• Bulletin of the Korean Chemical Society
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• v.22 no.11
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• pp.1192-1196
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• 2001

A diaphorase enzyme electrode for the catalytic reduction of NAD+ , the oxidized form of nicotinamide adenine dinucleotide, has been prepared. The enzyme layer grew spontaneously over an aminoethanethiol self assembled monolayer on a go ld plate electrode. The growth was accomplished by simply dipping the electrode covered by the aminoethanethiol monolayer into a solution containing both glutaraldehyde and diaphorase. We suggested that the glutaraldehyde as a cross-linking reagent was attached to the amino groups of the aminoethanethiol monolayer and the diaphorase enzyme molecules were bound to free aldehyde groups of the glutaraldehyde. Further attachments of the enzyme molecules over the bound enzyme molecules continued with the bridging of the glutaraldehyde. In frequency measurements with a quartz crystal microbalance, the frequency decrease was much more than it was for that of the enzyme monolayer formation, and an enzyme layer thicker than a monolayer was formed. The modified electrode was employed to reduce NAD+ , using diffusional methyl viologen as an electron transfer mediator. The NAD+ was electrocatalytically reduced, and the catalytic current was almost equivalent to that with the multilayered electrode of ten enzyme layers.

• Journal of Acupuncture Research
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• v.21 no.5
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• pp.1-12
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• 2004

Objective : This study was to investigate the effect of acupuncture on NADPH-diaphorase and nNOS in the brain stem and cerebellum of spontaneously hypertensive rats. Methods : The experimental groups were divided into four groups : Normal, Choksamni(ST36), Kokchi(LI11), arbitrary group. Thereafter we evaluated changes in NADPH-diaphorase positive neurons histochemically and changes in nNOS neurons immunohistochemically. Results : 1. The optical densities of NADPH-diaphorase positive neurons of all the Choksamni & Kokchi groups were significantly different in SuG, DLPAG, IP, Pr, Gi areas of brain stem and cerebellum as compared to normal & arbitrary groups. In PPTg only Choksamni group was significantly different as compared to normal and arbitrary groups. 2. The optical densities of nNOS-positive neurons of Choksamni & Kokchi groups were significantly different in SuG, DLPAG areas of brain stem as compared to normal group. In IP, Pr only Kokchi group was significantly different as compared to normal group. The optical densities of nNOS-positive neurons of Choksamni & Kokchi groups were significantly different in SuG, DLPAG, PPTg, Pr, Gi areas of brain stem as compared to arbitrary group. In IP, Pr only Kokchi group was significantly different as compared to arbitrary group. 3. The optical densities of nNOS-positive neurons of all the Choksamni & Kokchi groups were not significantly different in cerebellum as compared to normal & arbitrary groups. Conclusions : We found out that acupuncture have effects on NADPH-diaphorase and nNOS in the brain stem and cerebellum of spontaneously hypertensive rats.

• Journal of Ginseng Research
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• v.17 no.2
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• pp.123-126
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• 1993

Effects of chronic administration of ginseng extracts (30 or 150 mg/kg/day for 52 days, p.o.) to mice on the activities of DT-diaphorase and glutathione S-transferase (GST) in the liver and the brain were studied. The DT-diaphorase activity in the liver was increased over 2-fold at the dose of both 30 and 150 mg/kg/day, while there was no change in the activity of the enzyme in the brain. The GST activity in the liver was increased in a dose-dependent fashion up to 142% of the control value at the dose of 150 mg/kg/day. while there was no change in the activity of the enzyme in the brain. The ginseng-induced increase in the activities of these hepatic phase II drug-metabolizing enzymes which are involved in the detoxification of carcinogens, is suggested to underlie, at least in part, the anticarcinogenic activity of Panax ginseng.

