• Biomedical Science Letters
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• v.3 no.2
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• pp.89-94
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• 1997

The authors characterized the proteins of the crude antigen obtained from Clonorchis sinensis worm and excretory-secretory and billis from rabbits, experimentally infected for 3 months. Protein composition was observed after adding a cysteine proteinase inhibitor E-64 and a serine proteinase inhibitor PMSF, respectively. SDS-PAGE of the crude antigen from C. sinensis recovered from the infected rabbits, the crude antigen from the adult worm excretory-secretory, and the crude antigen from billis of the rabbits resolved 26, 27 and 19 profiles between 200-9 kDa, respectively. When E-64 supplemented 29, and 22 bands, respectively. More study should be carried out in the future on the immunological characteristics and the monoclonal antibody of the each antigen.

• Microbiology and Biotechnology Letters
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• v.29 no.2
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• pp.90-95
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• 2001

Kinetic Analysis of Cathepsin B Inhibitor Using a Spectrophotometric Assay. Han, Kil-Hwan and SangDal Kim*. Department of Applied MicrobioJ0f5Yt Yeungnam UniversitYt Kyongsan 77 2-749, Korea - The KHS 10, C4Hl10~6 formula produced from Streptomyces luteogriseus KT-] 0 effectively inhibited a lysosomal cysteine proteinase, cathepsin B. It inhibited the enzyme activity of cathepsin B competitively when the N a-CBZ-Llysine p-nitrophenyl ester HC] (CLN) was used as a substrate. The inhibition const:mt (Ki) of KHS 1 0 for cathepsin B detennined by spectrophotometeric assay was 430 nM. The effective inhibition of cathepsin B was observed at $25^{\circ}C$ :md pH 6.0. The cathepsin B inhibitor, KHSlO needed a preincubation of cathepsin B with the inhibitor for over 5 min. The KHS 10 preserved over 80% inhibition activity even after heat-treatment at $100^{\circ}C$ for ] hr.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.21
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• pp.9511-9515
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• 2014

Cholangiocarcinoma (CCA) is a cancer of the bile duct epithelial cells. The highest incidence rate of CCA with a poor prognosis and poor response to chemotherapy is found in Southeast Asian countries, especially in northeastern Thailand and Lao PDR. Cathepsin B is a lysosomal cysteine protease which is regulated by cysteine proteinase inhibitors such as cystatin C. Elevation of cathepsin B levels in biological fluid has been observed in patients with inflammatory diseases and many cancers. We aimed to investigate the serum cathepsin B and cystatin C levels of CCA patients to evaluate the feasibility of using cathepsin B and cystatin C as markers for the diagnosis of CCA. Fifty-six sera from CCA patients, 17 with benign biliary diseases (BBD) and 13 from controls were collected and the cathepsin B and cystatin C levels were determined. In addition, cathepsin B expression was investigated immunohistochemically for 9 matched-pairs of cancerous and adjacent tissues of CCA patients. Serum cathepsin B, but not cystatin C, was significantly higher in CCA and BBD patient groups compared to that in the control group. Consistently, all cancerous tissues strongly expressed cathepsin B while adjacent tissues were negative in 7 out of 9 cases. In contrast, serum cystatin C levels were comparable between CCA and control groups, although serum cystatin C levels in the BBD group was higher than that in the control or CCA groups. When the serum cathepsin B to cystatin C ratio was calculated, that of the CCA group was significantly higher than that of the control group, and, although statistically not significant, the ratio of CCA group showed a trend to be higher than that of the BBD group. Thus, the cathepsin B to cystatin C ratio might be used as an alternative marker for aiding diagnosis of CCA.

