• Title, Summary, Keyword: nitroblue tetrazolium

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Partial Purification and Characterization of Superoxide Dismutase from Tomato (Lycopersicon esculentum) Fruit

  • Kumar, Sunil;Dhillon, Santosh;Singh, Dharam;Singh, Randhir
    • Preventive Nutrition and Food Science
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    • v.9 no.3
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    • pp.283-288
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    • 2004
  • Superoxide dismutase (SOD) from tomato (Lycopersicon esculentum Mill.) fruit was purified by ammonium sulphate precipitation, Sephadex G-100 and DEAE-cellulose column chromatographies. A 22 fold purification and an overall yield of 44% were achieved. The purified enzyme was a homodimer with Mr 37.1 kDa and subunit Mr 18.2 kDa as judged by SDS-PAGE. SOD showed $K_{m}$ values of 25 ${\times}$ 10$^{-6}$ M and 1.7 ${\times}$ 10$^{-6}$ M for nitroblue tetrazolium (NBT) and riboflavin as substrates, respectively. The enzyme was thermostable upto 5$0^{\circ}C$ and exhibited pH optima of 7.8. The effect of metal ions and some other compounds on enzyme activity was studied. $Co^{2+}$ and $Mg^{2+}$ were found to enhance relative enzyme activities by 27 % and 73 %, respectively, while M $n^{2+}$ inhibited the SOD activity by 64%. However, $Ca^{2+}$ and C $u^{2+}$ had no effect on enzyme activity. Other compounds like $H_2O$$_2$ and Na $N_3$ inhibited enzymatic activities by 60% and 32%, respectively, while sodium dodecyl sulphate (SDS), chloroform plus ethanol and $\beta$-mercaptoethanol had no effect on the activity of SOD. of SOD.

In vitro Effect of Water Extract of Medicinal herbs on Antimicrobial Activity against Fish Pathogenic Bacteria and Superoxide Production of Kidney Phagocytes in Oliver Flounder, Paralichthys olivaceus (식물성 생약재 열수추출물이 어병 원인세균에 대한 항균활성 및 넙치 (Paralichthys olivaceus)식세포의 활성산소 생산에 미치는 in vitro 효과)

  • Jung, Sung-Hee;Sohn, Young-Chan;Kim, Yi-Cheong
    • Journal of fish pathology
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    • v.14 no.1
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    • pp.3-10
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    • 2001
  • The present study was to obtain a basic research data about medicinal herbs by screening in vitro antimicrobial activity and the production of superoxide anion($O_2^-$) from the head kidney phagocytes of olive flounder, Paralichthys olivaceus. The following fourteen kinds of medicinal herbs extracted by boiling water were used : Gosam, Gwijeonu, Gujeolcho, Bagha, Bangpung, Yeongyo, Yagssug, Jiyu, Sambaegcho, Samjiguyeobcho, Sangbaegpi, Sohwehyang, Pyeonchug, Palgag. Antimicrobial activity against fish pathogenic bacteria, Listonella anguillarum, Vibrio sp., Vibrio alginolyticus, Edwardsiella tarda, Streptococcus sp. and Lactococcus garvieae, and the production of superoxide in kidney macrophage of olive flounder were examined by disk method and nitroblue tetrazolium(NBT) reaction, respectively. Among the tested herbs, Yagssug showed the highest antimicrobial activity against those fish pathogenic bacteria and stimulation of $O_2^-$ production.

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Transition Metal Induces Apoptosis in MC3T3E1 Osteoblast: Evidence of Free Radical Release

  • Chae, Han-Jung;Chae, Soo-Wan;Kang, Jang-Sook;Yun, Dong-Hyeon;Bang, Byung-Gwan;Kang, Mi-Ra;Kim, Hyung-Min;Kim, Hyung-Ryong
    • The Korean Journal of Physiology and Pharmacology
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    • v.4 no.1
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    • pp.47-54
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    • 2000
  • Transition metal ions including $Se^{2+},\;Cd^{2+},\;Hg^{2+}\;or\;Mn^{2+}$ have been thought to disturb the bone metabolism directly. However, the mechanism for the bone lesion is unknown. In this study, we demonstrated that MC3T3E1 osteoblasts, exposed to various transition metal ions; selenium, cadmium, mercury or manganese, generated massive amounts of reactive oxygen species (ROS). The released ROS were completely quenched by free radical scavengers-N-acetyl cysteine (NAC), reduced glutathione (GSH), or superoxide dismutase (SOD). First, we have observed that selenium $(10\;{\mu}M),$ cadmium $(100\;{\mu}M),$ mercury $(100\;{\mu}M)$ or manganese (1 mM) treatment induced apoptotic phenomena like DNA fragmentation, chromatin condensation and caspase-3-like cysteine protease activation in MC3T3E1 osteoblasts. Concomitant treatment of antioxidant; N-acetyl-L-cysteine (NAC), reduced-form glutathione (GSH), or superoxide dismutase (SOD), prevented apoptosis induced by each of the transition metal ions. Catalase or dimethylsulfoxide (DMSO) has less potent inhibitory effect on the apoptosis, compared with NAC, GSH or SOD. In line with the results, nitroblue tetrazolium (NBT) stain shows that each of the transition metals is a potent source of free radicals in MC3T3E1 osteoblast. Our data show that oxidative damage is associated with the induction of apoptosis in MC3T3E1 osteoblasts following $Se^{2+},\;Cd^{2+},\;Hg^{2+}\;or\;Mn^{2+}$ treatment.

