• Title/Summary/Keyword: Enzymatic assay

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Hydrogen Peroxide produced by Two Amino Acid Oxidases Mediates Antibacterial Actions

  • Zhang Hongmin;Yang Qiuyue;Sun Mingxuan;Teng Maikun;Niu Liwen
    • Journal of Microbiology
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    • v.42 no.4
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    • pp.336-339
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    • 2004
  • The antibacterial actions of two amino acid oxidases, a D-amino acid oxidase from hog kidney and a L-amino acid oxidase from the venom of Agkistrodon halys, were investigated, demonstrating that both enzymes were able to inhibit the growth of both Gram-positive and Gram-negative bacteria, and that hydrogen peroxide, a product of their enzymatic reactions, was the antibacterial factor. However, hydrogen peroxide generated in the enzymatic reactions was not sufficient to explain the degree to which bacterial growth was inhibited. A fluorescence labeling assay showed that both of these two enzymes could bind to the surfaces of bacteria. To the best of our knowledge, this is the first report regarding the antibacterial activity of the D-amino acid oxidases.

Inhibition of Enzymatic Browning of Apple Juices by Benzoic Acid Isolated From Peach (Prunus persica Batsch) Seeds (복숭아씨로부터 분리된 안식향산에 의한 사과주스의 효소적갈변억제)

  • 이준영;홍순갑;최상원
    • Food Science and Preservation
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    • v.7 no.1
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    • pp.103-107
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    • 2000
  • Previously , the methanolic extract of peach sees was found to have a strong tyrosinase inhibitory activity in an in vitro assay. Several phenolic compunds were isolated from the seeds by solvent fractionation , Sephadex LH-20 chromatography, and preparative HPLC , and one of them showing strong tyrosinase inhibition was identified as benzoic acid by UV, IR, $^1$H/$^1$$^3$C-NMR, and EI-MS spectrsopy. Benzoic acid (IC50= 250$\mu\textrm{g}$/ml) and L-ascorbic acid (IC50=28$\mu\textrm{g}$/ml), well-known tyrosinase inhibitors. In particular , benzoic acid inhibited markedly the enzymatic browing (melanosis) of apple juices at low concentration of 0.01% and 0.05, comparable to that of L-ascorbic acid (P<0.05). these results suggest that benzoic acid, one of an effective food preservatives, may be potentially useful as a functional alternative to sulfites for the control of melanosis in fruit juices.

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A cost-effective and simple culture method for primary hepatocytes

  • Adaya, Sezin;Hasircib, Nesrin;Gurhana, Ismet Deliloglu
    • Animal cells and systems
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    • v.15 no.1
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    • pp.19-27
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    • 2011
  • Hepatocytes, the major epithelial cells of the liver, maintain their morphology in culture dishes coated with extracellular matrix (ECM) components such as collagen and fibronectin or biodegradable polymers (e.g. chitosan, gelatin). In these coated dishes, survival of cells and maintaining of liver-specific functions may increase. The aim of this study was to determine a suitable, cost-effective and simple system for hepatocyte isolation and culture which may be useful for various applications such as in vitro toxicology studies, hepatocyte transplantation and bioartificial liver (BAL) systems. In order to obtain primary cultures, hepatocytes were isolated from liver by an enzymatic method and cultured on plates coated with collagen, chitosan or gelatin. Collagen, gelatin-sandwich and gelatin-cell mixture methods were also evaluated. Morphology and attachment of the cells were observed by inverted microscope and scanning electron microscope (SEM). An MTT assay was used to determine cell viability and mitochondrial activity.

Antioxidative and Cytoprotective Effects of Isoflavones Isolated from Pueraria thunbergiana Flowers (갈화에서 분리한 이소플라본의 항산화 및 세포보호효과)

