• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.983-989
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• 2017

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of $NADP^+$ in the affinity chromatography process. In the present study, the rat NPR clone containing a $6{\times}$ Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using $Ni^{2+}$-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.141-146
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• 2012

Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7-ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

• Toxicological Research
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• v.20 no.4
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• pp.307-313
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• 2004

The present study was undertaken to investigate the effects of mistletoe (Viscum album var. coloratum) growing on Carpinus laxiflora BL. on proliferation and differentiation of HL-60 acute promyelocytic leukemia cells. Aqueous extract and its $(NH_2)_2SO_4$ saturated fractions of the mistletoe exhibited potent anti-proliferation activity against HL-60 cells. Moreover, when HL-60 cells were treated with 0~30% and 30～70% $(NH_2)_2SO_4$ saturated fractions of the mistletoe, HL-60 expressed CD 66b or CD 14 cell surface antigens and showed activity to reduce nitroblue tetrazolium, indicating that mistletoe induces the differentiation of HL-60 into granulocytes or monocytes. To understand how mistletoe induces the differentiation, we investigated the expression of molecules for modulating the proliferation and differentiation of leukemia cells, such as c-Myc and myeloblastin. The 0~30% $(NH_2)_2SO_4$ saturated fraction of the mistletoe reduced the mRNA levels of c-Myc and myeloblastin in a time-dependent manner. The results indicate that the mistletoe induces the differentiation of HL-60 cells via the decrease of c-Myc and myeloblastin expressions. Thus, it is suggested that mistletoe has a therapeutic potential for the treatment of acute promyelocytic leukemia.

• Journal of fish pathology
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• v.21 no.3
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• pp.219-228
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• 2008

The effects of dietary yeast β-glucan administration on growth, nonspecific immune responses, serum lysozyme, skin mucous lysozyme, NBT (nitroblue tetrazolium) reduction by phagocytes, and disease resistance against Edwardsiella tarda in Japanese eel, Anguilla japonica were evaluated. Fish were fed the diets supplemented with 0%, 0.1% and 0.5% of yeast β-glucan to a commercial diet for 6 weeks. The body weight gain from the fish fed on the 0.5% supplemented diet for 6 weeks was significantly higher than the control. Both serum and skin mucous lysozyme were significantly higher in the all experimental groups except 2 weeks of 0.5% group. The bactericidal activity of serum was slightly increased at 6 weeks. Also, The intracellular superoxide anion production of kidney phagocytes was significantly higher in the all experimental groups. The diet supplemented with 0.1% were also found to raise the relative percent survival (RPS) of Japanese eel after an artificial challenge with 1×107 cells of Edwardsiella tarda per fish. The results suggested the potential of yeast β-glucan to activate some innate immune responses and to improve the growth in Japanese eel.

• Fisheries and Aquatic Sciences
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• v.20 no.9
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• pp.20.1-20.8
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• 2017

The objective of this study was to examine the effect of dietary supplementation of taurine for juvenile olive flounder (Paralichthys olivaceus) at low water temperature ($16.4{\pm}0.36^{\circ}C$). Fish meal (FM)-based diet was used as the control diet. Four other experimental diets were prepared by adding taurine to FM-based diet at 0.25, 0.50, 1.00, and 1. 50% (T1, T2, T3, and T4, respectively). Each experimental diet was fed to triplicate groups of fish (initial mean body weight, 19.5 g) for 10 weeks. At the end of the feeding trial, growth performance and feed utilization, hematological parameters, non-specific immune responses, whole-body proximate composition, and liver mRNA expression of insulin-like growth factor-1 (IGF-1) were investigated. Feed conversion ratio was significantly reduced while protein efficiency ratio was significantly increased in taurine-supplemented groups. Hematocrit and hemoglobin were also significantly increased while plasma cholesterol levels were decreased in taurine-supplemented groups than those in the control group. Nitroblue-tetrazolium, myeloperoxidase and lysozyme activities, and plasma immunoglobulin level were significantly increased by taurine supplementation. These results suggest that dietary taurine supplementation is effective in improving growth performances, feed utilization, and innate immunity of olive flounder in low water temperature season.

• The Korean Journal of Zoology
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• v.34 no.2
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• pp.173-180
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• 1991

The objective of this experiment was to examine the effect of polyamine depletion by polyamine biosynthesis inhibitors on microbicidal activity and tumoricidal activity in mouse mac-rophages. $\alpha$ -Difluoromethylomithine (DFMO), inhibitor of putrescine and spernidine biosynthesis, treatment in vivo for 6-8 days reduced chemiluminescence levels in thioglycollate-, lipo-polysaccharide (LPS), and BCG-treated mouse macrophages. An DFMO treatment in vitro inhibited production of tumor necrosis factor (TNF), in dose-dependent manner, and tumoricidal activity by macrophages. The effect of polyamine depletion by MO on ThF production and tumoricidal activity could be reversed by the addition of exogenous putrescine. These result indicated that the obserbed effect of DFMO on macrophage activities were mediated through inhibition of polyamines are, must be, required for optimal activities of macrophages.

