• Molecules and Cells
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• 제43권7호
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• pp.632-644
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• 2020

The molecular mechanism underlying autophagy impairment in the retinal pigment epithelium (RPE) in dry age-related macular degeneration (AMD) is not yet clear. Based on the causative role of poly(ADP-ribose) polymerase 1 (PARP1) in RPE necrosis, this study examined whether PARP1 is involved in the autophagy impairment observed during dry AMD pathogenesis. We found that autophagy was downregulated following H₂O₂-induced PARP1 activation in ARPE-19 cells and olaparib, PARP1 inhibitor, preserved the autophagy process upon H₂O₂ exposure in ARPE-19 cells. These findings imply that PARP1 participates in the autophagy impairment upon oxidative stress in ARPE-19 cells. Furthermore, PARP1 inhibited autolysosome formation but did not affect autophagosome formation in H₂O₂-exposed ARPE-19 cells, demonstrating that PARP1 is responsible for impairment of late-stage autophagy in particular. Because PARP1 consumes NAD⁺ while exerting its catalytic activity, we investigated whether PARP1 impedes autophagy mediated by sirtuin1 (SIRT1), which uses NAD⁺ as its cofactor. A NAD⁺ precursor restored autophagy and protected mitochondria in ARPE-19 cells by preserving SIRT1 activity upon H₂O₂. Moreover, olaparib failed to restore autophagy in SIRT1-depleted ARPE-19 cells, indicating that PARP1 inhibits autophagy through SIRT1 inhibition. Next, we further examined whether PARP1-induced autophagy impairment occurs in the retinas of dry AMD model mice. Histological analyses revealed that olaparib treatment protected mouse retinas against sodium iodate (SI) insult, but not in retinas cotreated with SI and wortmannin, an autophagy inhibitor. Collectively, our data demonstrate that PARP1-dependent inhibition of SIRT1 activity impedes autophagic survival of RPE cells, leading to retinal degeneration during dry AMD pathogenesis.

• Natural Product Sciences
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• 제7권2호
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• pp.38-44
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• 2001

NAD(P)H:quinone reductase (QR), known as DT-diaphorase, is a kind of detoxifying phase II metabolic enzyme catalyzing hydroquinone formation by two electron reduction pathway from quinone type compounds, and thus facilitating excretion of quinoids from human body. With the usefulness of QR induction activity assay system for the modulation of toxicants, in the course of searching for cancer chemopreventive agents from natural products, the methanolic extracts of approximately two hundreds of oriental medicines were primarily evaluated using the induction potential of quinone reductase (QR) activity in cultured murine Hepa1c1c7 cells. As a result, several extracts including Hordeum vulgare, Momordica cochinchinensis, Strychnos ignatii, Houttuynia cordata, and Polygala japonica were found to significantly induce QR activity. In addition, the methylene chloride fraction of H. vulgare, one major dietary food source, showed potent induction of QR activity $(CD=6.4{\mu}g/ml)$. Further study for isolation of active principles from these lead extracts is warranted for the discovery of novel cancer chemopreventive agents.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.239.1-239
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• 1995

• Molecules and Cells
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• 제46권8호
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• pp.473-475
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• 2023

• Journal of Microbiology
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• 제42권1호
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• pp.20-24
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• 2004

Cloned myo-inositol-1-phosphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His-affinity column. The purified INOS required NAD$\^$+/ for the conversion of glucose-6-phosphate to inositol-1-phosphate. The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40$^{\circ}C$. The molecular weight of the native enzyme, as determined by gel filtration, was approximately M$\_$r/ 271,000${\pm}$15,000. A single subunit of approximately M$\_$r/ 62,000${\pm}$5,000 was detected upon SDS-polyacrylamide gel electrophoresis. The Michaelis ($K_{m}$) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD$\^$+/ these were 0.42 and 0.4 mM, respectively.

• 대한수의학회지
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• 제56권1호
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• pp.15-21
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• 2016

NAD(P)H-quinone oxidoreductase-1 (NQO1) is a down-stream target gene of nuclear factor erythroid 2-related factor 2 (Nrf2), and performs diverse biological functions. Recently, NQO1 is recognized as an effective gene for the cytotoxic inserts with its diverse biological functions, which is focused on antioxidant properties. The aim of present study was to assess the impact of NQO1 knockdown on cytoprotection-related protein expression in cisplatin cytotoxicity by using small interfering (si) RNA targeted on NQO1 gene. Cytotoxicity of cisplatin on ACHN cells was assessed in a dose- and time-dependent manner after siScramble or siNQO1 treatment. After cisplatin treatment, cells were subjected to cell viability assay, western-blot analysis, and immunofluorescence study. The cell viability was decreased in the siNQO1 cells (50%) than the siScramble cells (70%) after 24 h of cisplatin ($20{\mu}M$) treatment. Moreover, cytoprotection-related protein expressions were markedly suppressed in the siNQO1 cells after cisplatin treatment. The expression of Nrf2 and Klotho were decreased by 20% and 40%, respectively, of that in siScramble cells. Nrf2 and Klotho activation were also decreased in cisplatin treated siNQO1 cells, confirmed by cytoplasm-tonuclear translocation. Our findings demonstrate that the increased cisplatin-induced cytotoxicity was accompanied by suppressed Nrf2 activation and Klotho expression in siNQO1 cells.

