• BMB Reports
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• v.34 no.1
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• pp.80-84
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• 2001

The human papillomavirus E7 protein can form a specific complex with a retinoblastoma tumor suppressor gene product (p105-Rb) that results in the release of the E2F transcription factor, which is critical for the growth-deregulation and transforming properties of the viral E7 oncoprotein. In an attempt to apply interaction between the E7 oncoprotein and a target cellular protein Rb for an in vitro screening system for drugs against human papillomavirus infection, we primarily investigated the E7Rb binding through a pull down assay and enzyme-linked immunosorbent assay. The pull down assay showed that both glutathione S-transferase-tagged E7 and His-tagged E7 immobilized on resins specifically produced complexes with bacterially expressed Rb in a dose-dependent manner, as determined by immunoblot analyses. This result coincided with that of an enzyme-linked immunosorbent assay, which is a useful system for the mass screening of potential drugs. Taken together, this screening system (based on the interaction between E7 and Rb) can be a promising system in the development of drugs against cervical cancers caused by human papillomavirus infection.

• Korean Journal of Veterinary Service
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• v.31 no.3
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• pp.315-329
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• 2008

A total of 710 bovine serum samples which are composed 532 bovine serum samples showed negative reaction and 178 bovine serum samples showed positive reaction with tube agglutination test (TAT) from North area of Gyeong-nam, Korea were tested using all the 3 assays which are Rose-Bengal test(RBT), tube agglutination test (TAT) and enzyme-linked immunosorbent assay (ELISA, two types) and analyzed for evaluation of specificity, sensitivity, reproducibility and predictive value. In the comparison of serum antibody titer agglutination test, RBT showed almost agreement with TAT. In the comparison of TAT and two types of ELISA method, they showed difference in specificity and sensitivity about 5%. But there is no significant difference in detecting sensitivity between two types of ELISA method and TAT. In serologic tests for bovine brucellosis, the new assay ELISA would be a good candidate for serologic survey for bovine brucellosis in Korea because it is efficient in detecting many test samples quickly. But the serum agglutination tests (RBT, TAT) are more economical and easy assay for detection. In the test of comparison of antibody titer between first day of finding and 10 days after finding by TAT, there was no change in 55% (76/139) of positive cattle.

• The Korea Journal of Herbology
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• v.21 no.4
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• pp.115-121
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• 2006

Objectives : For the purpose of developing new anti-HIV agents from natural sources, the extracts of Actinidia arguta were tested for their inhibitory effects on essential enzymes as the reverse transcriptase (RT), protease and ${\alpha}-\;glucosidase$. And we predicted inhibition activity of major compounds of Actinidia arguta using Quantitative Structure Activity Relationships (QSAR). Methods : In this assay the activity of HIV-1 reverse transcriptase is measured as the formation of a strand of copy-DNA (cDNA) using RNA as a template. The activity of HIV-1 protease is measured as the cleavage of an oligopeptide by HIV-1 protease. Results : In the anti-HIV-1 RT using Enzyme Linked Oligonucleotide Sorbent Assay (ELOSA) method, water extracts (100ug/ml) of stem and leaf showed strong activity of 93.9% and 91.9%, respectively. In the HIV-1 protease inhibition assay, aqueous stem extract inhibited the activity of the enzyme to cleave an oligopeptide, resembling one of the cleavage sites in the viral polyprotein which can only be processed by HIV-1 protease with 56.8%. In the ${\alpha}-glucosidase$ inhibition assay, aqueous stem extract showed activity of 73.1%. Conclusion : We found out this result, for these samples it is possible that the inhibition of the viral replication in vitro is due to the inhibition at least one of RT and ${\alpha}-glucosidase$. It would be of great interest to identify the compounds which are responsible for this inhibition, since all therapeutically useful agent up to date are RT, PR and ${\alpha}-glucosidase$ inhibitors.