• Parasites, Hosts and Diseases
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• v.55 no.2
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• pp.115-120
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• 2017

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.131-131
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• 2017

Though the Platycodon grandiflorum, has a broad range of pharmacologic properties, but the mechanisms underlying these effects remain unclear. In order to profile proteins from the nodal segment, callus, root and shoot, high throughput proteome approach was executed in the present study. Two-dimensional gels stained with CBB, a total of 84 differential expressed proteins were confirmed out of 839 protein spots using image analysis by Progenesis SameSpot software. Out of total differential expressed spots, 58 differential expressed protein spots (${\geq}2-fold$) were analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. Out of 58 differential expressed protein, 32 protein spots were up-regulated such as ribulose-1,5-bisphosphate carboxylase, endoplasmic oxidoreductin-1, heat stress transcription factor A3, RNA pseudourine synthase 4, cysteine proteinase, GntR family transcriptional regulator, E3 xyloglucan 6-xylosyltransferase, while 26 differential protein spots were down-regulated such as L-ascorbate oxidase precursor, late embryogenesis abundant protein D-34, putative SCO1 protein, oxygen-evolving enhancer protein 3. However, the frequency distribution of identified proteins using iProClass databases, and assignment by function based on gene ontology revealed that the identified proteins from the explants were mainly associated with the nucleic acid binding (17%), transferase activity (14%) and ion binding (12%). Taken together, the protein profile may provide insight clues for better understanding the characteristics of proteins and its metabolic activities in various explants of this essential medicinal plant P. grandiflorum.

• Parasites, Hosts and Diseases
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• v.35 no.2
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• pp.87-94
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• 1997

Clonorchis sinearis is a liver fluke which is the most prevalent helminth of humans in Korea. The better diagnostic measure of clonorchiasis is required for its nationwide control program. The present study observed antigenic bands of C. sinensis and reacting immunoglobulins in serum of infected residents. Adult C. sinensis were recovered from experimentally infected rabbits and soluble crude extract of the worms was used as the antigen after supplementation of E-64, a cysteine proteinase inhibitor. SDS- PAGE of the crude antigen resolved more than 20 protein bands between 200 and 14 kDa. The sera of infected humans collected at an endemic village showed specific IgG and IgE antibodies but little IgM and IgA antibodies. The protein bands of 94, 80, 72, 68, 52, 47, 43, 37, 34, and 28-25 kDa strongly reacted with serum Ig(GMA) or IgG antibody and 64, 62, 52, 47,44, 34,28, and 26 kDa bands reacted with serum IgE antibody. However, the 94, 80, 72, 68, 64, 62, 52, 47, and 40 kDa bands of C. sinensis antigen were found non specific. The protein bands of 43, 34, and 28-25 kDa of C. sinensis are primary target molecules of further analysis.

• The Journal of Korean Medicine
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• v.23 no.4
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• pp.98-104
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• 2002

Objectives: Calpain, a calcium-dependent cysteine proteinase, may be one of the proteolytic enzymes that mediate cartilage degradation associated with rheumatoid arthritis. The object of this study is to ascertain immunohistochemically whether calpain is present in the inflamed joints of collagen-induced arthritis of rats, and examine the effect of Cortex Acanthopanacis Senticosi on the expression of calpain. Methods: Male Lewis rats, around 200g of body weight, were immunized with bovine type II collagen. After 3 weeks from first immunization, rats were divided into arthritic control (n=6) group and Cortex Acanthopanacis Senticosi-treated (n=6) group. Non-immunized rats served as the normal (n=6) group. All animals were sacrificed at 15 days post-treatment and tibiotarsal joints were removed. Calpain immunohistochemistry was performed on the midsagittal section of the tibiotarsal joint. Results: All animals of the control and treated groups showed ankylosing osteoarthritis. However, the animals of the treated group showed alleviation in the fibrous ankylosis, destruction of articular cartilage and destruction of subchondral bony tissue compared with the animals of the control group. Calpain was expressed in the chondrocyte lacunae of growing articular cartilage, in the skeletal muscle fibers, in the peripheral nerves, and in the vessel walls around the joints of all groups. In the control and treated groups, calpain was also expressed in proliferating synovial epithelia, subsynovial stroma cells, surface of articular cartilage, and fibrous pannus around destructive subchondral bony tissue. However, the expression density of calpain in the treated group was diminished compared with the control group, especially in surface of articular cartilage and fibrous pannus. Conclusions: These observations indicated that calpain plays an important role in the destruction of cartilage and bone in collagen-induced arthritis of rats, and also indicated that Cortex Acanthopanacis Senticosi inhibits the development of arthritis by decreasing the expression of calpain.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.60 no.1
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• pp.97-106
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• 2015