• Archives of Pharmacal Research
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• 제23권6호
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• pp.554-558
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• 2000

Synthesized 5-arylamino-2-methyl-4,7-dioxobenzothiazoles 3a-3o were evaluated for modulation of NAD(P)H: quinone oxidoreductase (NQOl) activity with the cytosolic fractions derived from cultured human lung cancer cells and their cytotoxicity in cultured several human solid cancer cell lines. The 4,7-dioxobenzothiazoles affected the reduction potential by NQOl activity and showed a potent cytotoxic activity against human cancer cell lines. The tested compounds 3a, 3b, 3g, 3h, 3n and 3o were considered as more potent cytotoxic agents, and comparable modulators of NQOl activity.

• 미생물학회지
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• 제14권4호
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• pp.176-185
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• 1976

An improved procedure for the rapid purification of glucose-6-phosphate dehydrogenase from extracts of Saccharomyces cerevisiae was developed by using affinity chromatography. Among six affinty media tested, NADP$^{+}$-agarose and Affi-gel Blue were more effective than others (i.e., Affi-gel Red, AMP-agarose, ATP-agarose, and NAD$^{+}$-agarose). Conditions to desorb the enzyme bound to the affinity media were examined to increase the purity as well as yield. The best result was obtained when the column was developed with a linear gradient of KCl (0-1.0M). In case of Affi-gel Blue, introduction of NAD$^{+}$ (15mM) washing step prior to the salt gradient was most effective to remove NAD$^{+}$-binding proteins. For a large scale preparation of G-6-P dehydrogenase higher recovery was obtained by Affi-gel Blue than NADP$^{+}$-agarose, however, the purity of the enzyme was decreased by 10 times if the former was used as the affinity medium. The capacity of Affi-gel Blue for G-6-P dehydrogenase was found to be 5 times higher than that of NADP$^{+}$-agarose. Furthermore Affi-gel Blue could be reused repeatedly and its preparation is relatively easier and less expensive than NADP$^{+}$-agarose.X> +/-agarose.

• Natural Product Sciences
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• 제3권2호
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• pp.81-88
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• 1997

NAD(P)H:quinone reductase (QR) is one of the protective phase II enzymes against toxicity that accomplishes the capacity of detoxification by modulating the effects of mutagens and carcinogens. The detoxification mechanism is that quinone reductase promotes the 2-electron reduction of quinones to hydroquinones which are less reactive. This study is to search new inducers of quinone reductase from natural products, which can be used as cancer chemopreventive agents. Plant extracts were evaluated by using quinone reductase generating system With Hepa 1c1c7 murine hepatoma cell lines for enzyme inducing properties and crystal violet staining method for the measurement of cytotoxicity provoked. We have tested approximately 106 kinds of natural products after partition into n-hexane, ethyl acetate and aqueous layers from 100% methanol extracts of natural products. The ethyl acetate fractions of Vitex rotundifolia $(fruits,\;2FC:\;12.7\;{\mu}g/ml)$, Cnidium officinale $(aerial\;parts,\;2FC:\;10.5\;{\mu}g/ml)$, Chrysanthemum sinese $(flowers,\;2FC:\;17.4{\mu}g/ml)$ and the hexane fractions of Angelica gigas $(roots,\;2FC:\;13.2\;{\mu}g/ml)$, Smilax china $(roots,\;2FC:\;l1.9\;{\mu}g/ml)$, Sophora flavescens $(roots,\;2FC:\;16.3\;{\mu}g/ml)$ revealed the significant induction of quinone reductase in a murine hepatic Hepa 1c1c7 cell culture system.

• Parasites, Hosts and Diseases
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• 제59권1호
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• pp.35-45
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• 2021

Adult echinostomes having 37 collar spines collected from the intestine of Pitalah ducks in Aceh Province, Indonesia in 2018 were morphologically and molecularly determined to be Echinostoma miyagawai Ishii, 1932 (Digenea: Echinostomatidae). Among 20 ducks examined, 7 (35.0%) were found to be infected with this echinostome, and the number of flukes collected was 48 in total with average 6.9 (1-17) worms per duck. The adult flukes were 7.2 (6.1-8.5) mm in length and 1.2 (1.0-1.4) mm in width (pre-ovarian or testicular level) and characterized by having a head collar armed with 37 collar spines (dorsal spines arranged in 2 alternating rows), including 5 end group spines, and variable morphology of the testes, irregularly or deeply lobed (3-5 lobes) at times with horizontal extension. The eggs within the worm uterus were 93 (79-105) ㎛ long and 62 (56-70) ㎛ wide. These morphological features were consistent with both E. miyagawai and Echinostoma robustum, for which synonymy to each other has been raised. Sequencing of 2 mitochondrial genes, cox1 and nad1, revealed high homology with E. miyagawai (98.6-100% for cox1 and 99.0-99.8% for nad1) and also with E. robustum (99.3-99.8% for nad1) deposited in GenBank. We accepted the synonymy between the 2 species and diagnosed our flukes as E. miyagawai (syn. E. robustum) with redescription of its morphology. Further studies are required to determine the biological characteristics of E. miyagawai in Aceh Province, Indonesia, including the intermediate host and larval stage information.