• The Korean Journal of Physiology and Pharmacology
• /
• v.20 no.4
• /
• pp.325-332
• /
• 2016

Resveratrol, a phytoalexin, is reported to activate AMP-activated protein kinase (AMPK) in vascular cells. The blood-brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. The aim of this study was to elucidate the effects of resveratrol and the role of AMPK in BBB dysfunction induced by lipopolysaccharide (LPS). Exposure of human brain microvascular endothelial cells (HBMECs) to LPS ($1{\mu}g/ml$) for 4 to 24 hours week dramatically increased the permeability of the BBB in parallel with lowered expression levels of occluding and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P) H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by resveratrol. Finally, upregulation of AMPK by either AMPK-CA or resveratrol abolished the levels of LPS-enhanced NAD(P)H oxidase subunits protein expressions. We conclude that AMPK activation by resveratrol improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs.

• Journal of Life Science
• /
• v.24 no.1
• /
• pp.98-103
• /
• 2014

NAD(P)H quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes the two electron reduction of diverse substrates, including quinones. It uses NADH or NADPH as a cofactor for enzymatic machinery. In the metabolism of quinones, NQO1 has two conflicting functions because of the different stability of converted hydroquinones. The stable form of hydroquinone is excreted from cells by conjugation with glutathione or glucuronic acid. The unstable form of hydroquinone induces cell death by induction of oxidative stress and DNA damage. Certain quinones known as bio-reductive agents have a cytotoxic function following reduction by NQO1. Bio-reductive agents, such as ${\beta}$-lapachone or mitomycin C, induce the depletion of NAD(P)H and the generation of oxidative stress in an NQO1-dependent manner. NQO1 is highly expressed in several cancer tissues. Therefore, NQO1 is a good therapeutic target for cancer treatment with bio-reductive agents.

• Korean Journal of Microbiology
• /
• v.41 no.1
• /
• pp.67-73
• /
• 2005

Photoautotrophic bacteria are promising candidates for the production of poly(3-hydroxybutyrate) (PHB) since they can address the critical problem of substrate costs. In this study, we isolated 25 Tn5-inserted mutants of the Synechocystis sp. PCC 6803 which showed enhanced PHB accumulation compared to the wild-type strain. After 5-days cultivation under nitrogen-limited mixotrophic conditions, the intracellular levels of PHB content in these mutants reached up to $10-30\%$ of dry cell weight (DCW) comparable to $4\%$ of DCW in the wild-type strain. Using the method of inverse PCR, the affected genes of the mutants were mapped on the completely known genome sequence of Synechocystis sp. PCC 6803. As a result, the increased PHB accumulation in 5 mutants were found to be resulted from defects of genes coding for NADH-ubiquinone oxidoreductase, O-succinylbenzoic-CoA ligase, photosystem II PsbT protein or histidine kinase, which are involved in photosystem in thylakoid inner membrane of the cell. The values of $NAD(P)H/NAD(P)^+$ ratio in the cells of these mutants were much higher than that of the wild-type strain as measured by using pulse-amplitude modulated fluorometer, suggesting that PHB synthesis could be enhanced by increasing the level of cellular NAD(P)H which is a limiting substrate for NADPH-dependent acetoacetyl-CoA reductase. From these results, it is likely that NAD(P)H would be a limiting factor for PHB synthesis in Synechocystis sp. PCC 6803.

• The Korean Journal of Physiology and Pharmacology
• /
• v.9 no.2
• /
• pp.109-115
• /
• 2005

Endothelial activation and subsequent recruitment of inflammatory cells are important steps in atherogenesis. The increased levels of cell adhesion molecules (CAM) have been identified in diabetic vasculatures, but the underlying mechanisms remain unclear. To determine the relationship among vascular production of superoxide, expression of CAM and diabetes, superoxide generation and expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E- and P-selectin in the aorta from control (C57BL/6J) and diabetic mice (ob/ob) were measured. In situ staining for superoxide using dihydroethidium showed an increased superoxide production in diabetic aorta, accompanied with an enhanced NAD(P)H oxidase activity. Immunohistochemical analysis revealed that the endothelial expression of ICAM-1 ($3.5{\pm}0.4$) and VCAM-1 ($3.8{\pm}0.3$) in diabetic aorta was significantly higher than those in control aorta ($0.9{\pm}0.5$ and $1.6{\pm}0.3$, respectively), accompanied with the enhanced expression of gp91phox, a membrane subunit of NAD(P)H oixdase. Furthermore, there was a strong positive correlation (r=0.89, P＜0.01 in ICAM-1 and r=0.88, P＜0.01 in VCAM-1) between ICAM-1/VCAM-1 expression and vascular production of superoxide. The present data indicate that the increased production of superoxide via NAD(P)H oxidase may explain the enhanced expression of CAM in diabetic vasculatures.

