• IMMUNE NETWORK
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• v.3 no.1
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• pp.23-28
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• 2003

Background: Intracellular antibody specific to hepatitis B virus X protein (HBx) might be useful for studying the role of HBx in hepatocellular carcinogenesis and HBV replication. Methods: With variable region genes for H7 monoclonal anti-HBx Ab, we constructed a vector for bacterial expression of single chain Ab (scFv) and a vector for eukaryotic cell expression of it. The expression of H7 scFv and its binding activity against HBx was examined by immunoblotting and immunofluorescence microscopy. Results: H7 scFv expressed in bacterial cells retained reactivity to HBx. We demonstrated its intracytoplasmic expression in CosM6 eukaryotic cells. Conclusion: This is the first study showing the expression of intracellular anti-HBx Ab in eukaryotic cells. H7 scFv may be a good tool to study the function of HBx in HBV infection.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.579-586
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• 2005

When subjected to hyperosmotic pressure by NaCl addition, H69K-NGD transfectoma, like KR12H-2 transfectoma, displayed decreased specific growth rate (${\mu}$) and increased specific antibody productivity ($q_{Ab}$): Elevation of medium osmolality from 280 mOsm/kg to 415 mOsm/kg decreased ${\mu}$ by $79\%$ in batch cultures of H69K-NGD transfectoma, while it increased $q_{Ab}$ by $103\%$. However, unlike KR12H-2 tranfectoma, enhanced $q_{Ab}$ of H69K-NGD transfectoma at hyperosmolalities was not due to elevated levels of Ig mRNAs. In hyperosmotic cultures of H69K-NGD transfectoma, heavy-chain mRNA per cell was not enhanced with increasing osmolality. Hyperosmotic pressure was found to preferentially enhance immunoglobulin (Ig) translation rates of H69K-NGD transfectoma. However, under hyperosmotic pressure, the translation rate of Ig polypeptides was not enhanced as much as $q_{Ab}$. This result suggests that hyperosmotic pressure also influences the post-translational process. Taken together, the results obtained show that intracellular response of transfectomas to hyperosmotic pressure, in regard to the main intracellular steps of the antibody secretory pathway, is cell-line dependent.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.14-25
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• 2001

Background: Bone marrow stromal cells (BMSCs) express many cell surface molecules, which regulate the proliferation and differentiation of immune cells within the bone marrow. Methods: To identify cell surface molecules, which can regulate cell proliferation through cell interaction, monoclonal antibodies (MoAbs) against BMSCs were produced. Among them, 1H8 MoAb, which recognized distinctly an 80 kDa protein, abolished myeloma cell proliferation that was induced by co-culturing with BMSCs. Results: IL-6 gene expression was increased when myeloma or stromal cells were treated with 1H8 MoAb. In addition, the expression of IL-6 receptor and CD40 was up-regulated by 1H8 treatment, suggesting that the molecule recognized by 1H8 MoAb is involved in cell proliferation by modulating the expression of cell growth-related genes. Myeloma cells contain high levels of reactive oxygen species (ROS), which are related to gene expression and tumorigenesis. Treatment with 1H8 decreased the intracellular ROS level and increased PAG antioxidant gene concomitantly. Finally, 1H8 induced the tyrosine phosphorylation of several proteins in U266. Conclusion: Taken together, 1H8 MoAb recognized the cell surface molecule and triggered the intracellular signals, which led to modulate gene expression and cell proliferation.