  • 이경태;손일철;공은아;김동현;최승기;최종원;박희준
    • YAKHAK HOEJI
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    • v.43 no.6
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    • pp.736-742
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    • 1999
  • Antioxidative and cytoprotective effects of tectorigenin and glycitein isolated from the pueraria thunbergiana and its derivative, genistein, were determined. Among these three compounds, tectorigenin and glycitein bearing 6-methoxyl groups in both isoflavones showed significant free radical scavenging activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and xanthine/xanthine oxidase (XOD) generating superoxide anion radical. Tectorigenin only showed a slight inhibitory effect on XOD. We further studied the inhibitory effects of these isoflavones on the lipid peroxidation in rat liver microsomes induced by enzymatic and non-enzymatic methods. Each of them exhibited inhibitory effect on both ascorbic $acid/Fe^{2+}-{\;}and{\;}ADP/NADPH/Fe^{+3}-induced$ lipid peroxidation. Moreover, tectorigenin exhibited the highest protection of hydrogen peroxide damage on HepG2 and Vero cells among the three isoflavones, in the cytoprotective assay. It was suggested that the pattern of antioxidative and cytoprotective effect of isoflavones could be crucially by the aromatic substitution of oxygen-containing groups.

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Influences of Peroxidase on Lysozyme Activity (Peroxidase가 Lysozyme 활성에 미치는 영향)

  • Lee, Sang-Goo;Kim, Hyung-Il;Kho, Hong-Seop
    • Journal of Oral Medicine and Pain
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    • v.33 no.1
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    • pp.1-8
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    • 2008
  • It is well known that many antimicrobial proteins in saliva interact with each other. The purpose of the present study was to investigate the interactions of lysozyme with peroxidase in the aspects of enzymatic activity in vitro. The interactions of lysozyme with peroxidase were examined by incubating hen egg-white lysozyme(HEWL) with bovine lactoperoxidase(bLP). The influence of peroxidase system on lysozyme was examined by subsequent addition of potassium thiocyanate and hydrogen peroxide. Lysozyme activity was determined by turbidity measurement of a Micrococcus lysodeikticus substrate suspension. Peroxidase activity was determined with an NbsSCN assay. The Wilcoxon signed rank test was used to analyze the changes of enzymatic activities compared with their controls. bLP at physiological concentrations enhanced the enzymatic activity of HEWL(P < 0.05) and its effect was dependent on the concentration of peroxidase. However, HEWL did not affect the enzymatic activity of bLP. Thiocyanate did not affect the enzymatic activity of HEWL, either. The addition of potassium thiocyanate and hydrogen peroxide did not lead to additional enhancement of the enzymatic activity of HEWL. The changes of hydrogen peroxide concentration in the peroxidase system did not affect the enzymatic activity of HEWL. Collectively, despite an in vitro nature of our study, the results of the present study provide valuable information on the interactions of lysozyme and peroxidase in the aspects of enzymatic activity in oral health care products and possibly in the oral cavity.

Investigation of Biological Activities of Enzymatic Hydrolysate of Spirulina (스피루리나 효소가수분해물의 생리활성 탐색)

  • Son, Min-Hee;Park, Keun-Hyoung;Choi, A-Reum;Yoo, Gui-Jae;In, Man-Jin;Kim, Dong-Ho;Chae, Hee-Jeong
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.38 no.2
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    • pp.136-141
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    • 2009
  • Biological activities of enzymatic hydrolysate of spirulina (EHS) were investigated. EHS showed no significant effects on the growth-stimulating activity for lactic-acid bacteria and antioxidant activity. EHS showed slight in vitro growth-inhibitory effects (15% at 1.42 mg/L) on a human cervical cancer cell line (HeLa). In addition, the anticoagulant activities of EHS were measured based on three different pathways: common, intrinsic and extrinsic pathways. As an indication of anticoagulant activity on common pathway, thrombin time (TT) of EHS (100 mg/L) was measured as 155.6 sec. Activated partial thromboplastin time (aPTT) for intrinsic pathway of EHS (1,000 mg/L) was measured as 95.8 sec. Prothrombin time (PT) based on extrinsic pathway of EHS (1,000 mg/L) was measured as 10.6 sec. These data showed that EHS have influences on anticoagulant factors of common pathway and intrinsic pathway. Consequently it was found that EHS could be used as a functional food for blood circulation.