Platycodon grandiflorum, commonly known as Doraji in Korea, has a wide range of pharmacologic properties, such as reducing adiposity and hyperlipidemia, and antiatherosclerotic effects. However, the mechanisms underlying these effects remain unclear. In order to profile proteins from the nodal segment, callus, root and shoot, high throughput proteome approach was executed in the present study. Two dimensional gels stained with CBB, a total of 84 differential expressed proteins were confirmed out of 839 protein spots using image analysis by Progenesis SameSpot software. Out of total differential expressed spots, 58 differential expressed protein spots (${\geq}$ 2-fold) were analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. Out of 58 differential expressed protein, 32 protein spots were up-regulated such as ribulose-1,5-bisphosphate carboxylase, endoplasmic oxidoreductin-1, heat stress transcription factor A3, RNA pseudourine synthase 4, cysteine proteinase, GntR family transcriptional regulator, E3 xyloglucan 6-xylosyltransferase, while 26 differential protein spots were down-regulated such as L-ascorbate oxidase precursor, late embryogenesis abundant protein D-34, putative SCO1 protein, oxygen-evolving enhancer protein 3. However, frequency distribution of identified proteins using iProClass databases, and assignment by function based on gene ontology revealed that the identified proteins from the explants were mainly associated with the nucleic acid binding (17%), transferase activity (14%) and ion binding (12%). In that way, the exclusive protein profile may provide insight clues for better understanding the characteristics of proteins and metabolic activity in various explants of the economically important medicinal plant Platycodon grandiflorum.

• Archives of Pharmacal Research
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• v.28 no.7
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 1997.06a
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• pp.73-101
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• 1997

Baculoviruses are characterized by large double-stranded circular DNA genomes and rod-shaped enveloped virions. Bombyx mori nucleopolyhedrovirus(BmNPV) is a major pathogen, which causes severe damage in sericulture. Currently, BmNPV is recogtnized as an improtant tool in molecular biology, especially for expression of useful genes in B.mori cells and silkworm larvae. Our laboratories have focused on the studies of the molecular mechanisms of BmNPV replication and the application of BmNPV to agriculture and medicine. The entire nucleotide sequence of the BmNPV genome has recently determined. The BmNPV genome possessed 135 putative genes and 7 homologous repeated sequence (hrs) regions. Relatively little space, a few to a few hundred base-pairs, was observed between the open reading frames and hrs. Termination codons often overlapped. These results showed a compactly packde BmNPV genome. Based on comparative sequence analyses, we speculated that the ancestor of BmNPV was a baculovirus similar to Autographa californica NPV(AcNPV). The function of the BmNPV genes were characterized by gene deletion analysis; p35 was found to be involved in blocking apoptosis and cysteine proteinase was found to be involved in horizontal virus transmission by degrading viral-infected larval host. By AcNPV and BmNPV coinfection experiments, we identified a BmNPV gene involved in expanding host specificity of AcNPV. The identified gene was likely encoded a DNA helicase based on the amino acid sequence analysis; a few amino acid substitutions in the putative DNA helicase gene resulted in the expansion of host range of AcNPV. These findings indicate that BmNPV evolved within a short period from an AcNPV-like ancestral virus due to rapid evolution including specific amino acid substitutions and gene deletions/insertions.