• Animal cells and systems
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• v.4 no.3
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• pp.299-304
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• 2000

In the present paper, the immunostimulatory effects of the extracellular products (ECP) from Mycobacterium spp. and various adjuvants on the non-specific immune responses of Nile tilapia, Oreochromis nilotica, were examined. Nile tilapia were immunized by injecting ECP of Mycobacterium spp. (strain TB40, TB267 or the type strain Mycobacterium marinum) into their swim bladders. A variety of adjuvants like as Freund s complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and Titremax were similarly injected into additional groups of tilapia. The number of nitroblue tetrazolium (NBT)-positive cells observed in the swim bladder of the immunized fish was signigicantly increased by the fourth day post-immunization. By day 8, the numbers of NBT-positive cells were fewer in fish immunized with ECP from mycobacteria strains TB40 or TB267 than those immunized with ECP from M. marinum or fish injected with FCA or FIA. The level of Iysozyme activity detected in the serum of fish 40 alter immunization with ECP from various Mycobacterium spp. was also significantly higher than that found in the serum of the control fish. Head kidney macrophages showed enhanced reduction of NBT when cultured in vitro with 1 $\mu$ g/ml of ECP. Concentrations greater than this (10 or 100 $\mu$g/ml) were found to suppress the reduction of NBT by the macrophages. ECP from Mycobacterium spp. and the various adjuvants used in the study all appear to be good activators of the non-specific immune responses of Nile tilapia.

• Biomolecules & Therapeutics
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• v.17 no.2
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• pp.156-161
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• 2009

The human cytochrome P450 4A11 is the major monooxygenase to oxidize the fatty acids and arachidonic acid. The production of 20-hydroxyeicosatetraenoic acid by P450 4A11 has been implicated in the regulation of vascular tone and blood pressure. Oxidation reaction by P450 4A11 requires its reduction partners, NADPH-P450 reductase (NPR). We report the functional expression in Escherichia coli of bicistronic constructs consisting of P450 4A11 encoded by the first cistron and the electron donor protein, NPR by the second. Typical P450 expression levels of wild type and several N-terminal modified mutants was observed in culture media and prepared membrane fractions. The expression of functional NPR in the constructed P450 4A11: NPR bicistronic system was clearly verified by reduction of nitroblue tetrazolium. Membrane preparation containing P450 4A11 and NPR efficiently oxidized lauric acid mainly to $\omega$-hydroxylauric acid. Bicistronic coexpression of P450 4A11 and NPR in E. coli cells can be extended toward identification of novel drug metabolites or therapeutic agents involved in P450 4A11 dependent signal pathways.

• Fisheries and Aquatic Sciences
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• v.24 no.4
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• pp.153-162
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• 2021

In this study, a fish metabolic accelerator (a combination of butaphosphan and cyanocobalamin [BPC]) was injected into the muscle of the olive flounder, Paralichthys olivaceus, to investigate its effect on immunity and stress in fish maintained at low temperatures. A single dose of BPC was injected (100 mg/kg body weight) into the olive flounder, and its immunity and stress were observed after one and two weeks. Immunity tests revealed the presence of lysozyme (LZM), nitroblue tetrazolium (NBT), myeloperoxidase (MPO), anti-protease (AP), glutathione peroxidase (GPx), and total immunoglobulin (TIg). BPC injection was found to increase immunity activity compared to the control group. In particular, there was significantly high GPx activity. There was similarly high activity for MPO and GPx in the first week following the injection, followed by significant differences between the BPC-injected and control groups in the second week. There was a reduced low water-temperature stress response in the BPC-injected fish, as evidenced by the cortisol and glucose levels of the control and BPC groups. Lower levels were also observed in the BPC group than the control group during the second week. Cortisol levels were significantly lower in the BPC group than the control group. Histological examinations were conducted in the first and second weeks after the intramuscular injection of the recommended BPC dose to confirm the safety of BPC in aquaculture. There were no abnormalities observed in any tissue samples. This study confirms that the injection of BPC is safe even when used in a culture situation. BPC helps relieve stress and improves non-specific immune responses (innate immunity) in the olive flounder.

• Proceedings of the Ginseng society Conference
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• 2006.05a
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• pp.3-12
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• 2006

1 The present work was performed to investigate the effects of ginsenoside Rh2 on proliferation, cell cycle-regulation and differentiation of human leukemia HL-60 cells as well as the underlying mechanisms for these effects. 2 Ginsenoside Rh2 potently inhibited the proliferation of HL-60 cells in both a dose- and time-dependent manner with an $IC_{50}$, $20{\mu}M$. 3 DNA flow-cytometry indicated that ginsenoside Rh2 markedly induced a $G_1$ phase arrest of HL-60 cells. 4 Among the $G_1$ phase cell cycle-related proteins, the levels of cyclin-dependent kinase(CDK)4, 6 and cyclin D1, cyclin D2, cyclin D3 were reduced by ginsenoside Rh2, whereas the steadystate levels of CDK2 and cyclin E were unaffected. 5 The protein levels of a CDK inhibitor p16, $p21^{CIP1/WAF1}$ and $p27^{KIP1}$ were markedly increased by ginsenoside Rh2. 6 Ginsenoside Rh2 markedly enhanced the binding of $p21^{CIP1/WAF1}$ and $p27^{KIP1}$ with CDK2 and CDK6, resulting in the reduced activity of both kinases and the hypophosphorylation of Rb protein. 7 We furthermore suggest that ginsenoside Rh2 is a potent inducer of the differentiation of HL-60 cells, based on observations such as a reduction of the nitroblue tetrazolium level, an increase in the esterase activities and phagocytic activity, morphology changes, and the expression of CD11b, CD14, CD64 and CD66b surface antigens. 8 In conclusion, the onset of ginsenoside Rh2-induced the $G_0/G_1$ arrest of HL-60 cells prior to the differentiation is linked to a sharp up-regulation of the $p21^{CIP1/WAF1}$ level and a decrease in the CDK2, CDK4 and CDK6 activities. This is the first report demonstrating that ginsenoside Rh2 potently inhibits the proliferation of human promyelocytic HL-60 cells via the $G_1$ phase cell cycle arrest and differentiation induction.