• IMMUNE NETWORK
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• v.3 no.3
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• pp.219-226
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• 2003

Background: Phage display is the most widely used technique among display methods to produce monoclonal antibody fragment with a specific binding activity. Having a large library for efficient antibody display/selection is quite laborious process to have more than $10^9$ members of transformants. To overcome these limitations, several in vitro selection approaches have been reported. Ribosome display that links phenotypes, proteins, directly to genotype, mRNA, is one of the in vitro display methods. Ribosome display can reach the size of scFv library up to $10^{14}$ molecules and it can be further diversified during PCR steps. To select the high affinity scFv from one pot library, we established ribosome display technique by modifying the previously reported eukaryotic translation system. Methods: To establish the antibody selection system by ribosome display, we used 3D8, anti-DNA antibody. A 3D8 scFv was synthesized in vitro by an in vitro transcription-translation system. The translated 3D8 scFv and the encoding 3D8 mRNA are connected to the ribosome. These scFv-ribosome-mRNA complexes were selected by binding to their specific antigens. The eluted mRNAs from the complexes are reverse transcribed and re-amplified by PCR. To apply this system, antibody library from immunized mouse with terminal protein (TP)-peptide of hepatitis B virus DNA polymerase TP domain was also used. This TP-peptide encompasses the 57~80 amino acid residues of TP. These mRNA/ribosome/scFv complexes by our system were panned three times against TP-peptide. The enrichment of antibody from library was determined by radioimmunoassay. Results: We specifically selected 3D8, anti-DNA antibody, against ssDNA as a model system. The selected 3D8 RNAs sequences from translation complexes were recovered by RT-PCR. By applying this model system, we enriched TP-peptide-specific scFv pools through three cycles of panning from immunized library. Conclusion: We show that our translating ribosome complexes are well maintained and we can enrich the TP-specific scFv pools. This system can be applied to select specific antibody from an antibody library.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2001.11a
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• pp.43-56
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• 2001

Colostrum contains various kinds of cytokines including TGF-${\beta}$ which is known to be multifunctional in immune response and act as an anti-inflammatory agent. First, we measured the amount of TGF-${\beta}$ in bovine and human colostrum. Expression pattern of TGF-${\beta}$ isotypes was dramatically different between human and bovine colostrial samples. Bovine colostrum collected on day 1 post-delivery retained $41.79{\pm}16.96ng/ml$ of TGF-${\beta}$ 1 and $108.4{\pm}78.65ng/ml$ of TGF-${\beta}$ 2 while in human, $284{\pm}124.75ng/ml$ of TGF-${\beta}$ 1 and $29.75{\pm}6.73ng/ml$ of TGF-${\beta}$ 2. Thus, TGF-${\beta}$ is the predominant TGF-${\beta}$ isotype in bovine colostrum and vice versa in human colostrum. Both TGF-${\beta}$ isotypes diminished significantly in human and bovine colostrum with time. Next, biological activity of colostrial samples was examined in vitro. Both human and bovine colostrum increased IgA synthesis by LPS-activated mouse spleen B cells, which is a typical effect of TGF-${\beta}$ on the mouse B cell differentiation. Futhermore, we found that anti-proliferative activity in MV1LU cells by colostrum samples disappeared by addition of anti-TGF-${\beta}$ 1 and anti-TGF-${\beta}$ 2 antibody. In conclusion, there are substantial amounts of biologically active TGF-${\beta}$ 1 and TGF-${\beta}$ 2 in bovine and human colostrum. The results that the colostrum can increase IgA expression has important implications since IgA is the major Ig class produced in the gastrointestinal tract. We have previously shown that the stimulatory effect of Bifidobacteria bifidum on spllen B cells was quite similar to that of LPS which is a well-known polyclonal activator for murine B cells. In the present study, we further asked whether B. bifidum regulate the synthesis of IgA by mucosal lymphoid cells present in Peyers patches (PP) and mesenteric lymph nodes (MLN). B. bifidum alone, but not C. perfringens, significantly induced overall IgA and IgM synthesis by both MLN and PP cells. This observation indicates that B. bifidum possesses a modulatory effect on the mucosal antibody production in vivo. We, therefore, investigated the mucosal antibody prodduction following peroral administration of B. bifidum to mice. Ingested B. bifidum significantly increased the numbers of Ig (IgM, IgG, and IgA) secreting cells in the culture of both MLN and spleen cells, indicating that peroally introduced B. bifidum enhances mucosal and systemic antibody response. Importantly, however, B. bifidum itself does not induce the own specific antibody responses, implying that B. bifidum do not incite any unwanted immune reaction. Subsequently, it was found that excapsulation of B. bifidum further augments the total IgA production by increasing the number of IgA-secreting cells in the culture of both MLN and spleen cells. Finally, we found that the immuno-stimulating activity of B. bifidum is due to its cell wall components but not due to any actively secreting component(s) from bacteria. Thus our data reveal that peroral administration of B. bifidum can enhance intestinal IgA production and that encapsulation of B. bifidum further reinforces the IgA production.