• Biomolecules & Therapeutics
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• v.3 no.1
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• pp.58-62
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• 1995

We investigated whether nitric oxide (NO) may serve a role in bladder function by immunohistochemical analysis of the distribution of intrinsic NADPH-diaphorase and functional study of isometric tension recordings via a photo-induced adequate nitric oxide (PIANO) generating system using rat bladder. Results suggest that a small number of NADPH-diaphorase-positive perikarya are present within the bladder wall and within adjacent small ganglia. Furthermore, NADPH-diaphorase-positive nerve fibers were observed in the adventitial and muscular layers, subjacent to the urothelium and perivascular fibers. Rat bladder strips precontracted with 3$\mu$M carbachol were reversibly relaxed upon NO generation by UV irradiation. PIANO-mediated relaxation was sensitive to oxygen free radicals. In addition, tissue cGMP levels were increased by the PIANO generating system and elevated cGMP levels were decreased by pretreatment of guanylate cyclase inhibitor, methylene blue. These results indicate that NO may serve a role in modulating bladder tone in the rat.

• Journal of Electrochemical Science and Technology
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• v.5 no.1
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• pp.19-22
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• 2014

Here we report a highly sensitive and stable detection of $NAD^+$ and NADH by the electrode on which diaphorase (DI) is covalently immobilized in viologen polymer network. The network is prepared by the covalent formation of the structure by mixing propylamine viologen (PAV), poly(ethylene glycol)(400) diglycidyl ether (PEGDGE), an diaphorase (DI). The PAV/PEGDGE/DI modified electrode has the sensitivity of $0.02nA{\cdot}{\mu}M$ and the detection limit of $3{\mu}M$ with a response time of 2 s ($t_{90%}$) for NADH sensing.

• Journal of Acupuncture Research
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• v.20 no.6
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• pp.148-159
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• 2003

Objective: This study examines the effects of acupoint combination on NADPH-diaphorase and neuronal nitric oxide synthase(nNOS) in the brainstem of spontaneously hypertensive rats. Methods: The changes of NADPH-d-positive neurons using a histochemical method and the changes of nNOS-positive neurons using an immunohistochemical method were evaluated. The optical densities of NADPH-d-positive neurons and nNOS-positive neurons of the Choksamni(ST36) Umnungchon(SP9) groups were significantly increased in all brainstem areas as compared to the Choksamni and Choksamni Kokchi(LI11) groups and decreased, with the exeption of the nNOS-positive neurons in the superficial gray of superior colliculus, as compared to the normal group. Results: Our results demonstrated that electroacupuncture changes the activity in the NO system in the brainstem of SHR and the acupoint combination is one of the important parameters for this effect.

• Toxicological Research
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• v.13 no.3
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• pp.251-256
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• 1997

Toluene inhalation increases glutamate level and its receptor in various brain regions. In this study, nitric oxide synthase (NOS) activities were investigated in various rat brain regions using NADPH diaphorase staining method which examined histochemical changes of NOS in the neural cells. Also, in vitro LDH leakage assay and MTT test were performed to investigate the toxic influences of toluene in cultured granule cell of rat cerebellum which was significantly affected with toluene in vivo. Rats were exposed to toluene of 10000 ppm for 3 days. 7 days and 14 days by 20 min $\times$ 2 times a day. NADPH diaphorase staining was processed in the different brain regions after inhalation. NADPH diaphorase staining density was not significantly changed at 3 days inhalation group, but the density decreased in proportion to the duration of toluene inhalation. Over 30% of staining density was decreased at 14 days group which was maximum duration of inhalation in this study. The tendency of staining density decrease was significant in granule cell of cerebellum. Cell death by toluene exposure was observed in cultured cerebellar granule cell. $EC_{50}$ measured with LDH leakage assay and MTT test were 43 mM and 72 mM respectively.

