• Asian-Australasian Journal of Animal Sciences
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• v.33 no.3
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• pp.515-524
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• 2020

Objective: Human mesenchymal stromal cells (MSCs) exhibit variable differentiation potential and can be divided accordingly into distinct subpopulations whose ratios vary with donor age. However, it is unknown whether the same is true in pigs. This study investigated MSC subpopulations in miniature pig and compared their characteristics in young (2 to 3 months) and adult (27 to 35 months) pigs. Methods: Osteogenic, chondrogenic, and adipogenic capacity of isolated MSCs was evaluated by von Kossa, Alcian blue, and oil red O staining, respectively. Cell surface antigen expression was determined by flow cytometry. Proliferative capacity was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Expression of marker genes was detected by quantitative real-time polymerase chain reaction. Results: Porcine MSCs comprised cells with trilineage and bilineage differentiation potential (tMSCs and bMSCs, respectively) and non-differentiating stromal cells (NDSCs). The tMSC and bMSC fractions were smaller in adult than in young pigs (63.0% vs 71.2% and 11.6% vs 24.0%, respectively, p<0.05); NDSCs showed the opposite trend (25.4% vs 4.8%; p<0.05). Subpopulations showed no differences in morphology, cell surface antigen expression, or proliferative capacity, but octamer-binding transcription factor 4 (OCT4) expression was higher in tMSCs than in bMSCs and NDSCs (p<0.05), whereas sex determining region Y-box 2 (SOX2) expression was higher in tMSCs and bMSCs than in NDSCs (p<0.05). Aging had no effect on these trends. Conclusion: Porcine MSCs comprise distinct subpopulations that differ in their differentiation potential and OCT4 and SOX2 expression. Aging does not affect the characteristics of each subpopulation but alters their ratios.

• Molecules and Cells
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• v.42 no.12
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• pp.850-857
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• 2019

The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa, has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand-binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF-oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.

• Childhood Kidney Diseases
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• v.20 no.1
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• pp.23-28
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• 2016

Purpose: We investigated whether serum levels of insulin growth factor-1 (IGF-1) and insulin growth factor binding protein-3 (IGFBP-3) are valuable in predicting clinical outcomes or are correlated with other laboratory findings in children with Henoch-$Sch{\ddot{o}}nlein$ purpura (HSP). Methods: We examined 27 children who were consecutively admitted to our hospital with HSP between January 2011 and February 2012. Blood tests (C-reactive protein, white blood cell count, platelet count, erythrocyte sedimentation rate, albumin, immunoglobulin A, complement C3, antineutrophil cytoplasmic antibody, IGF-1, IGFBP-3) and urine tests were performed upon admission. IGF-1 and IGFBP-3 were resampled in the recovery phase. Controls included 473 children whose IGF-1 and IGFBP-3 were sampled for evaluating their growth, at the outpatient department of pediatric endocrinology in our hospital. IGF-1 and IGFBP-3 were compared between the HSP children and controls, and between the acute and recovery phases in HSP children. The ability of these values to predict clinical outcomes including renal involvement was analyzed using bivariate logistic regression analysis (BLRA). Results: IGF-1 and IGFBP-3 were not different between the HSP children and controls ($148.7{\pm}117.6$ vs. $69.2{\pm}96.9$, P=0.290: $3465.9{\pm}1290.9$ vs. $3597.2{\pm}1,127.6$, P=0.560, respectively). There was no significant difference in IGF-1 or IGFBP-3 between acute and recovery phases. Based on the BLRA, no variable, including IGF-1 and IGFBP-3, could predict clinical outcomes including the presence of nephritis Conclusion: We concluded that IGF-1 and IGFBP-3 do not predict clinical outcomes of HSP, including renal involvement, in this study.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.537-546
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• 2015

Classical swine fever (CSF) is a highly contagious disease of pigs caused by CSF virus (CSFV). E2 is the major viral envelope protein of immune dominance that induces neutralizing antibodies and confers protection against CSFV infection. The B/C domains of E2 are variable among CSFV isolates, which could affect immunogenicity and binding to antibodies. We attempted to characterize the epitope recognized by a monoclonal antibody 2B6 (mAb-2B6) raised against the E2 B/C domains of the vaccine C-strain and to examine if mutations in the epitope region would affect antibody binding and viral neutralization. The epitope specific for mAb-2B6 recognition is linear, spanning five residues ⁷⁷⁴DGXNP⁷⁷⁸ in the B/C domains. The residue N777 is indispensable for the specificity. The epitope exists only in group 1 strains, but not in those of group 2. The recombinant viruses containing individual mutations on the epitope region lost the reactivity to mAb-2B6. The mutant virus RecC-N777S had low replication potential, about 10-fold decrease in the yield of progeny virus particles, whereas the mutant virus RecC-P778A reverted to proline upon continuous passaging. The mutations on the mAb-2B6 epitope region did not affect neutralization by anti-C-strain polyclonal sera from pigs. Deletion from aa774 covering the mAb-2B6 epitope, but not that from aa781, also affected binding with the polyclonal antibodies from vaccinated pigs, although the major binding region for the vaccinated antibodies is aa690-773.