• Parasites, Hosts and Diseases
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• v.42 no.4
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• pp.185-193
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• 2004

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of $Ca^{2+}$ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and $Ca^{2+}$ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the $Ca^{2+}$ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular $Ca^{2+}$ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.93-93
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• 1997

Analysis of protein is often frustrated by the inability to isolate large amounts of purified protein from a native source. To overcome this problem, fusion protein expression systems such as pGEX system have been widely used. Using pGEX system, the desired protein could be easily obtained in a large amount in E. coli, and then the fusion protein could be used for the study of the function of the given protein. To analyze and purify the GST fusion protein, anti-GST antibody could be used as one of the system of choice. However, the production and characterization of monoclonal anti-GST antibody has not been studied extensively yet. To produce monoclonal anti-GST antibody, GST was purified from E. coli transformed with pGEX-cs, one of the pGEX system and was used as an antigen. The monoclonal antibody was produced by fusion of the immunized spleen cells with SP2-0 myeloma cells. The antibody was characterized by ELISA, western blotting, etc. The monoclonal antibody produced in this study (mAb-GSTA) showed strong and specific immunoreactivity against not only GST but also GST-fusion proteins. Also, mAb-GSTA was successfully used for the immunoaffinity purification of the GST ${\beta}$-Rc.-third intracellular-loop fusion protein. The results of the present study suggest that mAb-GSTA may be used for the identification and purification of GST fusion proteins.

• Journal of Physiology & Pathology in Korean Medicine
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• v.16 no.4
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• pp.680-683
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• 2002

Bovine aortic endothelial cells(BAEC) produce two variant forms of laminin with a subunit composition of AB1B2 and A'B1B2. Analyses of the intracellular assembly of these subunits revealed that the B1B2 dimer formed first, and that A or A' joined to form the AB1B2 or A'B1B2 trimer. Angiostatic steroids shifted the relative size of the A and A' monomer pool in BAEC, and competition between the A and A' subunits in joining the B1B2 dimer produced AB1B2 and A'B1B2 in different ratios. This result suggests that subunit replacement is the general mechanism for producing laminin variants by various cells for tissue morphogenesis. When laminin subunits in BAEC were cross-linked with dithio-bis-succinimidylpropionate(DSP) and immunoprecipitated with anti-Iaminin antiserum, monomeric A,A',B1 and B2 monomers and the B1B2 dimer migrated as extremely large molecules in sodium dodecyl sulfate gel electrophoresis under nonreducing conditions. When the crosslinking disulfide bonds were cleaved under reducing conditions, they migrated as the usual subunits. This result suggests that molecular chaperones were involved in the process of the assembly and replacement of laminin subunits.

• The Korean Journal of Zoology
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• v.19 no.1
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• pp.25-32
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• 1976

To observe the changes of mucosubstances of the mucous secreting cells, stomach tissues in frog tadpoles at each stage of metamorphosis were fixed in 10% buffered formalin at $4^{\circ}C$, embedded in paraffin, sectioned to 4 $\mu$m thickness and stained with periodic acid-Schiff(PAS) and alcian blue (AB) of pH 2.5 and pH 1.0. The results obtained were as follows: 1. The reactivities of the surface mucous cells, which exhibited strong PAS-positivity and weak alcianophilia at both pH 2.5 and 1.0, were not changed in metamorphosis stages and the intracellular contents of neutral mucosubstances in the surface mucous cells increased significantly in XXIV and XXV stages of metamorphosis. 2. In the foveolar mucous cells, which appear after metamorphosis XXI, the staining reactivities to PAS, AB of pH 2.5 and 1.0 were the same as that of surface mucous cells during metamorphosis and the alcianophilia were stronger at pH 1.0 than at pH 2.5. 3. THe mucous neck cells, which appear after metamorphosis XXIV, exhibited a strong PAS-positive reaction and weak alcianophilia at metamorphosis XXIV but at metamorphosis XXV weak reactivity to PAS and strong alcianophilia at pH 1.0.