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Comparative study of Photobacterium damselae subsp. damselae and Vibrios on pathogenicity in vivo (In vivo에서 Photobacterium damselae subsp. damselae와 Vibrio 속 세균의 병원성 비교)

  • Kwon, Mun-Gyeong;Cho, Byoung-Youl;Park, Soo-Il
    • Journal of fish pathology
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    • v.22 no.2
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    • pp.115-124
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    • 2009
  • Photobacterium damselae subsp. damselae and 4 Vibrio spp, V. anguillarum, V. splendidus, V. harveyi and V. ordalii, were isolated from the diseased olive flounders, Paralichthys olivaceus. The pathogenicity of the isolates were compared to mortality, blood biochemical contents, such as alanineaminotransferase(ALT), aspatate aminotransferase(AST), and cortisol level, and non-specific immune responses, nitroblue tetrazolium (NBT) reduction of macrophages and lysozyme activities of serum. The mortalities and levels of ALT, AST and cortisol of fishes infected with P. damselae were higher than those of others but significantly low in non-specific immune responses, NBT and lysozyme activities. These results suggest that P. damsela might produce damselysin having high hemolytic and phospholipase activities, correlated with the pathogenicity. P. damsela could also make an obstruction of internal organs, following increasing in the level of ALT and AST, and depression in host immunity caused by induced high levels of cortisol.

The Detection of Superoxide Dismutase Activity and Isozyme Pattern of Panax ginseng C.A. Meyer Leaves (인삼엽에서 Superoxide Dismutase Activity 측정 및 Isozyme Pattern 검정)

  • Yang, Deok-Jo;Kim, Myeong-Sik;Lee, Seong-Jong
    • Journal of Ginseng Research
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    • v.11 no.1
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    • pp.24-31
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    • 1987
  • We studied a assay method on the measurement of superoxide dismutase (SOD Superoxide : superoxide oxidoreductase, EC. 1. 15. 1. 1) activity with photoreduced flavin and nitroblue tetrazolium (NBT) as superoxide (${O_2}^{-}$) source and detector, respectively. The $\Delta$E (1000 ng SOD$.$$min.)^{-1}$ of photoreduced flavin-NBT system was 0.08, whereas that of xanthine-xanthine-cytochrome system used broadly in experiments was 0.014. Therefore, the new method was regarded more simple and utilizable than xanthine-xanthine cytochrome system method. In the present paper, we also carried out to investigate the SOD activity and isozyme pattern for the parpose of study of leaf-burning disease in ginseng (Panax ginseng C.A. Meyer) leaves.

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The antioxidative compounds of the Aster tataricus

  • Choi, Doo-Youn;Moon, Young-Hee;Woo, Eun-Rhan
    • Proceedings of the PSK Conference
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    • pp.207.1-207
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    • 2003
  • The Aster tataricus is a chinese traditional medicine called “Ziwan” which has an expectorative and remediable cough action. The anti-oxidant activities of A. tataricus were investigated. The MeOH extract of A. tataricus showed strong anti-oxidant activity in the NBT(nitroblue tetrazolium) method system, and thus fractionated with several solvents in to the EtOAc, n-BuOH, CH$_2$Cl$_2$, H$_2$O fraction. The EtOAc soluble fraction exhibiting strong anti-oxidant activity was further purified by repeated silica gel and sephadex LH-20 column chomatography. (omitted)

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Costunolide Induces Differentiation of Human Leukemia HL-60 Cells

  • Choi, Jung-Hye;Seo, Bo-Rim;Seo, Seong-Hoon;Lee, Kyung-Tae;Park, Jae-Hoon;Park, Hee-Juhn;Choi, Jong-Won;Yoshie-Itoh;Miyamoto, Ken-Ichi
    • Archives of Pharmacal Research
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    • v.25 no.4
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    • pp.480-484
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    • 2002
  • Costunolide has been reported to be a cytotoxic and chemopreventive agent. This work investigated the mechanism of the anti proliferative effect of costunolide and determined that it induced differentiation of the human leukemia cell line HL-60. Costunolide exhibited a potent antiproliferative activity against HL-60 cells. It was also found to be a potent inducer of differentiation in human leukemia derived HL-60 cells through the examination of differentiation markers, as assessed by the reduction of nitroblue tetrazolium, the increase in esterase activities and phagocytic activity, morphology change and the expression of CD14 and CD66b surface antigens. These results, accompanied by a decline in the expression of c-myc protein, suggest that costunolide induces differentiation of human leukemia cells to granulocytes and monocytes/macrophages lineage.