• Korean Journal of Plant Tissue Culture
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• v.28 no.3
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• pp.165-171
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• 2001

New visible and fast assay system have been developed for tobacco transformant introduced with adenosine deaminase (ADA) marker gene, which converts cytotoxic adenosine analogues to non-toxic inosine analogues and ammonia. Ammonia was changed to blue color in the solution of phenol-nitoprusside and alkaline-hypochlorite. It was possible to detect activity of ADA visibly on the holes of 96 well plate using tiny explant of transgenic tobacco leaves within 1 hour incubation time. As substrates of ADA enzyme from transgenic plant on the plate, a number of adenosine analogues such as 9-D-arabinofuranosyl adenine, cordycepin, 2'-deoxyadenosine, adenosine and xylofuranosyl adenine were possible for detection of ADA activity. Optimal condition of substrate for ADA enzyme was each 10 mM and pH 7.5 in adenosine solution. Especially, transgenic plant did not convert adenosine to inosine and ammonia in the presence of ADA inhibitor deoxycoformycin, which means that ammonia produced from transgenic plant is due to expression of ADA gene. Now, we show that this detection system can be easily, sensitively, fast and cheaply as well as visibly assayed in vitro as GUS gene system with very small size of transformant explant.

• Perinatology
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• v.29 no.4
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• pp.165-169
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• 2018

Objective: Highly sensitive haptoglobin measurement should be used in neonates because the haptoglobin concentration in neonates is lower than that of adults. The aim of this study was to establish the reference values of haptoglobin levels in the cord blood of uninfected neonates. Methods: The cord blood of 29 preterm and 51 term babies was collected, and data from the mother and the newborn were recorded. The haptoglobin concentrations of 80 cord blood samples were simultaneously measured by enzyme-linked immunosorbent assay (ELISA; Assaypro, St Charles, MO, USA) and immunoturbidimetry assay (Roche Diagnostics, Basel, Switzerland). C-reactive protein (CRP) was also measured by immunoturbidimetry assay (Roche Diagnostics, Switzerland). Results: Mean values of CRP and ELISA haptoglobin were not significantly different between preterm and term babies. The 2.5 percentile and 97.5 percentile values of ELISA haptoglobin concentration were as follows: 80 neonates, 0.01 mg/dL and 0.59 mg/dL; 29 preterm babies, 0.08 mg/dL and 0.18 mg/dL; and 51 term babies, 0.07 mg/dL and 0.23 mg/dL. There were no differences in ELISA haptoglobin concentration according to maternal underlying diseases, delivery method, usage of antibiotics or steroids before delivery, gestational age, gender of baby, or twin gestation. Conclusion: A highly sensitive haptoglobin method should be used to determine the haptoglobin concentration in Korean newborns because the reference values of cord blood haptoglobin concentration in Korean newborns are less than the lower detection limit for commonly used immunoturbidimetric haptoglobin measurement methods.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.32 no.3
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• pp.1-12
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• 2019

Objectives: Amygdalin is abundant in the seeds of bitter almond and apricots of the Prunus genus and other rosaceous plants. Amygdalin is known to have antitussive and anticancer activities. Apoptosis, also known as programmed cell death, is an important mechanism in cancer treatment. Methods: In the present study, we investigated whether the aqueous extract of Amygdalin induces apoptotic cell death in ME-180 cervical cancer cells. For this study, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, terminal deoxynuclotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, flow cytometric analysis, DNA fragmentation assay, Western blot, and caspase-3 enzyme assay were performed on ME-180 cervical cancer cells. Results: Through morphological and biochemical analyses, it was demonstrated that ME-180 cells treated with Amygdalin exhibit several apoptotic features. The treatment of Amygdalin increased the Bax expression and caspase-3 enzyme activity and decreased Bcl-2 expression. Here, we have shown that Amygdalin induces apoptotic cell death in ME-180 cervical cancer cells through Bax-dependent caspase-3 activation. These results suggest the possibility that Amygdalin exerts anti-tumor effect on human cervical cancer.