• Journal of Life Science
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• v.29 no.2
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• pp.256-264
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• 2019

Lymphotoxin ${\beta}$ receptor ($LT{\beta}R$), a member of the tumor necrosis factor receptor family, plays an important role in lymphoid tissue's architecture and organogenesis. In contrast, MLCK and ROCK play critical roles in the regulation of stress fiber (SF) formation in cells. To determine whether $LT{\beta}R$ stimulation in fibroblastic reticular cells (FRCs) is involved in these signaling pathways, myosin light chain kinase inhibitor-7 (ML-7) was used to inhibit them. ML7-treated FRCs completely blocked SFs and showed retraction and shrinkage processes comparable to those observed in agonistic anti-$LT{\beta}R$ antibody-treated cells. The inhibition of ROCK activity with Y27632-induced changes in actin cytoskeleton organization and cell morphology in FRCs. Actin bundles rearranged into SFs, and phospho-myosin light chain (p-MLC) co-localized in FRCs. We checked the level of Rho-guanosine diphosphate (RhoGDP)/guanosine triphosphate (GTP) exchange activity using FRC lysate. When $LT{\beta}R$ was stimulated with agonistic anti-$LT{\beta}R$ antibodies, Rho-GDP/GTP exchange activity was markedly reduced. Regarding $LT{\beta}R$ signaling with a focus on MLCK inhibition, we showed that the phosphorylation of MLCs was reduced by $LT{\beta}R$ stimulation in FRCs. Cytoskeleton components, such as tubulin, b-actin, and phospho-ezrin proteins acting as membrane-cytoskeleton linkers, were produced in de-phosphorylation, and they reduced expression in agonistic anti-$LT{\beta}R$ antibody-treated FRCs. Collectively, the results suggested that MLCK and ROCK were simultaneously responsible for SF regulation triggered by $LT{\beta}R$ signaling in FRCs.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4423-4428
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• 2014

A novel monoclonal antibody (mAb), known as AC10364, was identified from an antibody library generated by immunization of mice with human carcinoma cells. The mAb recognized proteins in lysates from multiple carcinoma cell lines. Cell cytotoxicity assays showed that AC10364 significantly inhibited cell growth and induced apoptosis in multiple carcinoma cell lines, including Bel/fu, KATO-III and A2780. Compared with mAb AC10364 or chemotherapeutic drugs alone, the combination of mAb AC10364 with chemotherapeutic drugs demonstrated enhanced growth inhibitory effects on carcinoma cells. These results suggest that mAb AC10364 is a promising candidate for cancer therapy.

• BMB Reports
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• v.28 no.4
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• pp.353-358
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• 1995

Epitope tagging is the process of fusing a set of amino acid residues that are recognized as an antigenic determinant to a protein of interest. Tagging a protein with an epitope facilitates various immunochemical analyses of the tagged protein with a specific monoclonal antibody. The monoclonal antibody H8 has subtype specificity for an epitope derived from the preS2 region of hepatitis B virus surface antigen. Previous studies on serial deletions of the preS2 region indicated that the preS2 epitope was located in amino acid residues 130~142. To test whether the amino acid sequence in this interval is sufficient to confer on proteins the antigenicity recognizable by the antibody H8, the set of amino acid residues in the interval was tagged to the amino terminal of ${\beta}$-galactosidase and to the carboxyl terminal of the truncated $p56^{lck}$ fragment. The tagged ${\beta}$-galactosidase, expressed in Escherichia coli, maintained the enzymatic activity and was immunoprecipitated efficiently with H8. The tagged $p56^{lck}$ fragment, synthesized in an in vitro translation system, was also immunoprecipitated specifically with H8. These results demonstrate that the amino acid sequence of the preS2 region can be used efficiently for the epitope tagging approach.

• Toxicological Research
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• v.18 no.2
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• pp.183-190
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• 2002

The mechanism by which catechin-mediated cytotoxicity against tumor cells remains to be elusive. To elucidate the mechanical mights of anti-tumor effects, (-)epigallocatechin-gallate (EGCG) of catechin was applied to human prostate cancer DU 145 cells. Cell viability was measured by crystal violet staining. Cell lysates were wed to measure the catalytic activity of caspases by using fluorogenic peptide: Ac-DEVD-AMC for caspase-3 protease, Z-IETD-AFC for caspase-8 protease, Ac-LEHD-AFC for caspase-9 protease as substrates. The equal amounts of protein from cell lysate was separated on SDS-PAGE and analyzed by western blotting with anti-Fas antibody, anti-FasL antibody, anti-BCL2 antibody and anti-Bax antibody. (-)EGCG induced the death of DUl45 cells, which was revealed as apoptosis shown by DNA fragmentation. (-)EGCG induced the activation of caspase family cysteine proteases including caspase-3, -8 and -9 proteases in DU145 cells. Also, (-)EGCG increased the expression of Fas and Fas ligand (FasL) protein in DU145 colls. The expression level of BCL2 was decreased in (-)EGCG treated DU145 cells, whereas Bax protein was increased in a time-dependent manner. We suggest that (-)EGCG-induced apoptosis of DU145 cells is mediated by signaling pathway involving caspase family cysteine protease, mitochondrial BCL2-family protein and Fas/FasL.

