• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1699-1707
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• 2013

During the fermentative production of 1,3-propanediol under high substrate concentrations, accumulation of intracellular 3-hydroxypropionaldehyde will cause premature cessation of cell growth and glycerol consumption. Discovery of oxidoreductases that can convert 3-hydroxypropionaldehyde to 1,3-propanediol using NADPH as cofactor could serve as a solution to this problem. In this paper, the yqhD gene from Klebsiella pneumoniae DSM2026, which was found encoding an aldehyde reductase (KpAR), was cloned and characterized. KpAR showed broad substrate specificity under physiological direction, whereas no catalytic activity was detected in the oxidation direction, and both NADPH and NADH can be utilized as cofactors. The cofactor binding mechanism was then investigated employing homology modeling and molecular dynamics simulations. Hydrogen-bond analysis showed that the hydrogen-bond interactions between KpAR and NADPH are much stronger than that for NADH. Free-energy decomposition dedicated that residues Gly37 to Val41 contribute most to the cofactor preference through polar interactions. In conclusion, this work provides a novel aldehyde reductase that has potential applications in the development of novel genetically engineered strains in the 1,3-propanediol industry, and gives a better understanding of the mechanisms involved in cofactor binding.

• Natural Product Sciences
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• 제24권1호
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• pp.59-65
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• 2018

An isoform of NADPH oxidase (NOX), NOX2 is a superoxide-generating enzyme involved in diverse pathophysiological events. Although its potential as a therapeutic target has been validated, there is no clinically available inhibitor. Herein, NOX2-inhibitory activity was screened with the constituents isolated from Schisandra chinensis, which has been reported to have antioxidant and reactive oxygen species (ROS)-scavenging effects. Among the partitions prepared from crude methanolic extract, a chloroform-soluble partition showed the highest NOX2-inhibitory activity in PLB-985 cell-based NOX2 assay. A total of twenty nine compounds (1 - 29) were identified from the chloroform fraction, including two first isolated compounds; dimethyl-malate (25) and 2-(2-hydroxyacetyl) furan (27) from this plants. Of these constituents, two compounds (gomisin T, and pregomisin) exhibited an NOX2-inhibitory effect with the $IC_{50}$ of $9.4{\pm}3.6$, and $62.9{\pm}11.3{\mu}M$, respectively. They are confirmed not to be nonspecific superoxide scavengers in a counter assay using a xanthine-xanthine oxidase system. These findings suggest the potential application of gomisin T (6) and other constituents of S. chinensis to inhibit NOX2.

• Korean Journal of Acupuncture
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• 제20권2호
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• pp.67-76
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• 2003

목적 : 오가피 약침이 알코올에 의해서 중독된 Sprague-Dawley(S-D)계 흰쥐의 해마 치상회에서 새로운 신경세포 생성 및 NOS발현에 미치는 영향을 조사하였다. 방법 : S-D계 흰쥐에 알코올을(2g/kg) 3일간 연속으로 투여한후, 5일간 인체의 중완혈에 상응하는 부위에 오가피 약침(30mg/kg) 치료를 시행하였다. 치료효과를 관찰하기 위해서 BrdU-면역조직화학 염색법 및 NADPH-d-조직화학염색법을 이용하였다. 결과 : 알코올 처치군에서는 BrdU-양성세포수 및 NADPH-양성세포수가 모두 정상군에 비해서 감소한 반면에 알코올 처치후 오가피 약침으로 치료한 군에서는 BrdU-양성세포수 및 NADPH-양성세포수 모두 알코올 처치군에 비해서 증가하였다. 결론 : 오가피 약침치료가 알코올에 의해서 중독된 S-D계 흰쥐 해마 치상회에서 새로운 신경세포의 생성을 증가시키는 것을 확인하였으며, 그 기전으로 산화질소가 관여할 것으로 사려된다.

