• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.96-96
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• 1993

간부분 절제술을 하지 않는 비수술적 방법으로서 D-galactosamine을 이용한 중기발암성 시험의 개발을 목적으로 F344 수괵 랫드를 이용하여 본 실험을 수행하였다. 실험 I 에서는 실험방법에 따라 3가지 모델로 구분하고, 각 모델에 처치군과 대조군을 두었다. 모델 1 에서는 실험개시시에 diethylinitrosamine (DEN)을 200 mg/kg body weight로 복강내로 1회 투여하고, 실험개시후 2및 5주에 D-galactosamine을 300 mg/kg body weight로 복강내로 각각 1회 투여하였다. 처치군에는 실험개시후 2주부터 6주간 2-acetylaminofluorene을 0.01%로 혼합한 사료를 급여하였으며, 대조군에는 기초사료를 계속 급여하였다. 모델 2에서는 모델 1의 4주차까지의 처치를 2회 반복하였다. 모델 3은 간부분 절제술을 하는 DEN-PH (diethylnitrosamine-partial hepatectomy) 모델과 같은 방법으로 처치하였다. 사육기간 중 매주 체중 및 사료소비량을 측정하였고, DEN 투여후 8주에 전동물을 부검하여 적출한 간의 중량을 측정하고, glutathione S-transferase placental form (GST-P) 양성 foci에 대한 면역조직화학적 염색표본을 만들어 GST-P 양성 foci의 수 및 면적을 측정하였다. 실험 II에서는 모델 1의 방법으로 phenobarbital(PB), 3-methylcholanthrene (3-MC), n-ethyl-n'-nitro-n-nitrosoguanidine 및 3,3'-diaminobenzidine외 GST-P 양성 foci의 발현정도를 조사하였다. 실험 I의 결과, 모델 1이 정상적인 체중 증가를 보여주었으며, 간조직의 GST-P 양성 foci 의 발현율이 가장 좋았다. GST-P 양성 foci의 면적은 큰것 부터 미상엽, 내측우엽, 외측우엽의 순으로 나타났으나 foci의 수는 모델별로 다르게 나타났다. 실험 II의 PB 투여군과 3-MC 투여군에서 GST-P 양성 foci의 수 및 면적의 유의성 있는 증가가 관찰되었다. 이와 같은 결과로 볼때, 비수술적 방법인 D-galactosamine 을 이용한 중기 발암성 검색법은 간부분 절제술을 이용한 중기발암성 검색법에 비하여 GST-P 양성foci의 발현능력이 동등하거나 더 우수하였으며, 간 및 간이외 장기의 발암물질에 대한 발암성 검색에 보다 유용할 것으로 생각된다.

• Biomolecules & Therapeutics
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• v.5 no.4
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• pp.344-350
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• 1997

The purpose of the present study was to produce and characterize a monoclonal antibody against human ${\beta}_2$-adrenergic receptor. Male BALB/c mice were immunized with glutathione S-transferase (GST) fusion protein of the C-terminal portion of the human ${\beta}_2$-adrenergic receptor which was expressed in E.Coli. The immunized splenocytes were fused with myeloma SP2/0-Agl4 cells. The resulting hybridomas were screened for the production of a monoclonal antibody which can recognize human ${\beta}_2$-adrenergic receptor, and then subcloned by limiting dilution. The resulting monoclonal antibody was named as mAb$\beta$CO2. The mono-clonal antibody $\beta$CO2 was determined as IgM subtype and then purified by anti-mouse IgM-agarose affinity chromatography. The results of ELISA, Western blot, and immunocytochemistry showed that mAb$\beta$CO2 recognized human ${\beta}_2$-adrenergic receptor in the ${\beta}_2$-adrenergic receptor-GST fusion protein and human spider-moid carcinoma cell line A431 with highly specific immunoreactivity, The monoclonal antibody $\beta$CO2 may provide useful tools for the study of the $\beta$-adrenergic receptor of human and other species including rats.