• YAKHAK HOEJI
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• v.37 no.4
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• pp.420-426
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• 1993

To effectively purify of IL-1 inhibitor from human febrile urine, we have established monoclonal antibody that reacts with human recombinant interleukin l$\beta$(IL-1$\beta$). The antibody, designated ON-1, was highly specific to IL-1$\beta$ and no cross-reaction with other cytokines(IL-l$\alpha$ and IL-4) was observed. As the results of ELISA inhibition assay and Western blotting method, it was further identified that ON-1 had high binding specificity with IL-1$\beta$. IL-1 receptor binding material from febrile patient urine was effectively purified with affinity column chromatography which conjugated with ON-1. This urinary material inhibited the thymocyte proliferation in a dosedependent manner. IL-l$\beta$ induced thymocyte proliferation activity was inhibited to 67.3% at 6 $\mu\textrm{g}$ of the purified urinary material. The result may suggest that this urinary material the purified urinary material. The result may suggest that this urinary material will have antagonic effect on IL-1 action mechanism and act IL-l$\beta$ inhibitor.

• Archives of Pharmacal Research
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• v.21 no.2
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• pp.128-134
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• 1998

Nitric oxide synthase, NOS (EC.1.14.13.39), was purified from bovine pancreas over 5,500-fold with a 7.6% yield using 30% ammonium sulfate precipitation, and $2^1$,$5^1$-ADP-agarose and calmodulin-agarose affinity chromatography. The purified bovine pancreatic NOS (bpNOS) showed a single band on SDS-PAGE corresponding to an apparent molecular mass of 160 kDa, whereas it was 320 kDa on non-denaturating gel-filtration. This indicated a homodimeric nature of the enzyme. The specific activity of the purified bpNOS was 31.67 nmol L-citrulline fored/mtn/mg protein and an apparent $K\textrm{m}$ for L-arginine was 15.72 $\mu\textrm{M}$, The enzyme activity was dependent on $Ca^{2+}$ and calmodulin, and to a lesser extent on NADPH, FAD and FMN. $H_4B$ was not required as a cofactor for the activity. In an inhibition experiment with L-arginine analogues, $N^G$-nitro-L-arginine (NNA) had the most potent inhibitory effect on bpNOS, and $N^{G}$, $N^{G1}$-dimethyl-L-arginine (symmetric; sDMA) did not have any inhibitory effect. Immunohistochemical analysis of the bovine pancreas using brain type NOS antibody (anti-bNOS antibody) revealed that acinar cells showed strong immunoreactivity against the antibody.

• Korean Journal of Pharmacognosy
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• v.42 no.3
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• pp.246-252
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• 2011

Korean mistletoe Lectin (KML-C) is composed of A and B sub-chain. B chain binds to carbohydrates on cell surface and A chain hinders translation and induces an apoptosis as a RIP (ribosome inactivating protein). KML-C has very strong biological activities, it has seriously limits to use as a cancer therapy or adjuvant because of its toxicity to normal cells. This study is therefore conducted to see if B chain of KML-C might have immunological activity, especially adjuvant activities with less toxicity. We isolated B chain from KML-C using the lactose affinity chromatography, and examined their immunoadjuvant activity. The isolated B-chain did not show any cytotoxicity against tumor cell, RAW264.7, and P388D1 while KML-C had a very strong toxicity. This non-toxic effect was observed also by in-vivo study. Both humoral and cellular immunities were observed ; the antibody titer was increased when the mice were immunized with B-chain used as adjuvant like Freund's adjuvant, indicating that B chain of mistletoe lectin alone might be used for adjuvant; it also increased DTH in cellular immunity. These results suggest that B-chain of KML-C might be used for adjuvant used for the production of antibody or vaccine with less toxicity.

• BMB Reports
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• v.55 no.3
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• pp.142-147
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• 2022

Human pluripotent stem cells (PSCs) have been utilized as a promising source in regenerative medicine. However, the risk of teratoma formation that comes with residual undifferentiated PSCs in differentiated cell populations is most concerning in the clinical use of PSC derivatives. Here, we report that a monoclonal antibody (mAb) targeting PSCs could distinguish undifferentiated PSCs, with potential teratoma-forming activity, from differentiated PSC progeny. A panel of hybridomas generated from mouse immunization with H9 human embryonic stem cells (hESCs) was screened for ESC-specific binding using flow cytometry. A novel mAb, K312, was selected considering its high stem cell-binding activity, and this mAb could bind to several human induced pluripotent stem cells and PSC lines. Cell-binding activity of K312 was markedly decreased as hESCs were differentiated into embryoid bodies or by retinoic acid treatment. In addition, a cell population negatively isolated from undifferentiated or differentiated H9 hESCs via K312 targeting showed a significantly reduced expression of pluripotency markers, including Oct4 and Nanog. Furthermore, K312-based depletion of pluripotent cells from differentiated PSC progeny completely prevented teratoma formation. Therefore, our findings suggest that K312 is utilizable in improving stem cell transplantation safety by specifically distinguishing residual undifferentiated PSCs.