• Applied Biological Chemistry
• /
• v.35 no.3
• /
• pp.157-164
• /
• 1992

In order to produce nicotinamide adenine dinucleotide (NAD) which is a pyridine nucleotide coenzyme, Saccharomyces sake KBA No. 6 having high NAD content was selected from 12 strains of yeast and various factors affecting the production of NAD were investigated. For NAD production, 4% of glucose was effective as a carbon source and 2% of bactopeptone was the best nitrogen source. The optimum pH and temperature was 5.0 and $30^{\circ}$, respectively. Also, when 4 mg/ml of nicotinamide and 3 mg/ml adenine were used as precursors simultaneously, NAD production was the best. To increase NAD production, 2 valence metal ions were used during cultivation and $Zn^{2+}$ was very efficient. Among the surface active agents, anionic sodium dodesyl sulfate (SDS) was effective. Under the optimum conditions, the maximum amount of produced NhD was 35 mg/100 ml medium after cultivation of 144 hrs and 89% of total NAD amount, 31 mg of NAD, was leaked into culture broth.

• Archives of Pharmacal Research
• /
• v.23 no.6
• /
• pp.554-558
• /
• 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.

• Animal cells and systems
• /
• v.9 no.4
• /
• pp.191-198
• /
• 2005

The effects of 6-aminonicotinamide (6-AN) on viability of IMR32 neuroblastoma cells in the presence of ATP or $NAD^+$ have been investigated. 6-AN caused marked reduction in cell viability and similar observations were also made with cells treated with 6-AN+ATP. However, cells treated with $6-AN+NAD^+$ showed cell viability similar to untreated cells. Morphologically, 6-AN and 6-AN+ATP treated cells showed loss of neurites, polyhedric shapes, shrinkage of cell bodies and formation of lysed cells, while $6-AN+NAD^+$ cells did not show any such changes. The flow cytometry analysis demonstrated that 6-AN increased cell population in $G_0/G_1$ phase and decreased cell population in Sand $G_2/M$ phase following a 72 h exposure. Western blot analysis showed that 6-AN stimulated a substantial increase in the level of the cdk inhibitor $p27^{kip1}$, but lowered the levels of cdk2, cyclin E and p-Rb. However, cdc25A and p53R2 were not significantly affected. Immunofluorscence staining of $p27^{kip1}$, cdk2, cyclin E and p-Rb revealed close correlation between the signal observed in the Western blot analysis. 6AN+ATP treated cells showed similar results obtained with 6-AN treated cells in expression of cdk2, cyclin E, p-Rb proteins and $p27^{kip1}$, $6-AN+NAD^+$ cells showed greater expression of cdk2, cyclin E and p-Rb than those in 6-AN and 6-AN+ATP treated cells. The results suggest that 6-AN induced the $G_0/G_1$ phase arrest in IMR32 neuroblastoma cell lines through the increase of $p27^{kip1}$ and the decrease of cdk2, cyclin E and p-Rb.

• Archives of Pharmacal Research
• /
• v.24 no.5
• /
• pp.390-396
• /
• 2001

Synthesized 6-arylamino-5,8-quinolinediones 4a-4j and 6-chloro-7-arylamino-5,8-isoquinolinediones 5a-5g were evaluated for effects on 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 5,8-quinolinediones 4 and 5,8-isoquinolinediones 5 affected the reduction potential by NQO1 activity and showed a potent cytotoxic activity against human cancer cell lines. The tested compounds 4a, 5c, 5f, and 5g were considered as more potent cytotoxic agents. The compounds 4d, 5b, 5c, 5e and 5g were comparable modulators of NQO1 activity.

• Microbiology and Biotechnology Letters
• /
• v.38 no.3
• /
• pp.227-234
• /
• 2010

Cytochrome P450 enzymes which require the supply of electrons from NAD(P)H have a great biotechnological impact as they catalyze valuable reactions on a vast variety of substrates. However, very limited biotechnological application has been reported so far due to their functional complexity, limited stability (instability) and, in most cases, low catalytic activity. In this present review, we introduce some possibilities for improving their defect by exploring electron transport system and substrate flexibility in field of Streptomyces cytochrome P450.

• Microbiology and Biotechnology Letters
• /
• v.41 no.2
• /
• pp.153-159
• /
• 2013

A mannitol dehydrogenase (MDH; EC 1.1.1.67) gene was cloned from the Sinorhizobium meliloti 1021 (KCTC 2353) genome and expressed in Escherichia coli. It was seen to have an open reading frame consisting of 1,485 bp encoding 494 amino acids (about 54 kDa), which shares approximately 35-55% of amino acid sequence identity with some known long-chain dehydrogenase/ reductase family enzymes. The recombinant S. meliloti MDH (SmMDH) showed the highest activity at $40^{\circ}C$, and pH 7.0 (D-fructose reduction) and pH 9.0 (D-mannitol oxidation), respectively. SmMDH could catalyze the oxidative/reductive reactions between D-mannitol and D-fructose in the presence of $NAD^+/NADH$ as a coenzyme, but not with NADP+/NADPH. These results indicate that SmMDH is a typical $NAD^+/NADH$-dependent mannitol dehydrogenase.