• Biomedical Science Letters
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• v.10 no.3
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• pp.211-217
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• 2004

For discrimination of ductal and ascinar cells, we isolated a single-chain variable domain fragment (scFv) antibody against ductal cells of salivary gland using phage display technique. From the spleen of a mouse immunized with ductal cell lysate, total RNA was prepared and used as a template for cDNA synthesis of antibody genes. The scFv genes were constructed with variable domain genes of heavy and light chain and were introduced into pCANTAB5E to construct phage scFv library. The phage particles specific for acinar cells were screened by subtraction using immunotubes coated with acinar and ductal cell lysate and enzyme-linked immunoabsorbance assay (ELISA). The characteristics of the scFv were determined by immunohistochemistry (IHC) and the result indicated that the isolated scFv has the specificity against ductal cells of salivary glands and tubules of kidney. And the scFv has an unique binding activity specific for Hashimoto's thyroiditis. The nucleotide sequence of isolated scFv gene was determined and revealed that V/sub H/ belongs to the mouse H-chain family subgroup IB and V/sub L/ to the mouse L-chain family subgroup III.

• Korean Journal of Veterinary Service
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• v.34 no.3
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• pp.201-208
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• 2011

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a pathogen of a variety of infections in horse. We studied biochemical and molecular characteristics of S. zooepidemicus isolated from the genital tract of Thoroughbred mares in Korea. Seventy-nine isolates were identified as S. zooepidemicus by biochemical and PCR method from 374 horses. The biochemical characteristics of S. zooepidemicus isolates were positive reaction of lactose and sorbitol. However, S. zooepidemicus isoltes were negative reaction of inulin, mannitol, raffinose, trehalose, aesculin hydrolysis, growth in 6.5% NaCl and variable reaction of maltose. Epidemiological investigations of S. zooepidemicus isolates were performed by fragment analysis of SzP (S. zooepidemicus protective protein) gene, CNE (collagen binding protein) gene and ISR (16s rRNA intergenic spacer region) gene using ABI Prism $3,130{\times}1$ Genetic Analyzer System. All isolates were shown single amplification size of 906 bp in CNE gene, but SzP and ISR gene were shown variable patterns of fragment size. The characteristics of S. zooepidemicus investigated in this study will be very useful for the prevention of infection and the studies of epidemiologic characteristics of S. zooepidemicus, causing the severe economic losses due to reproductive failures.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1186-1190
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• 2008

Single-chain Fv (scFv) antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells owing to the important biological roles of c-Met in the proliferation of malignancies. Here, we show that the productivity of scFv against c-Met in Escherichia coli is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders (i.e., $V_L$-linker-$V_H$ and $V_H$-linker-$V_L$), expressed them in the cytoplasm of E. coli trx/gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with $V_H/V_L$ orientation was more than five times higher than that with $V_L/V_H$ format. Coexpression of DsbC enhanced the yield of soluble amounts of anti-c-Met scFv protein for both constructs. The purified scFv antibodies of the two different formats exhibited almost the same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with $V_H/V_L$ or $V_L/V_H$ formats and obtained similar results to the case of scFv antibodies.

• Journal of Microbiology and Biotechnology
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• v.21 no.5
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• pp.529-535
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• 2011

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.571-578
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• 2007

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The $IC_{50}$ value by IC-ELISA with scFv antibody was 4.8ng/ml, compared with 1.6ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.

• BMB Reports
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• v.51 no.11
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• pp.572-577
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• 2018

Recent studies showed that the PD-1/PD-L1 checkpoint blockade is a dramatic therapy for melanoma by enhancing antitumor immune activity. Currently, major strategies for the PD-1/PD-L1 blockade have mainly focused on the use of antibodies and compounds. Seeking an alternative approach, others employ endogenous proteins as blocking agents. The extracellular domain of PD-1 (ePD1) includes the binding site with PD-L1. Accordingly, we constructed a PD-1-based recombinantly tailored fusion protein (dFv-ePD1) that consists of bivalent variable fragments (dFv) of an MMP-2/9-targeted antibody and ePD1. The melanoma-binding intensity and antitumor activity were also investigated. We found the intense and selective binding capability of the protein dFv-ePD1 to human melanoma specimens was confirmed by a tissue microarray. In addition, dFv-ePD1 significantly suppressed the migration and invasion of mouse melanoma B16-F1 cells, and displayed cytotoxicity to cancer cells in vitro. Notably, dFv-ePD1 significantly inhibited the growth of mouse melanoma B16-F1 tumor cells in mice and in vivo fluorescence imaging showed that dFv-ePD was gradually accumulated into the B16-F1 tumor. Also the B16-F1 tumor fluorescence intensity at the tumor site was stronger than that of dFv. This study indicates that the recombinant protein dFv-ePD1 has an intensive melanoma-binding capability and exerts potent therapeutic efficacy against melanoma. The novel format of the PD-L1-blocked agent may play an active role in antitumor immunotherapy.