• 한국식품영양학회지
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• 제10권1호
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• pp.37-43
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• 1997

본 연구는 지질의 과산화에 대한 항산화 효과를 조사하기 위하여 엉겅퀴(Silybum marianum)로부터 silymarin 및 silybin을 정제하여 실험하였다. Silymarin 및 silybin은 xanthine oxidase system에서 superoxide anion의 생성을 억제하였다. 쥐의 간 mitochondria에서는 silymarin 및 silybin은 reduced nicotinamide adenine dinucleotide phosphate(NADPH)에 의해 효과적 또는 ascorbic acid 또는 Fenton's reagent에 의하여 비효소적으로 유도되는 지질의 과산화를 억제하였다. 또 mitochondria의 지질과산화도 silymarin 및 silybin에 의하여 억제되었고 NADPH 의존 cychrome P-450 reductase에 의한 Fe2+의 산화도 silymarin 및 silybin에 의하여 억제되었다. Silymarin 및 silybin은 microsome의 효소 시스템 및 linoleic acid hydroperoxide induced peroxidation system에서 지질의 과산화의 연쇄반응에서 유리기의 억제효과가 있었다.

• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.218-224
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• 2013

The aldo-keto reductases catalyze reduction reactions using various aliphatic and aromatic aldehydes/ketones. Most reductases require NADPH exclusively as their cofactors. However, NADPH is much more expensive and unstable than NADH. In this study, we attempted to change the five amino acid residues that interact with the 2'-phosphate group of the adenosine ribose of NADPH. These residues were selected based on a docking model of the YOL151W reductase and were substituted with other amino acids to develop NADH-utilizing enzymes. Ten mutants were constructed by site-directed mutagenesis and expressed in Escherichia coli. Among them, four mutants showed higher reductase activities than wild-type when using the NADH cofactor. Analysis of the kinetic parameters for the wild type and mutants indicated that the $k_{cat}/K_{m}$ value of the Asn9Glu mutant toward NADH increased 3-fold. A docking model was used to show that the carboxyl group of Glu 9 of the mutant formed an additional hydrogen bond with the 2'-hydroxyl group of adenosine ribose. The Asn9Glu mutant was able to produce (R)-ethyl-4-chloro-3-hydroxyl butanoate rapidly when using the NADH regeneration system.

• 약학회지
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• 제31권6호
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• pp.361-369
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• 1987

The four azo dyes such as Amaranth (FD & C Red No. 2), Tartrazine (FD & C Yellow No. 4), sunset Yellow (FD & C Yellow No. 5) and Allura red (FD & C Red No. 40) are currently employed as a food additives in Korea. In this study, the effects of these azo dyes on the hepatic microsomal mixed function oxidase systems in Rats. (i.e., Cyt. P-450, Cyt. b$_5$, NADPH cyt. c-reductase and azo reductase) were investigated. Furthermore, to determine the relationship among the electron transport systems, each level of azo reductase, Cyt. P-450 and NADPH cyt. c-reductase was measured upon the administration of phenobarbital (known as an inducer of Cyt. P-450), 3-methylcholanthrene (Known as an inducer of Cyt. P-448), CoCl$_2$ (inhibitor on Cyt. P-450) or $CCl_4$ (inhibitor on Cyt. P-450). The results of these studies are as follows; (1) The levels of Cyt. P-450 and Cyt. b$_5$ were decreased upon the administration of these azo dyes. (2) When the level of Cyt. P-450 was decreased, the azo reductase activity was also decreased. (3) These azo dyes did not show any significant effect on the level of NADPH cyt. c-reductase. (4) The administration of 3-methylcholanthrene resulted in the elevation of azo reductase activity. The 3-methylcholanthrene may be responsible for the induction of CO-insensitive electron transport system.