• Parasites, Hosts and Diseases
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• v.46 no.4
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• pp.209-216
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• 2008

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

• Korean Journal of Microbiology
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• v.29 no.6
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• pp.392-396
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• 1991

Mu d1(Ap lac) bacteriophage can be used to search for genes which are members of a common regulatory network without having to know the functions of the genes in advance. Aim was for obtaining the loci in the SOS network as well as temperature inducible loci. For this purpose, recA441 allele was used. This allele encodes a thermosensitive recA gene product; thus, the recA441 allele can be activated upon temperature upshift without by external DNA damage. Approximately 10, 000 colonies were screened, and then searched for the colonies which expressed .betha.-galactosidase higher level at 42.deg.C than at 30.deg.C. The strains identified fell into two dlasses; (i) ones in which the increased expression was $recA^{+}$ $lexA^{+}$ -dependent, that is, din(damage-inducible) genes which were due to the activation of recA441 allele and (ii) ones in which the increased expression was $recA^{+}$ $lexA^{+}$ -independent and only temperature-inducible, tin genes. Rough mapping position was obtained for these genes.

• Journal of Biosystems Engineering
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• v.30 no.6 s.113
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• pp.333-339
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• 2005

The pesticide is raising public interest in the world, because it causes damage to an environmental pollution and the human health remaining agricultural products and an ecosystem, in spite of the advantages. Particularly, each country restricts the residual pesticide and induces observance about the safety and usage standard so that they can control the amount of pesticide used and defend the safety of agricultural products. The habitual practice for the analysis of the residual pesticide depends on GC (gas chromatography), HPLC (high performance liquid chromatography) and GC/MS (gas chromatography/mass spectroscopy), which triturate the fixed quantity of samples, abstract and purify as a suitable organic solvent. These methods have the highly efficient in aspects of sensitivity and accuracy. On the other hand, they need the high cost, time consuming, much effort, expensive equipment and the skillful management. Carbofuran is highly toxic by inhalation and ingestion and moderately toxic by dermal absorption. As with other carbamate compounds, it is metabolized in the liver and eventually excreted in the urine. The half-life of carbofuran on crops is about 4 days when applied to roots, and longer than 4 days if applied to the leaves. This research was conducted to develop immunoassay for detecting carbofuran residue quickly on the basis of surface plasmon resonance and to evaluate the measurement sensitivity. Gold chip used was CM5 spreaded dextran on the surface. An applied antibody to Immunoassay was GST (glutathione-s-transferase). The association and the dissociation time were 176 second and 215 second between GST and carbofuran. The total analysis time using surface plasmon resonance was 13 minutes including regeneration time, on the other hand HPLC and GC/MS was 2 hours usually. The minimum detection limit of a permissible amount for carbofuran in the country is 0.1 ppm. The immunoassay method using surface plasmon resonance was 0.002 ppm.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.237-242
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• 2009

Pregnancy is a unique event in which a fetus develops in the uterus despite being genetically and immunologically different from the mother, and the underlying mechanisms remain poorly understood. To analyze the differential gene expression profiles in nonpregnant and 7 days post coitus (dpc) pregnant uterus of mice, we performed a global proteomic study by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The uterine proteins were separated using 2-DE, Approximately 1,000 spots were detected on staining with Coomassie brilliant blue. An image analysis using Melanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between pregnant and nonpregnant uterus. Twenty-one spots were identified as differentially expressed proteins, of which 10 were up-regulated proteins such as alpha-fetoprotein, chloride intracellular channel 1, transgelin, heat-shock protein beta-1, and carbonic anhydrase II, while 11 were down-regulated proteins such as X-box binding protein, glutathione S-transferase omega 1, olfactory receptor Olfr204, and metalloproteinase-disintegrin domain containing protein TECADAM. Most of the identified proteins appeared to be related with catabolism, cell growth, metabolism, regulation, cell protection, protein repair, or protection. Our results uncovered key proteins of mouse uterus involved in pregnancy.

• Korean Journal of Environmental Biology
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• v.26 no.2
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• pp.109-114
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• 2008

To evaluate the extent of Sea-Nine 211 (4,5-dichloro-2-n-octyl-3(2H) isothiazolone), one of the alternating organic booster compound used in antifouling paint to replace TBT, on marine benthic bivalve, we injected Sea-Nine 211 to clam, Pseudocardium sachalinensis, and then determined some xenobiotics metabolizing enzyme activities, especially EROD (ethoxyresorufin deethylase) and MROD (methoxyresorufin demethylase), in digestive gland during 4 day-exposure period. Moreover, the results were compared with those of TBT exposed clam. CYP1A1 dependant EROD activity in both the Sea-Nine 211 and the TBTC exposure groups showed no significant differences compared to those of the solvent control group. CYP1A2 dependant MROD activity in Sea-Nine 211 exposure group was significantly induced, but no significant difference was obtained in the TBTC exposure group. These results indicate that Sea-Nine 211 demonstrated a tendency to induce MROD activity, while TBTC inhibits the activities of this enzyme.

