• Journal of Microbiology and Biotechnology
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• 제31권1호
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• pp.154-162
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• 2021

L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in Lactococcus lactis for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 ㎍/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing folE encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 ㎍/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5-MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 ㎍/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 ㎍/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 ㎍/l. To the best of our knowledge, this titer is the highest achieved by L. lactis. This study lays the foundation for further engineering of L. lactis for efficient 5-MTHF biosynthesis.

• Animal Bioscience
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• 제35권2호
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• pp.155-165
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• 2022

Objective: This study was performed to examine whether the porcine glutamic-oxaloacetic transaminase 1 (GOT1) gene has important functions in regulating adipocyte differentiation. Methods: Porcine GOT1 knockout and overexpression vectors were constructed and transfected into the mouse adipogenic 3T3-L1 cells. Lipid droplets levels were measured after 8 days of differentiation. The mechanisms through which GOT1 participated in lipid deposition were examined by measuring the expression of malate dehydrogenase 1 (MDH1) and malic enzyme (ME1) and the cellular nicotinamide adenine dinucleotide phosphate (NADPH) content. Results: GOT1 knockout significantly decreased lipid deposition in the 3T3-L1 cells (p<0.01), whereas GOT1 overexpression significantly increased lipid accumulation (p<0.01). At the same time, GOT1 knockout significantly decreased the NADPH content and the expression of MDH1 and ME1 in the 3T3-L1 cells. Overexpression of GOT1 significantly increased the NADPH content and the expression of MDH1 and ME1, suggesting that GOT1 regulated adipocyte differentiation by altering the NADPH content. Conclusion: The results preliminarily revealed the effector mechanisms of GOT1 in regulating adipose differentiation. Thus, a theoretical basis is provided for improving the quality of pork and studies on diseases associated with lipid metabolism.

• 약학회지
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• 제39권4호
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• pp.450-460
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• 1995

The acute toxicity of fthalide in rat was studied in vivo by the observations of the changes in hematogram, serological parameters, content of cytochrome p-450, activities of NADPH-cytochrom c reductase, glucose-6-phosphatase, and the contents of cholinesterase and carboxylesterase in liver. Fthabde is a practically non-toxic substance(LD50 is 3.86g/kg), but rats were intoxicated with fthabde at a oral dose of 100 mg/kg for 12 days. WBC were significantly decreased and activities of ALT and LDH, on the cotrary, the content of glucose in serum were slightly increased. Cytochrome p-450 and lipid peroxide in liver were significantly increased in the fthalide-intoxicated rats. The longer administration of fthalide showed further increase of carboxylesterase activity in liver and serum, but decrease of activities of glucose-6-phosphatase and cholinesterase in liver and serum. These results show that fthatide can induce the hepatocellular injury and neurotoxicity.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.771-774
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• 2001

NADPH has been known as a regulating factor the biosynthesis of polyhydroxyalkanote(PHA), and the flux of NADPH for PHA biosynthesis could be enforced by the amplification of zwf gene encoding glucose 6-phosphate dehydrogenase. The recombinant plasmid pCZWF harboring PHA synthase, phbC from R. eutropha and zwf from E. coli were constructed, and were transformed to R. eutropha by electroporation. The biosynthesis of P(3HB-3HV) copolymer were carried out in transformant R. eutropha through the two-stage cultivation method using valerate as a precursor. The biosynthesis rate and PHA content of transformant R. eutropha harboring pCZWF were increased compared with transformant R. eutropha harboring only phbC. Especially, the molar fraction of 3HV was increased from 68% to 74% due to amplification of zwf gene. And the biosynthesis P(3HB-3HV) and P(3HB-4HB) carried out using propionate and ${\gamma}-butyrolactone$ as a precursor, respectively. But the rate, content, and molar fraction of biosynthesis copolymers were not influenced appreciably. This may be due to the reduced availability of NADPH.

• IMMUNE NETWORK
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• 제14권5호
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• pp.241-248
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• 2014

It is debatable whether AMP-activated protein kinase (AMPK) activation is involved in anti-apoptotic or pro-apoptotic signaling. AICAR treatment increases AMPK-${\alpha}1$ phosphorylation, decreases intracellular reactive oxygen species (ROS) levels, and significantly increases Annexin V-positive cells, DNA laddering, and caspase activity in human myeloid cell. AMPK activation is therefore implicated in apoptosis. However, AMPK-${\alpha}1$-knockdown THP-1 cells are more sensitive to apoptosis than control THP-1 cells are, suggesting that the apoptosis is AMPK-independent. Low doses of AICAR induce cell proliferation, whereas high doses of AICAR suppress cell proliferation. Moreover, these effects are significantly correlated with the downregulation of intracellular ROS, strongly suggesting that AICAR-induced apoptosis is critically associated with the inhibition of NADPH oxidase by AICAR. Collectively, our results demonstrate that in AICAR-induced apoptosis, intracellular ROS levels are far more relevant than AMPK activation.