• Journal of Microbiology and Biotechnology
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• v.12 no.4
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• pp.628-634
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• 2002

A microfilament-based motility in Toxoplasma gondii (T. gondii) Is involved in host cell invasion, yet the exact mechanism has not yet been determined. Accordingly, the current study examined the localization of actin and tubulin in T gondii using immunofluorescent (IF) and immunogold staining for electron microscopy. Indirect immunofluorescence (IF) staining using anti-actin and anti-tubulin monoclonal antibodies (mAbs) revealed localization of fluorescence on the entire surface of the tachyzoites. The actin in T. gondii was observed by immunogold staining, and the gold particles were seen on the surface, especially at the anterior end and in the cytoplasm of the parasite. However, there were no gold particles in the nucleus, rhoptries, and dense granules. The tubulin in T gondii was located on the surface and in the cytoplasm of the tachyzoites in the extracellular parasite, compared with anterior part of tachyzoites in the intracellular parasite. The antigens of T gondii recognized by anti-actin mAb were 107 kDa, 50 kDa, 48 kDa, and 40 kDa proteins, while those recognized by anti-tubulin mAb were 56 kDa, 52 kDa, and 34 kDa proteins. Tachyzoites of T gondii pretreated with the actin inhibitor, cytochalasin D (20 $\mu\textrm{g}$/ml), and tubulin inhibitor, colchicine (2$\times$10$\^$-6/ M), for 30 min at 37$\^{C}$ were used to infect the isolated mouse macrophages (tachyzo ites:macrophage=2:1). Pretreatment with the inhibitors resulted in lower multiplication of tachyzoites within the macrophages than in the untreated group 18 h post infection (p<0.05). Therefore, the present results suggest that actin and tubulin appear to be involved in the invasion of and multiplication in host cells.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.736-744
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• 2023

The introduction of the toxT-139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in most Vibrio cholerae strains. Such V. cholerae strains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in these V. cholerae strains is secreted into the culture medium. In this study, V. cholerae strains that can express intracellular CTB under the control of the toxT-139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking the ctxAB promoter to ctxB without ctxA and confirmed CTB expression from the plasmid in V. cholerae containing the toxT-139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids-from the 7^th to the 20^th amino acid-of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructed V. cholerae strains in which chromosomal ctxAB is replaced by ntrctxB or ntrctxB-dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.

• Animal Bioscience
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• v.36 no.7
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• pp.1120-1129
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• 2023

Objective: This study aimed to determine the protective efficacy of Buddha's Temple (BT) extract against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in Gallus gallus chicken embryo fibroblast cell line (DF-1) and its effects on the cell lipid metabolism. Methods: In this experimental study, Gallus gallus DF-1 fibroblast cells were pretreated with BT 10^-7 for 24 hours, followed by their six-hour exposure to t-BHP (100 μM). Water-soluble tetrazolium salt-8 (WST-8) assays were performed, and the growth curve was computed. The intracellular gene expression changes caused by BT extract were confirmed through quantitative polymerase chain reaction (qPCR). Flow cytometry, oil red O staining experiment, and thin-layer chromatography were performed for the detection of intracellular metabolic mechanism changes. Results: The WST-8 assay results showed that the BT pretreatment of Gallus gallus DF-1 fibroblast cell increased their cell survival rate by 1.08%±0.04%, decreased the reactive oxygen species (ROS) level by 0.93%±0.12% even after exposure to oxidants, and stabilized mitochondrial activity by 1.37%±0.36%. In addition, qPCR results confirmed that the gene expression levels of tumor necrosis factor α (TNFα), TIR domain-containing adapter inducing IFN-beta (TICAM1), and glucose-regulated protein 78 (GRP78) were regulated, which contributed to cell stabilization. Thin-layer chromatography and oil red O analyses showed a clear decrease in the contents of lipid metabolites such as triacylglycerol and free fatty acids. Conclusion: In this study, we confirmed that the examined BT extract exerted selective protective effects on Gallus gallus DF-1 fibroblast cells against cell damage caused by t-BHP, which is a strong oxidative inducer. Furthermore, we established that this extract significantly reduced the intracellular ROS accumulation due to oxidative stress, which contributes to an increase in poultry production and higher incomes.