• Environmental Mutagens and Carcinogens
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• v.20 no.1
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• pp.26-33
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• 2000

Genetic polymorphisms of metabolizing enzymes to chemical carcinogens have been recognized as a major important host factors in human cancers. To datermine the frequencies of genotypes of CYP1A1 and GSTM1 metabolizing enzymes in healthy controls and head and neck cancer patients in Korean and to identify the relative high risk genotypes of these metabolizing enzymes to head and neck cancer, we have analyzed 133 head and neck cancer patients and corresponding healthy controls matched in age and sex using polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP). In analysis of CYP1A1, the Val/Val genotype of exon 7 polymorphism and m2/m2 genotype of Msp 1 polymorphism were associated with higher relative risks to head and neck cancers (Odds ratio : 2.34, 95% CI : 0.79-6.96 and 1.27, 95% CI : 0.59-2.73, respectively). In combined genotyping of CYP1A1 and GSTMI enzymes polymorphisms, the patients with Val/Val ad GSTM1(-), and m1/m21 and GSTM1(-) combined genotypes had higher relative risks than the patients with each base genotype of combined genotypes (Odds ratio : 4.57, 95% CI : 0.5-41.25 and 1.65, 95% CI L 0.73-3.77, respectively). These results sugget the combined genotyping of metabolizing enzymes could be useful for predicting individual genetic susceptibility and screening the high risk subpopulation to head and neck cancer in Korea.

• Journal of Ginseng Research
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• v.36 no.4
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• pp.418-424
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• 2012

This study focused on the enzymatic biotransformation of the major ginsenoside Rb1 into Rd for the mass production of minor ginsenosides using a novel recombinant ${\beta}$-glucosidase from Flavobacterium johnsoniae. The gene (bglF3) consisting of 2,235 bp (744 amino acid residues) was cloned and the recombinant enzyme overexpressed in Escherichia coli BL21(DE3) was characterized. This enzyme could transform ginsenoside Rb1 and gypenoside XVII to the ginsenosides Rd and F2, respectively. The glutathione S-transferase (GST) fused BglF3 was purified with GST-bind agarose resin and characterized. The kinetic parameters for ${\beta}$-glucosidase had apparent $K_m$ values of $0.91{\pm}0.02$ and $2.84{\pm}0.05$ mM and $V_{max}$ values of $5.75{\pm}0.12$ and $0.71{\pm}0.01{\mu}mol{\cdot}min^{-1}{\cdot}mg$ of $protein^{-1}$ against p-nitrophenyl-${\beta}$-D-glucopyranoside and Rb1, respectively. At optimal conditions of pH 6.0 and $37^{\circ}C$, BglF3 could only hydrolyze the outer glucose moiety of ginsenoside Rb1 and gypenoside XVII at the C-20 position of aglycon into ginsenosides Rd and F2, respectively. These results indicate that the recombinant BglF3 could be useful for the mass production of ginsenosides Rd and F2 in the pharmaceutical or cosmetic industry.

• Journal of Life Science
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• v.25 no.4
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• pp.385-389
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• 2015

In this study, it is reported that a large polyprotein can be stoichiometrically cleaved by the use of caspase-3-dependent proteolysis. Previously, it has been shown that the proteolytic IETD motif was partially processed when treated with caspase-3, while the DEVD motif was completely cleaved. The cleavage efficiency of the DEVD-based substrate was approximately 2.0 times higher than that of the IETD substrate, in response to caspase-3. Based on this, 3 protein genes of interest were genetically linked to each other by adding two proteolytic cleavage sequences, DEVD and IETD, for caspase-3. Particularly, glutathione-S transferase (GST), maltose binding protein (MBP), and red fluorescent protein (RFP) were chosen as model proteins due to the variation in their size. The expressed polyprotein was purified by immobilized metal ion affinity chromatography (IMAC) via a hexa-histidine tag at the C-terminal end, showing 93 kDa of a chimeric GST:MBP:RFP fusion protein. In response to caspase-3, cleavage products, such as MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa), were separated from a large precursor GST:MBP:RFP (93 kDa) via SDS-PAGE. The results obtained from this study indicate that a multi-protein can be stoichiometrically produced from a large poly-protein by using proteolytic recognition motifs, such as DEVD and IETD tetra-peptides, for caspase-3.