. Salmonella detection was made by measuring resonant frequency shift owing to a mass change by specific binding of microbial cells to the gold surface of the PZ crystal. The PZ antibody sensor was operated optimally at 0.1M phosphate buffer, pH 7.2 and $35^{\circ}C$. The sensor was quite specific to Salmonella spp. The obtained frequency shift was correlated with the Salmonella concentration in the range of $10^5{\sim}10^6\;CFU/mL$. The frequency shift increased further by addition of polystyrene beads. The Salmonella detection which is indicated by a steady-state microbial adsorption to the quartz crystal was accomplished within 50min.

• Molecules and Cells
• /
• v.40 no.5
• /
• pp.355-362
• /
• 2017

The ${\beta}2$ integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of ${\beta}2$ integrin, ${\alpha}M{\beta}2$ and ${\alpha}X{\beta}2$, share the leukocyte distribution profile and integrin ${\alpha}X{\beta}2$ is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. ${\underline{R}}eceptor$ for ${\underline{a}}dvanced$ ${\underline{g}}lycation$ ${\underline{e}}nd$ ${\underline{p}}roducts$ (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and ${\alpha}X{\beta}2$ play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of ${\alpha}X{\beta}2$, we characterize the binding nature and the interacting moieties of ${\alpha}X$ I-domain and RAGE. Their binding requires divalent cations ($Mg^{2+}$ and $Mn^{2+}$) and shows an affinity on the sub-micro molar level: the dissociation constant of ${\alpha}X$ I-domains binding to RAGE being $0.49{\mu}M$. Furthermore, the ${\alpha}X$ I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of ${\alpha}X$ I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to ${\alpha}X$ I-domain. In conclusion, the main mechanism of ${\alpha}X$ I-domain binding to RAGE is a charge interaction, in which the acidic moieties of ${\alpha}X$ I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.

• Korean Journal of Microbiology
• /
• v.55 no.3
• /
• pp.199-205
• /
• 2019

Yeast two-hybrid (Y2H) technique has been used to study protein-protein interactions, but its application particularly to a large-scale analysis of protein interaction networks, is limited by the fact that the technique is labor-intensive, based on scoring colonies on plate. Here, we develop a new reporter for the measurement of the protein-protein interactions by flow cytometry. The yeast harboring interacting proteins can also be enriched by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). When two interacting proteins are present in the same yeast cell, a reporter protein containing 10 tandem repeats of c-myc epitope becomes localized on the surface of the cell wall, without affecting cell growth. We successful measured the surface display of c-myc epitope upon interacting p53 with SV40 T antigen by flow cytometry. Thus, the newly developed Y2H assay based on the display of c-myc repeat on yeast cell wall could be used to the simultaneous analysis of multiple protein-protein interactions without laborious counting colonies on plate.

• Parasites, Hosts and Diseases
• /
• v.61 no.2
• /
• pp.138-146
• /
• 2023

Toxoplasma gondii is an intracellular protozoan parasite which can infect most warm-blooded animals and humans. Among the different mouse models, C57BL/6 mice are more susceptible to T. gondii infection compared to BALB/c mice, and this increased susceptibility has been attributed to various factors, including T-cell responses. Dendritic cells (DCs) are the most prominent type of antigen-presenting cells and regulate the host immune response, including the response of T-cells. However, differences in the DC responses of these mouse strains to T. gondii infection have yet to be characterized. In this study, we cultured bone marrow-derived DCs (BMDCs) from BALB/c and C57BL/6 mice. These cells were infected with T. gondii. The activation of the BMDCs was assessed based on the expression of cell surface markers and cytokines. In the BMDCs of both mouse strains, we detected significant increases in the expression of cell surface T-cell co-stimulatory molecules (major histocompatibility complex (MHC) II, CD40, CD80, and CD86) and cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-12p40, IL-1β, and IL-10) from 3 h post-T. gondii infection. The expression of MHC II, CD40, CD80, CD86, IFN-γ, IL-12p40, and IL-1β was significantly higher in the T. gondii-infected BMDCs obtained from the C57BL/6 mice than in those from the BALB/c mice. These findings indicate that differences in the activation status of the BMDCs in the BALB/c and C57BL/6 mice may account for their differential susceptibility to T. gondii.

