• Journal of Marine Bioscience and Biotechnology
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• v.1 no.4
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• pp.298-304
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• 2006

In this study, we establish a thin-layer chromatography (TLC) immunostaining method for detecting starfish gangliosides. A new monoclonal antibody (MAb) against AG-2, the major gangliosides molecular species of Acanthaster planci, was produced by fusing hybridoma with splenocytes immunized to liposomal AG-2. BALB/c male mice were injected with liposomal AG-2 antigen, and immunized. Their splenocytos were isolated and fused with hypoxanthine-aminopterine-thimidine (HAT)-sensitive mouse myeloma cells. Hybridomas producing MAb reactive to AG-2 were cloned using the limited dilution method. Established hybridomas were cultured in eRDF medium. Crude MAb produced from clone 8D4 was purified with a magnesium pyrophosphate column. Enzyme immunoassay and TLC immunostaining of AG-2 were performed using the purified MAb. Structurally related gangliosides did not cross-react with anti-AG-2 antibodies. The detection limit of TLC immunostaining was 50 ng of AG-2. The newly established immunostaining method was further developed for detecting AG-2 distribution and qualitative analysis in tissues and/or organs. Our results show that the majority of AG-2 is present in the stomach of male A. planci, while AG-2 is distributed not only in the stomach but also in the the pyloric caeca of female A. planci.

• Animal cells and systems
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• v.10 no.4
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• pp.227-231
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• 2006

Micro-deletions at specific loci of the Y chromosome have been observed frequently in male infertility patients, suggesting that genes in these regions are involved in male germ cell development. DAZ is a representative male infertility gene at the AZFc locus of the Y chromosome. Since DAZ contains an RNA binding motif along with so-called a DAZ domain, it was proposed to participate in RNA metabolism during spermatogenesis. A mouse gene homologous to the human DAZ gene has been cloned and named Dazl (DAZlike). Dazl is autosomal and expressed in the testis and also at a low level in the ovary. Male mice homozygous for the Dazl null allele have small testes with a few spermatogonia and almost complete absence of germ cells beyond the spermatogonial stage, suggesting the requirement of Dazl for entry or progression through meiosis. However, its exact cellular functions have not been understood yet. In order to investigate cellular functions of Dazl, we decided to isolate candidate interacting protein genes of the mouse Dazl, using yeast two-hybrid screening. A number of candidate Dazlinteracting proteins have been isolated, such as Bprp, Acf, Hgs, Murr1, Nbak3 and Ranbp9, but dynein light chain 1 (Dlc1) was most predominant. A strong interaction of Dazl with Dlc1 suggests that Dazl might function as an mRNA adaptor to the dynein motor complex.

• International Journal of Oral Biology
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• v.43 no.1
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• pp.5-11
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• 2018

Recent findings indicate that Type 2 taste receptors (T2Rs) are expressed outside the gustatory system, including in the gastrointestinal tracts and the exocrine glands, such as the submandibular (SM), parotid (P), lacrimal (L) glands and pancreas (PC). Specifically, T2Rs are found in some of the gastrointestinal endocrine cells, and these cells secreted peptide hormones in response to stimulation by bitter-tasting compounds. The results show that T2Rs may have significant physiological roles besides bitter taste reception. The functions of the T2Rs in the exocrine glands remain poorly understood. An expression levels analysis of T2Rs will help to determine those functions in the exocrine glands. The expression levels of the T2Rs in the exocrine glands were discovered via the qPCR. C57BL/6J mice of 42~60-day-old were used. Messenger RNAs were extracted from S, P, L and PC. Cloned DNAs were synthesized by reverse transcription. Quantitative PCRs were performed using the SYBR Green method. The expression levels of the T2Rs were calculated as relative expression levels to that of the GAPDH. The statistical significance among the observed exocrine glands was tested using the variance analysis (ANOVA test). Tas2r108, out of murine 35 T2Rs, was the most highly expressed in every observed exocrine gland. This finding was similar to previous results from tongue papillae, but the expression levels were lower than those of the tongue papillae. Tas2r137 of SM, P, L and PC were expressed a little lower than that of tongue papillae. The T2Rs in the exocrine glands may play slightly different roles from those in the tongue. We suggest that physiological studies such as a patch clamp and functional $Ca^{2+}$ imaging of acinar cells are necessary for understanding the Tas2r108 functions.

• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.719-727
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• 2011

Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) antigenically diverse neurotoxins (BoNTs). BoNTs are the most poisonous substances known to humans, with a median lethal dose ($LD_{50}$) of approximately 1 ng/kg of body weight. Owing to their extreme potency and lethality, they have the potential to be used as a bioterrorism agent. The mouse bioassay is the gold standard for the detection of botulinum neurotoxins; however, it requires at least 3-4 days for completion. Attempts have been made to develop an ELISA-based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. The present study was designed using a synthetic gene approach. The synthetic gene encoding the catalytic domain of BoNT serotype B from amino acids 1-450 was constructed with PCR overlapping primers (BoNT/B LC), cloned in a pQE30 UA vector, and expressed in an E. coli M15 host system. Recombinant protein production was optimized at 0.5 mM IPTG final concentration, 4 h post induction, resulting in a maximum yield of recombinant proteins. The immunogenic nature of the recombinant BoNT/B LC protein was evaluated by ELISA. Antibodies were raised in BALB/c mice using various adjuvants. A significant rise in antibody titer (p<0.05) was observed in the Alum group, followed by the Titermax Classic group, Freund's adjuvant, and the Titermax Gold group. These developed high-titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.