Diagnosis of Potato Leafroll Virus with Digoxigenin-labeled cRNA Probes (Digoxigenin으로 표지된 cRNA 프로브를 이용한 감자잎말림바이러스(PLRV)의 짐단)

  • 서효원;함영일;오승은;신관용;최장경
    • Korean Journal Plant Pathology
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    • v.14 no.6
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    • pp.636-641
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    • 1998
  • Digoxigenin (DIG) was used to prepare nucleic acid probe for the detection of RNA of potato leafroll virus (PLRV) in the potato leaf extracts. The 0.6 kb coat protein (CP) gene cDNA of PLRV in plasmid pSPT 18 vector was labeled with digoxigenin by in vitro run-off transcription and then used for cRNA probe. In the several buffers tested for increase the total RNA extraction efficiency AMES buffer was the most suitable for this detection method. The RNA extracts from potato leaves shown symptoms of PLRV were dot blotted onto nylon membrane and hybridized with labeled RNA probes. After hybridization, labeled RNA bound to PLRV RNA on membrane was detected with anti-digoxigenin alkaline phosphatase. 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (NBT) salt and CSPD were used as substrate for colorimetric and film exposure detection, respectively. These detection methods were very sensitive allowing for detection of 1/32 diluted total RNA extract from 100 mg leaf tissue.

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Korean Mistletoe (Viscum album Coloratum) Extract Induces Eel (Anguilla japonica) Non-specific Immunity

  • Yoon, Taek-Joon;Park, Kwan-Ha;Choi, Sang-Hoon
    • IMMUNE NETWORK
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    • v.8 no.4
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    • pp.124-129
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    • 2008
  • Background: The immunomodulatory effects of Korean mistletoe (Viscum album Coloratum) on the innate immune responses of eel (Anguilla japonica) were studied. Methods: Mistletoe, Freund’s complete adjuvant (FCA), or phosphate-buffered saline (PBS) as a control was injected into eel peritoneal cavities. Results: Nitroblue tetrazolium (NBT)-positive cells in the head kidney of eel were significantly augmented by the second day post-injection of mistletoe. Reactive oxygen intermediates (ROI) were more produced in mistletoe-injected fish kidney leucocytes than in FCA-injected ones. The level of lysozyme activity in the serum of fish 2 days after injection with mistletoe was also significantly higher than that in the serum of the control fish. The optimal concentration of mistletoe in inducing the highest serum lysozyme activity was revealed to 500${\mu}$g/200 g of fish. In phagocytic activity assay, mistletoe-sensitized eel kidney phagocytes captured more zymosan than did the control fish. Conclusion: Korean mistletoe appeared to be a good activator of the non-specific immune responses of eel.

Effect of Pyrroloquinoline Quinone on Osteoclast Generation and Activity (Pyrroloquinoline quinone이 파골세포의 생성 및 활성에 미치는 영향)

  • Ko, Seon-Yle;Han, Dong-Ho;Kim, Jung-Keun
    • Journal of Oral Medicine and Pain
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    • v.30 no.3
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    • pp.329-336
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    • 2005
  • We examined the effect of PQQ, as a scavenger of superoxide, on osteoclast-like cell formation and on mature osteoclast function. To determine whether PQQ scavenges the superoxide, nitroblue tetrazolium (NBT) staining, which is a method to detect superoxide, was performed on HD-11 cells which are a chick myelomonocytic cell line having tartrate-resistant acid phosphatase (TRAP) activity in response to 1,25-dihydroxyvitamin $D_3\;[1,25(OH)_2D_3]$. Histochemical study of TRAP was also performed on HD-11 cells. PQQ inhibited the TRAP-positive multinucleated cell formation of chicken bone marrow cells was also examined. The addition of 20 ${\mu}M$ PQQ inhibited the formation of TRAP-positive multinucleated cell. When chicken osteoclasts were cultured on dentin slices, treatment of 20 ${\mu}M$ PQQ resulted in a significant decrease in dentin resorption by osteoclasts in terms of total resorption area and number of resorption pits. The present data suggest that PQQ, possibly as a scavenger of superoxide ion, inhibits the osteoclastic differentiation and bone resorption.