• Journal of Korean Ophthalmic Optics Society
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• v.20 no.1
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• pp.75-81
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• 2015

Purpose: The present study was conducted to investigate whether the changes in structure and activity of antioxidative enzymes, superoxide dismutase(SOD) and catalase(CAT) present in the eyes appeared when they were repeatedly exposed to UV-A, and reveal the correlation of these changes. Methods: Each enzyme solution was prepared from the standardized SOD and CAT, and repeatedly exposed to UV-A of 365 min under the condition of 30 minutes, 1 hour and 2 hours a day over 1, 2, 3, 4 and 5 days. Structural denaturation of SOD and CAT induced by repeat UV-A irradiation was confirmed by the electrophoretic analysis, and their enzyme activity was determined by the colorimetric assay using the proper assay kit. Results: SOD exposed repeatedly to UV-A showed the polymerization pattern through the electrophoretic analysis when it was repeatedly exposed under the condition of at least 1 hour a day however, the change of its activity was found to be less than 12%. On the other hand, CAT repeatedly exposed to UV-A showed reduced size of the electrophoretic band which indicated a structure denaturation and its activity was significantly decreased. In the case of that the repeat exposure time was longer, CAT activity was completely lost even though some enzyme band was shown in the electrphoretic analysis. Conclusions: From these results, it was revealed that the degree and pattern in structural denaturation of antioxidative enzymes differently appeared according to the type of enzyme, and the degree of structural denaturation was not always consistent with the reduction in enzyme activity.

• Biomedical Science Letters
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• v.2 no.1
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• pp.121-126
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• 1996

Specific monoclonal antibodies(mAbs) against bis-indole alkaloids related to vinblastine were established to develop a simple and specific immunoassay system for the quantitation of Vinca alkaloids. Vinca alkaloids were extracted from tissue cultured cells of periwinkle plant (Vinca rosea L.). Spleen cells from Balb/c mice immunized with vinblastine-bovine serum albumin(VBL-BSA) conjugate as immunogen were fused with myeloma cells(Sp2/0-Ag.14) in the presence of polyethylene glycol. In the preliminary experiments, 32 clones which highly reacted with VBL-BSA conjugate were selected by ELISA(Enzyme-linked immunosorbent assay).These clones were further analyzed by inhibition assay of ELISA. The results obtained with two typical monoclonal antibodies, KN-1 and KN-2, were described. KN-1 exhibited considerable reactivities with soluble dimeric bis-indole alkaloids, whereas no cross reacted with monomeric bis-indole alkaloids. However KN-2 showed cross reactivity with mono- and di-meric bis-indole alkaloids. Furthermore, KN-1 was applied to the immunoassay system for determining the VBL amounts of in vitro cultured cell extracts. This assay system could detect dimeric vinca alkaloid as low as 0.05 nM.

• Molecules and Cells
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• v.23 no.3
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• pp.370-378
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• 2007

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.

• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.388-394
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• 2017

Although several analytical methods for measuring total antioxidant capacity (TAC) have been applied to biological samples, there were often dissimilar results due to the different principles of methods applied. Thus, this study aimed to validate four conventional analytical methods for measuring plasma TAC, including the ABTS assay, DPPH assay, FRAP assay, and ORAC assay, by comparing with urinary 8-isoprostane concentration. In addition, TAC results were compared with antioxidant enzyme activities including superoxide dismutase (SOD) and glutathione peroxidase in erythrocyte, and catalase in plasma. Plasma TAC measure by ABTS assay was strongly correlated with the result by FRAP assay. Plasma TAC by FRAP and ORAC assays were negatively correlated with erythrocyte SOD activity. The agreement among the four TAC assay methods and 8-isoprostane was determined using 95% prediction limits of linear regression, expressed as the mean of 8-isoprostane ${\pm}95%$ prediction limits. The ABTS method better agreed with 8-isoprostane than the other methods, demonstrating narrow prediction of limits. Furthermore, only plasma TAC determined by the ABTS assay was inversely correlated with urinary 8-isoprostane (r = -0.35, p < 0.05). In summary, the ABTS assay would be an appropriate method to measure overall plasma antioxidant capacity and predict the body's antioxidant status.