• Journal of Microbiology and Biotechnology
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• v.33 no.8
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• pp.1091-1100
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• 2023

Human papillomavirus (HPV) types 16 and 18 are the major causes of cervical lesions and are associated with 71% of cervical cancer cases globally. However, public health infrastructures to support cervical cancer screening may be unavailable to women in low-resource areas. Therefore, sensitive, convenient, and cost-efficient diagnostic methods are required for the detection of HPV16/18. Here, we designed two novel methods, real-time ERA and ERA-LFD, based on enzymatic recombinase amplification (ERA) for quick point-of-care identification of the HPV E6/E7 genes. The entire detection process could be completed within 25 min at a constant low temperature (35-43℃), and the results of the combined methods could be present as the amplification curves or the bands presented on dipsticks and directly interpreted with the naked eye. The ERA assays evaluated using standard plasmids carrying the E6/E7 genes and clinical samples exhibited excellent specificity, as no cross-reaction with other common HPV types was observed. The detection limits of our ERA assays were 10⁰ and 10¹ copies/µl for HPV16 and 18 respectively, which were comparable to those of the real-time PCR assay. Assessment of the clinical performance of the ERA assays using 114 cervical tissue samples demonstrated that they are highly consistent with real-time PCR, the gold standard for HPV detection. This study demonstrated that ERA-based assays possess excellent sensitivity, specificity, and repeatability for HPV16 and HPV18 detection with great potential to become robust diagnostic tools in local hospitals and field studies.

• Journal of the Korean Society of Food Science and Nutrition
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• v.44 no.12
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• pp.1912-1917
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• 2015

Pancreatic lipase is a potential therapeutic target for the treatment of diet-induced obesity in humans. As part of our continuing search for novel bioactive compounds, the convenient enzymatic transformation of caffeic acid into neolignans as well as related oxidized-products enhanced pancreatic lipase inhibitory activity. Enzymatic transformation of caffeic acid (1) using polyphenol oxidase originating from Korean pear yielded four oxidized metabolites, which were identified by different spectroscopic techniques ($^1H$,$^{13}C$ NMR, DEP/T, $^1H-^1H$ COSY, HMBC, HMQC, and NOESY). The anti-obesity efficacy of caffeic acid reactant was tested by in vitro porcine pancreatic lipase assay. All tested samples showed dose-dependent pancreatic lipase inhibitory activities. Four oxidative products including phellinsin A (2), caffeicinic acid (3), isocaffeicinic acid (4), and 7,8-erythro-caffeicin (5) were isolated and identified. The major metabolites (2~5) were evaluated for their pancreatic lipase inhibitory activity, and oxidized-products (2~3) improved potency against pancreatic lipase when compared to original caffeic acid. This result suggested that the neolignans isolated from oxidative transformation of caffeic acid might be beneficial in the treatment of obesity and relevant diseases, and the convenient enzymatic transformation by polyphenol oxidase may be a valuable method for structural modification and enhancement of activity.

• Journal of Pharmacopuncture
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• v.17 no.1
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• pp.44-50
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• 2014

Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

• Journal of Pharmacopuncture
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• v.21 no.3
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• pp.177-184
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• 2018

Objectives: Auraptene, a natural citrus coumarin, found in plants of Rutaceae and Apiaceae families. In this study, we investigated the effects of auraptene on tumor migration, invasion and matrix metalloproteinase (MMP)-2 and -9 enzymes activity. Methods: The effects of auraptene on the viability of A2780 and Hela cell lines was evaluated by MTT assay. Wound healing migration assay and Boyden chamber assay were determined the effect of auraptene on migration and cell invasion, respectively. MMP-2 and MMP-9 activities were analyzed by gelatin zymography assay. Results: Auraptene reduced A2780 cell viability. The results showed that auraptene inhibited in vitro migration and invasion of both cells. Furthermore, cell invasion ability suppressed at $100{\mu}M$ auraptene in Hela cells and at 25, $50{\mu}M$ in A2780 cell line. Gelatin zymography showed that for Hela cell line, auraptene suppressed MMP-2 enzymatic activity in all concentrations and for MMP-9 at a concentration between 12.5 to $100{\mu}M$ in A2780 cell line. Conclusion: Auraptene inhibited migration and invasion of human cervical and ovarian cancer cells in vitro by possibly inhibitory effects on MMP-2 and MMP-9 activity.

• Archives of Pharmacal Research
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• v.23 no.2
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• pp.182-186
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• 2000

Chondroitin sulfates proteoglycans were isolated from human placenta. For the identification of enzymatic digestion products of isolated proteoglycan, strong anion exchange-high performance liquid chromatography (SAX-HPLC) was performed. By the action of chondroitin ABC and chondroitin B lyase, three unsaturated disaccharides 2-acetamide-2-deoxy-3-O-($\beta$-D-gluco-4-enepyranosyluronic acid)-D-galactose ($\delta$Di-OS), 2-acetamide-2-deoxy-3-O-($\beta$-D-gluco-4-enepyranosyluronic acid)-6-O-su lfo-D-galactose ($\delta$Di-6S) and 2-acetamide-2-deoxy-3-O-($\beta$-D-gl uco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose ($\delta$Di-4S) were produced from the human placenta proteoglycan. The anticoagulant activity of chondroitin sulfate proteoglycan was evaluated by activated partial thromboplastin time (aPTT) assay and thrombin time (TT) assay. The clotting times of aPTT and TT were increased from 72 to 144 sec and 19 to 27 sec, respectively. The Immune-modulating activity of chondroitin sulfate proteoglycan was examined by cell proliferation assay and these results suggest that it may play a role in suppression of the function of immune-related cells.