• Journal of Acupuncture Research
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• 제24권2호
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• pp.187-201
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• 2007

Objectives: This study was to investigate the effect of high frequency electro-acupuncture (EA) at $ST_{36}$ acupoint on the Freund's Complete Adjuvant (FCA)-induced arthritis in rats. Methods : Arthritis was induced by intradermal injection of FCA into base of tail. Experimental groups were divided into 4 groups; Normal, Control, $ST_{36}$ and Non-Acupoint (NA). $ST_{36}$ group was treated by 120Hz EA at $ST_{36}$ acupoint. Body weight, paw edema volume and ankle joint thickness were measured after treatment. And we investigated the effects of 120Hz EA via WBC count, segmen neurophil, lymphocyte, PGE2 assay and NADPH-d histochemistry. Results and Conclusions : The mean of body weight and ankle joint thickness of $ST_{36}$ group was increased compared with control group. The mean of paw edema volume, WBC count, segment neutrophil and lymphocyte of were not decreased with control group. The mean of PGE2 concentration ($1,851.00{\pm}160.11$) and NADPH-d positive neurons ($13.12{\pm}1.23$) were changed significantly (p

• BMB Reports
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• 제29권6호
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• pp.507-512
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• 1996

The NADPH oxidase of phagocytes catalyzes the reduction of oxygen to $O_{2}^{-}$ at the expense of NADPH The enzyme is dormant in resting neutrophils and hecomes activated on stimulation. During activation. $p47^{phox}$ (phagocyte oxidase factor), a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303-S379. Although the biochemical role of phosphorylation is speculative, it has been suggested that phosphorylation could neutralize the strongly cationic C-terminal which may result in the change of conformation of $p47^{phox}$ and subsequent translocation of this protein and other cytosolic components to the membrane. In order to mimic the effect of phosphorylation in terms of neutralizing the positive charges, recombinant $p47^{phox}$ was treated with phenylglyoxal, which removes positive charges of arginine residues. Modification of recombinant $p47^{phox}$ resulted in the activation of oxidase in a cell-free translocation system as well as a conformational change in recombinant $p47^{phox}$ which may be responsible for the activation of the enzyme.

• 약학회지
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• 제59권5호
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• pp.201-206
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• 2015

The aim of the present study was to optimize in vitro method for the prediction of drug-induced liver injury using human liver microsomes (HLM). Cytotoxicity test of cyclophosphamide and acetaminophen in HepG2 cells cultured with HLM showed that the newly established condition using 0.375 mg/ml HLM for 24 hr incubation was comparable or more sensitive than the previously established condition using 0.75 mg/ml HLM for 12 hr incubation. Although the cytotoxic effect of troglitazone was completely attenuated by 0.75 mg/ml HLM, it was augmented by 0.375 mg/ml HLM in the presence of the NADPH-generating system. The cytotoxic effect of chlormezanone, a withdrawn drug due to hepatotoxicity in human, was increased by HLM in the presence of the NADPH-generating system. In contrast, the cytotoxic effect of methapyrilene, a withdrawn drug due to hepatotoxicity in rats, was decreased by HLM in the presence of the NADPH-generating system. The present study suggests that the optimized in vitro method using HLM can be useful for the prediction of drug-induced hepatotoxicity.

• Archives of Pharmacal Research
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• 제8권3호
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• pp.119-132
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• 1985

The apparent effectiveness of 1-naphthyl-N-methyl carbamate (carbaryl) against a wide variety of insects motivated the study of its mammalian toxicity. In this toxicological study of carbaryl, mature male rats inhaled carbaryl at a mean concentration of 112mg, 168mg and 224 mg/$m^{3}$ for one hour. After inhalation, pentobarbital sleeping time, Nadph-cytochrome c reductase activity, cytochrome p-450 and protein content in liver microsomes, various tissue residues, cholinesterase inhibition in plasma and histopathological findings at autopsy were observed. The pentobarbital sleeping time was prolonged in rats inhaled with carbaryl for one day while the sleeping time was shortened in the 3 days inhaled group. The changes of cytochrome p-450 content and NADPH-cytochrome c reductase activity exhibited biphasic response showing the decrease in the one day inhaled group and the increase in the 3 days inhaled group. The marked depression of plasma ChE activity was observed in rats inhaled with carbaryl at 112 mg/$m^{3}$, however no more progressive effect was observed at the higher concentration of the compound. The main observations in histopathological finding were ciliary detachment, epithelial swelling and subepithelial inflammatory cellular infiltration in trachea due to the irritation.