• Journal of Electrochemical Science and Technology
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• v.2 no.3
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• pp.163-167
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• 2011

Nicotinamide adenine dinucleotide, $NAD^+$, and its reduced form, NADH, play important roles as coenzymes in many enzymatic reactions. Electrochemical methods for $NAD^+$ or NADH detection or generation are drawn attention because it can provide the simple and low cost platform with fairly good sensitivity. In this study, the polysiloxane viologen polymer/diaphorase/hydrophilic polyurethane (PSV/DI/HPU) modified electrodes were simply prepared and demonstrated for bio-electrocatalytic $NAD^+$ sensors. The electrodes were co-immobilized with diaphorase and polysiloxane viologen polymer as an electron mediator followed by the overcoating with HPU membrane. The mixture of the enzyme and the electron mediator was well stabilized within HPU membrane and exhibited good reversibility and stability. The sensitivity was 0.2 $nA{\cdot}{\mu}M^{-1}$ and the detection limit was 28 ${\mu}M$ with a response time of 50 s ($t_{90%}$). The capability for NADH sensor was also observed on the PSV/DI/HPU electrode.

• Journal of Nutrition and Health
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• v.33 no.5
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• pp.491-496
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• 2000

Food intake is regulated by both central and peripheral mechanisms. In the central nervous, the hypothalamus acts for autonomic and endocrine homeostasis. The paraventricular nucleus(PVN) of hypothalamus is an imprtant site of interaction in central feeding pathways. Neuroepetide Y(NPY)is one of the most powerful neurochemical stimulants of food intake known. Also brain nitric oxide(NO), known as neurotransmitter, is involved in the mechanisms that regulate food intake. In this experiment, 24h fasting mice and anorexia mutant mice have been to examine the expression of NPY, which is the major neuropeptide increasing food intake. Double staining with NPY and nicotinamide-adenine-dinucleotide-phosphate diaphorase(NADPH-d), followed by immunohistochemical method and image analysis, have been used to observe coexisting neurons and the level of expression of each neurons. The results were as follows. 1) NPY-immunoreactivitys reduced immune response of the hypothalamus, particularly paraventricular nucleus(PVN), in anorexia mutant mice. Decreased level of NPY is assumed to be a major pathological factor in anorexia mutant mice. On the other hand, PVN in hypothalamus of fasting mice showed increased immunoreactivity which is in agreement of other researchers. 2) NPY and NADPH-d double staining revealed coexisting neurons in the cerebral cortex. Fasting mice had a tendency to have increased level of coexisting neurons compared to the control group. Compared to the control group, fasting mice express is not increase level of NPY-immunoreactivity, while anorexia mutant mice tended to have a decreased level.

• Journal of Pharmacopuncture
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• v.6 no.2
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• pp.57-65
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• 2003

Objective : This study was carried out to systemically investigate the effect of Joongwan(中脘; CV12) acupuncture in cerebrum and cerebellum of indomethacin-induced gastrointestinal disease in SD-rats. Method : 1. We induced gastrointestinal disease by indomethacin oral administration in SD-rats. 2. We selected Joongwan(中脘; CV12) acupuncture point that generally have been used to treat gastrointestinal disease. 3. We categorized SD-rats into three groups as followings. (1) Normal group : The group without any management (2) Control group : The group with indomethacin-induced gastrointestinal disease (3) Treated group : The group that Joongwan(中脘; CV12) acupuncture was performed after inducing gastrointestinal disease 4. We figured out the effect of acupuncture by analyzing staining degree of NADPH-diaphorase in cerebrum and cerebellum. Results : 1. Cerebrum (1) Normal group : The degree of staining was very low. (2) Control group : NADPH-diaphorase was mainly stained in cerebral cortex and the stained region was wider than Normal group. (3) Treated group : The degree and region of staining was higher and wider than the other goups. Sometimes the intensively stained regions were observed. 2. Cerebellum In both cases of Control group and Treated group, the regions in cortex were stained mainly. But, between Control group and Treated group, there was no remarkable difference. Conclusion : In case of cerebellum, there was no remarkable result. On the other hand, in case of cerebrum, there were certain differences among three groups. Through those results, we could conclude that Joongwan(中脘; CV12) acupuncture treatment was able to affect NADPH-diaphorase expression in the cerebrum of SD-rats that have gastrointestinal disease with indomethacin-inducing.