• Korean Journal of Food Science and Technology
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• v.22 no.6
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• pp.625-631
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• 1990

This study was carried out to investigate the antimutagenic effects of five kinds of apple enzymatic browning reaction products(AEBRP) on mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide(4NQO), benzo(${alpha}$)pyrene(B(${alpha}$)P) and 3-amino-1,4-dimethyl-5H-pyri-do [4,3-b]indol (Trp-P-1). In spore rec-assay using B. subtilis Hl7($rec^+$) and M45($rec^-$), homocatechol-AEBRP and hydroquinone-AEBRP showed strong antimutagenic effects on MMC and MNNG as the concentration of AEBRP increased. In the Ames test using S. typhimurium TA98 and TA100, hydroxyhydroquinone-AEBRP and pyrogallol-AEBRP showed strong antimutagenic effects on Trp-p-1 and B(${alpha}$)p in TA98 and TA100 in the presence of S-9 mix. Most of AEBRPs suppressed about 50% to 80% the mutagenesis in S. typhimurium TA98 induced by MNNG, however, AEBRPs except hydroxyhydroquinone-AEBRP showed antimutagenic effects of about 94% in TA100. Antimutagenic effects of the five kinds of AEBRPs on 4-NQO were more or less weak, in particular homocatechol-AEBRP exhibited the inhibitory effect of about 48% in TA98, and homocatechol-AEBRP and hydroquinone-AEBRP showed inhibitory effects of about 46% to 58% in TA 100.

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.495-500
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• 1986

Enzymatic micro-assay methods were studied those were capable of determining ammonia down to 10$^{-5}$M(0.01 $\mu$mole/ml) in the presence of other nitrogenous compounds such as protein and amino acid. Microquantities of ammonia (0.01-0.1 $\mu$mole) could be determined indirectly by measuring phosphorous, one of the products of the enzymatic reaction catalyzed by glutamine synthetase. In this reaction, L-glutamate, ATP and ammonium chloride were used as substrates, and phosphorous was formed in propotion to the concentration of ammonium chloride In the reaction mixture. Another procedure was examined in which glutamine synthetase reaction coupled with pyruvate kinase and lactate dehydrogenase reactions was used. One mililiter of the assay mixture contained; phosphoenol pyruvate, 3 mM, L-glutamate, 10 mM; ATP, 1mM: MgSO$_4$, 20 mM: KCl, 75mM: NADH, 0.2mM: Tris-HCl buffer(pH 7.0), 100mM; pyruvate kinase, 10 U: lactate dehydrogenase, 12 U and glutamine synthetase, 4 U. After preincubation for 20 min at 3$0^{\circ}C$, NH$_4$Cl was added and the rates of NADH oxidation were followed at 340nm. The effective range of this method was proved to be from 0.01 to 0.05 $\mu$mole/$m{\ell}$. Glutamine synthetase reaction coupled with glutamate synthase reaction could also be effectively used for determining microquantities of ammonia. The one mililiter assay mixture contained; ATP, 5mM: L-glutamate, 5mM; L-ketoglutarate, 5mM; MgCl$_2$, 15mM; NADPH, 0.15mM; Tris-HCl buffer(pH 7.0); 100mM; glutamine synthetase, 1U and glutamate synthase, 0.5U. After preincubation for 20min at 3$0^{\circ}C$ NH$_4$Cl was added and the rates of NADPH oxidation were followed at 340nm. The effective range of this procedure was appeared to be from 0.01 to 0.05$\mu$mole/$m{\ell}$.

• Bulletin of the Korean Chemical Society
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• v.32 no.5
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• pp.1650-1656
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• 2011

A series of potent tricyclic derivatives with a non-aromatic amide as potent PARP-1 inhibitors were successfully synthesized and their PARP-1 inhibitory activity was evaluated. Among the derivatives, 2-(1-propylpiperidin-4-yloxy)-7,8,9,10-tetrahydrophenanthridin-6(5H)-one 23c displayed potent activity in a PARP-1 enzymatic assay and cell-based assay ($IC_{50}$ = 0.142 ${\mu}M$, $ED_{50}$ = 0.90 ${\mu}M$) with good water solubility. Further, molecular modeling studies confirmed the obtained biological results.

• Microbiology and Biotechnology Letters
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• v.41 no.4
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• pp.456-461
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• 2013

Using the different adsorption properties of ssDNA and dsDNA to GO, this study used a real time and efficient fluorescence assay to detect the enzymatic activity of the Klenow fragment with the adsorbed DNA to GO. Results showed that adsorption of fluorescein-tagged ssDNA to GO resulted in fluorescence quenching and DNA was released from GO by adding complementary DNA. In addition, fluorescence restoration was increased through a polymerization reaction by the Klenow fragment in the presence of a fluorescein-attached template, GO, and primer. Gel electrophoresis was conducted to confirm the hybridization and DNA polymerization reactions on GO.

• Journal of the Korean Chemical Society
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• v.19 no.4
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• pp.246-251
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• 1975

A spectrophotometric assay technique is descriead for the measurement of free SH-groups in the enzyme reaction mixture. The method utilizes a new substrate, S-hippuryl-thioglycolyl-glycine(S-Hip-thioglycol-Gly) which is the basis for a convenient assay of angiotensin-converting enzyme and other dipeptidyl carboxypeptidases. This substrate contains an appropriately located thioester linkage that is hydrolyzed by the converting enzyme and other dipeptidyl carboxypeptidases. One of the products, thioglycolyl glycine, is readily measured by reaction with Ellman's reagent, 5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB, to produce 5-thio-2-nitrobenzoic acid which has a strong absorption band at 410 nm. The method is sensitive (${\varepsilon}M = 1.36{\times}10^4$ at 412 nm) and can be applied as a continuous recording with DTNB present in the enzymatic reaction mixture.