• IMMUNE NETWORK
• /
• v.5 no.2
• /
• pp.124-129
• /
• 2005

Background: CM1 (centrocyte/-blast marker 1) is originally defined as a germinal center B cell marker. It is known that CM1 plays a critical role on B cell development in germinal center. In addition, we have found that CM1 is expressed on lymphoma cell lines, such as Raji, Ramos and IM-9. This means that CM1 might be served as a tumor marker as well. In the present study, we examined the expression of CM1 on the surface of the other tumors and the possibility of the development of tumor screening ELISA kit by using CM1. Methods: First, we have examined the expression of CM1 on stomach cancer and hepatoma, which are predominantly (discovered) occurred in Korean, by flow cytometry analysis. After purifying of CM1 antigen from Raji and Ramos, the optimal ELISA condition was determined. And then we compared the level of CM1 between normal individuals and cancer patients by ELISA. To decrease the non-specific binding of anti-CM1 mAb with serum components except CM1 and to enhance the diagnostic accuracy, albumin depletion spin column was used. Results: CM1 was highly expressed on stomach cancer and hepatoma cell lines. In addition, we have also confirmed the increased CM1 expression on cancer patients. The difference of CM1 expression between normal individuals and cancer patients were more clearly observed, after deletion of serum albumin by using albumin depletion spin column. Conclusion: Based on the results from this study, CM1 might be a useful molecule for the early diagnosis of cancer. In addition, further studies for the increase of ELISA sensitivity and appropriate albumin depletion methods should be needed.

• Journal of the Korean Society for Nondestructive Testing
• /
• v.31 no.5
• /
• pp.487-493
• /
• 2011

Alpha fetoprotein(AFP), which is serological marker for hepatocellular carcinoma, was quantitatively measured by its normal concentration, 10 ng/ml, with a label-free piezoelectric microcantilever mass sensor. The principle of detection is based on changes in the resonant frequency of the piezoelectric microcantilever before and after target molecules are attached to it, and its resonant frequency is measured electrically using a conductance spectrum. The resonant frequency of the developed sensor is approximately 1.34 MHz and the mass sensitivity is approximately 175 Hz/pg. The sensor has high reliability as mass sensor by reducing the effect of surface stress on resonant frequency due to attached proteins. 'Dip and dry' technique was used to react the sensor with reagents for immobilizing AFP antibody on the sensor and detecting AFP antigen. The measured mass of the detected AFP antigen was 6.02 pg at the concentration of 10 ng/ml, and 10.67 pg at 50 ng/ml when the immunoreaction time was 10 min.

• The Journal of the Korean Society for Microbiology
• /
• v.21 no.3
• /
• pp.399-406
• /
• 1986

This study was performed to establish human plasmacytoma cell line as the partner cells for producing human hybridoma. Bone marrow cells from a multiple myeloma patient from Seoul National University Hospital, Korea were cultured and established as the cell line, named as HMC-BM4. HMC-BM4 cells were cultivated in RPMI 1640 media containing 8-azaguanine(8-AG; gradually increasing concentration from $1\;{\mu}g/ml$ to $20\;{\mu}g/ml$). 8-AG resistant cells were collected and cloned by limiting dilution. Each clone was divided and tested to die in hypoxanthine, aminopterine and thymidine (HAT) selection media. Finally one clone was selected and named as HMC-AR, which was sensitive to HAT selection media. HMC-AR cells showed typical morphology of plamacytoma in Wright staining. No cell formed the rosette with sheep erythrocytes. Surface membrane $\mu$ heavy chain was detected in 20% of HMC-AR cells and cytoplasmic $\mu$ heavy chain in 90% of them by direct immunofluorescent staining. Ia-like antigen was found in 90% of HMC-AR cells by indirect immunofluorescent staining using anti-Ia-like antigen monoclonal antibody, 1BD9-2. And about $1.0\;{\mu}g/ml$ of human $\mu$ heavy chain was detected in the 3-day culture supernatant of HMC-AR cells. 88% of cells contained 46 chromosomes. Mycoplasma was not detected in HMC-AR cells by Hoechst 33258 staining. This cell line would be used for making hybridomas secreting human monoclonal antibody.