Rec-assay and DNA-breaking Action on the Engymatic Browning Reaction Products -Influence of Cupric Ion Concentration- (효소적(酵素的) 갈변반응(褐變反應) 생성물(生咸物)에 대(對)한 Rec-assay 및 DNA 절단(切斷) 작용(作用) -동(銅) Ion 농도(濃度)의 영향(影響)-)

  • Ham, S.S.;Park, B.K.;Lee, S.Y.;Lee, J.H.;Kang, C.K.;Lee, D.S.;Omura, Hirohisa
    • Applied Biological Chemistry
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    • v.27 no.4
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    • pp.264-270
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    • 1984
  • In order to obtain mutagenic data of enzymatic browning reaction products, we investigated their mutagenicity. The rec-assay with Bacillus subtilis strains $H17(rec^+)$ and $M45(rec^-)$ were carried out with their spores. Detection of double strand breakage in calf-thymus DNA was investigated into sample solution with and without $Cu^{2+}$ by the agarose slab gel electrophoresis. In the rec-assay, catechol, pyrocatechol, DL-dopa, 3,4-dihydroxytoluene, hydroquinone, hydroxyhydroquinone with and without $Cu^{2+}$ in 0.05M ana 0.1M at the enzymatic browning reaction products stowed mutagenic action. And also browning solution of 0.05M hydroxyhydroquinone and catechol with $Cu^{2+}$, hydroquinone with and without $Cu^{2+}$ of 0.1M at the enzymatic browning reaction products were strong mutagenic action. The DNA breakability of the enzymatic browning reaction products of 0.1M pyrogallol was stronger than that of 0.05M pyrogallol browning solution with $Cu^{2+}$ and 3,4-dihydroxytoluene browning solution was stronger than that of 0.01M 3,4-dihydroxytoluene browning solution.

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Activity of Protein Kinase C in Abnormally Proliferated Vascular Endothelial Cells (비정상적인 세포증식이 유도된 혈관 내피세포에서 Protein Kinase C에 대한 활성 분석)

  • Bae, Yong Chan;Park, Suk Young;Nam, Su Bong;Moon, Jae Sul;Choi, Su Jong
    • Archives of Plastic Surgery
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    • v.34 no.1
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    • pp.13-17
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    • 2007
  • Purpose: To understand the pathogenesis of the disease that presents abnormally proliferated vascular endothelial cells, a model of DMH(1,2-dimethylhydrazine)-induced abnormal proliferation of HUVECs(Human Umbilical Vein Endothelial Cells) was made. We indirectly determined that Protein Kinase C(PKC) restricts the cellular proliferation and inhibits the manifestation of growth factor by using several inhibiting substances of the transmitter through our previous studies. Thereupon, we attempted to observe direct enzymatic activities of PKC and its correlation with the abnormal proliferation of vascular endothelial cells. Methods: $10^5$ HUVECs cells were applied to 6 individual well plates in three different groups; A control group cultured without treatment, a group concentrated with $0.75{\times}10^{-8}M$ DMH only, and a group treated with DMH & $5{\times}10^{-9}M$ Calphostin C, inhibitor of PKC. In analyzing the formation of intracellular PKC enzyme, protein separation was performed, and separated protein was quantitatively measured. PKC enzyme reaction was analyzed through Protein Kinase C Assay System (Promega, USA), and the results were analyzed according to Beer's law. Results: Enzymatic activity of PKC presented the highest in all reaction time of a group concentrated only with DMH, and the lowest in the control group. The group treated with DMH and the inhibitor revealed statistically lower enzymatic activity than group only with DMH in all reaction time, although higher than the control group. Conclusion: From the enzymatic aspect, most active and immediate reaction of the PKC was observed in the group concentrated with DMH only. The group treated with DMH & PKC inhibitor showed meaningful decrease. Accordingly, PKC holds a significant role in DMH-induced abnormal proliferation of vascular endothelial cells.

Enzymatic Studies on capsaicin, The hot component of capsicum annum III Studies on the Capsicin in Kochuzang (고추의 신미성분 Capsacin에 대한 효소화학적구 (제 3 보) 고추장중 Capsaicin 소장에 관하여)

  • 한구동;이상섭;최순진
    • YAKHAK HOEJI
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    • v.4 no.1
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    • pp.60-62
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    • 1959
  • We found a fact, when Kochuzang goes on ferment, that capsaicin in Kochuzang breaks down to other substances. And those substances are less hot than original capsaicin. The degradation of capsaicin depends on the concentration of table salt in Kochuzang. Nevertheless, these is rather no difference between a Kochuzang which contains no salt and the other one which contains sufficient salt when we assay them by Electrophotometry. We can explain this contradiction by making an assumption that the degradation might be happened at the acid radical part of capsaicin and not at Vanillyl radical.

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