• The Journal of the Korean Society for Microbiology
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• v.22 no.3
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• pp.197-208
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• 1987

Chlamydia trachomatis has now shown that this interesting intracellular parasite is a cause of nongonococcal urethritis, infantile pneumonia, pelvic inflammatory disease and epididymitis, in addition to lymphogranuloma venerum and inclusion conjunctivitis. There are several diagnostic methods for C. trachomatis, but the method using monoclonal antibody is the most sensitive and specific. The hybride cell were prepared by fusion of myeloma cell($P_3X_{63}\;Ag_8{\cdot}V_{653}$) of mouse and lymphocyte of mouse(BALB/c) that were immunized with formalin killed C. trachomatis serotype D. The cell mixtures after fusion were dispensed into 640 wells of the 96 well culture plates and continuously cultured in HAT medium for 2 weeks. The supernatants of culture media in 83(13%) wells were reacted with C. trachomatis, which were determined by enzyme-linked immunosorbent assay in 96 well microplate. The clones that secreted antibody to C. trachomatis were cloned by limiting dilution. Only six monoclones secreted antibody to C. trachomatis. The antibody titer of ascitic fluid that collected from same BALB/c mice bearing hybridoma cells was above 1:100,000. These monoclonal antibodies that were IgG reacted with elementary and reticulate bodies of all serotypes(Ba, D, E, F, G, H, J and LGV type-I) using ELISA and indirect immunofluorescence stain, but there were no cross reaction with other bacteria(coagulase negative Staphylococcus, Proteus and E. coli). We concluded these six monoclones secreted the same monoclonal antibody to C. trachomatis. The sensitivity and specificity of the monoclonal antibody compared with Microtrak(confirmatory test of C. trachomatis, Syva) was 100%, respectively.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.19-19
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• 2001

Mammalian urothelium undergoes unique membrane specialization by making the asymmetric unit membrane (AUM) that is covered with the apical cell surface during terminal differentiation. The AUM contains several major integral membrane proteins including uroplakin Ia, Ib, II and III. The genes for uroplakins have been cloned from humans and mice, but not from porcine. In this study, we report the cloning of the UPII genomic DNA, which codes for the full length open reading frame for the uroplakin II protein. The deduced amino acid sequence encodes of a hydrophobic NH$_2$-terminal peptide, a prosequence, and a mature protein. The prosequence contains three potential N-glycosylation sites and a RGRR cleavage site that may be involved in uroplakin II processing and maturation. Northern and immunohistochemistry analyses showed that the porcine UPII gene is only expressed in urothelium and that the protein was specifically localized in urothelial superficial cells. A 2kb of upstream in the promoter sequence contains multiple transcription factor binding sites, including GC-box, SPI, AP2, and GATA-box sites, but not for TATA or CAAT-box sequences. Comparison of the porcine UPII promoter sequence with that of the murine by MEME system presented two conserved motifs, suggesting a cis-acting regulatory role for the conserved sequences. Sequence homology between two species in motif A and B was 79% and 80% respectively, although their relative locations were different. During the gestation, mouse bladder at estrus stages and day 10 after parturition showed higher UPII expression, while showed lower expression at peri-implantation stage. Taken together, our results showed that the porcine UPII gene was expressed highly and specifically in the bladder urothelium and that steroid hormones for implantation changed the expression of UPII in the bladder, although the biological significance of UPII remains to be not determined.

• BMB Reports
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• v.38 no.6
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• pp.703-708
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• 2005

We cloned and expressed human pyridoxal-5'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin $B_6$, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin $B_6$ abnormalities.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.95-95
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• 1997

Among the various receptor molecules discovered so far the ${\beta}$2-adrenergic receptors have been regarded as excellent model systems for the so called 7 transmembrane helix receptor and have been the focus of extensive studies. For the analysis of receptor structure and function a monoclonal antibody plays a crucial role, thus providing useful tools for the study of receptor. However, because of the minute quantity of receptor molecules which could be obtained from natural sources, the generation of specific monoclonal antibody against receptor molecules from the purified receptors has been regarded as virtually impractical in consideration of cost and experimental times. The purpose of the present study was to generate and characterize a monoclonal antibody against human ${\beta}$2-adrenergic receptor. For the production of antibody, C-terminal regions of the human ${\beta}$2-adrenergic receptor was produced as a fusion protein with Glutathion S-transferase (GST) in E. Coli. The expression of the fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein was purified to an apparent homogeniety by affinity chromatography with Glutathion Sepharose CL-4B and used as an antigen for the immunization of BALB/C mice. The Production of monoclonal antibody was achieved by fusion of the immunized spleen cells and SP/2-0 myeloma cells. Positive hybridomas were screened by ELISA and were cloned by two consecutive rounds of limiting dilution. The monoclonal antibody produced in this study (mAb${\beta}$C02) was IgM type and purified by immunoaffinity chromatography using anti-mouse IgM agarose as an affinity matrix. MAb${\beta}$C02 showed strong and specific immunoreactivity against both the fusion protein and human ${\beta}$2-adrenergic receptor in ELISA and Western blot. The molecular weight of immunoreactive band was 64 kDa and exactly coincided with the previously reported molecular weight of ${\beta}$2-adrenergic recepters. The results of the present study suggest that mAb${\beta}$C02 may be used for the study of receptor function and regulation in normal or nonphysiological status.

• IMMUNE NETWORK
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• v.3 no.2
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• pp.126-135
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• 2003

Background: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the $65^{th}$ residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. Methods: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains $57{\sim}80^{th}$ amino acid residues of TP domain. After isolation of mRNA of heavy-variable region ($V_H$) and light-chain variable region ($V_L$) from the spleen of the immunized mouse, DNA of $V_H$ and $V_L$ were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. scFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. Results: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. Conclusion: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.97-97
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• 2002

Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2${\times}$10$\^$6//${\mu}\ell$. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6${\times}$10$\^$6//${\mu}\ell$. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.