• Journal of Microbiology
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• 제34권2호
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• pp.137-143
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• 1996

We isolated a promoter inducible by paraquat, a superoxide-generating agent, from Escherichia coli using a promoter-probing plasmid pRS415. From sequence analysis we found out the promoter is for fpr ENCODING nadph : ferredoxin oxidoreductase. We constructed on operon fusion of lacZ gene with fpr promoter to monitor the expression of the gene in the single-copy state. LacZ expression generators, menadione and plumbagin, also induced the expression of .betha.-galactosidase in the fusion strain. On the other hand, no significant induction was observed by treatment with hydrogen peroxide, ethanol, and heat shock. Induction of .betha.-galactosidase was significantly reduced by introducing a .DELTA. sox 8 :: cat of soxS3 :: Tn10 mutation into the fusion strain, indicating that fpr gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis. Possible roles of fpr induction in superoxide stress were discussed.

• 대한약리학회지
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• 제25권1호
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• pp.75-85
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• 1989

면역 보체가 결합되어 있는 zymosan에 의하여 활성화된 다형핵 백혈구에서 세포 투과성 물질인 N-ethylmaleiamide과 $Hg^{++}$은 superoxide 라디칼 생성, NADPH oxidase 활성도 및 lysosomal enzyme (lactic dehydrogenase, ${\beta}-glucuronidase$)의 유리를 억제하였다. 세포막 단백에 특이적인 p-chloromercuribenzoic acid와 p-chloromercuribenzenesulfonic acid는 superoxide 라디칼 생성에 영향을 주지 않았으나 NADPH oxidase 활성도와 lysosomal enzyme의 유리를 억제하였다. 식작용 중에 세포막과 세포내의 sulfhydryl기는 반응시간에 따라 점진적으로 감소하였다. N-ethylmaleiamide와 $Hg^{++}$은 세포막과 세포내의 sulfhydryl기를 모두 감소시켰다. P-Chloromercuribenzoic acid와 p-chloromercuribenzenesulfonic acid는 세포막의 sulfhydryl기를 유의하게 감소시켰으나 세포내 용해성 sulfhydryl기에는 영향을 주지않았다. Cysteine과 mercaptopropionylglycine는 superoxide 라디칼의 생성과 lysosomal enzyme의 유리를 억제하였다. Gluthathione은 superoxide생성에 영향을 주지 않았으나 뚜렷하게 lactic dehydrogenase의 유리를 억제하였다. N-ethylmaleiamide에 의한 superoxide 생성의 억제는 cysteine과 mercaptopropionyl-glycine에 의하여 반전되었으나 gluthathione의 영향은 없었다. N-ethylamleiamide에 의한 NADPH oxidase의 비활성화는 gluthathione, cysteine과 mercaptopropionylglycine에 의하여 저해되었다. Carbachol에 의하여 항진된 superoxide 라디칼 생성은 N-ethylamleiamide에 의하여 완전히 억제되었고, atropine에 의하여 길항되었다. 그러므로, 외부 자극에 대한 다형핵 백혈구 반응의 표현은 sulfhydryl기의 양의 변화와 연관이 있을 것으로 시사되었다. Lysosomal enzyme 유리는 세포막과 세포내의 sulfhydryl기에 의하여, 이에 반하여 superoxide생성은 세포내 sulfhydryl기에 의해서 영향받을 것으로 추정되었다.

• Journal of Acupuncture Research
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• 제21권2호
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• pp.205-222
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• 2004

Objective: To investigate the effects of acupointed Choksamni(ST36), Kokchi(LI11) and Arbitrary acupoint on NADPH-diaphorase, neuronal nitric oxide synthase(nNOS), neuropeptide Y(NPY) and vasoactive intestinal peptide(VIP) in the cerebral cortex of spontaneously hypertensive rats. Methods: The experimental groups were divided into four groups: Normal, Choksamni(ST36), Kokchi(LI11), arbitrary group. Needles were inserted into acupoints at the depth of 0.5 cm with basic insertion method. Such stimulation was applied continuously for 10 minutes, every other day, for 10 sessions of treatments. Thereafter we evaluated changes in NADPH-d-positive neurons histochemically and changes in nNOS, NPY and VIP-positive neurons immunohistochemically. Results : The optical densities of NADPH-d-positive neurons of all the Choksamni & Kokchi groups were significantly different in all areas of cerebral cortex as compared to arbitrary group. In motor1, sensory2, cingulate2, insular, peripheral, visual cortex there was a significant difference between Choksamni & Kokchi group. The optical densities of nNOS-positive neurons of Choksamni group were significantly different in all areas except for auditory, visual and pisiform cortex and Kokchi group in all areas except for auditory and pisiform cortex as compared to arbitrary group. And there was a significant difference in cingulate1, cingulate2, ectohinal, visual cortex between Choksamni & Kokchi group. The optical densities of NPY neurons of Choksamni group were significantly different in cingulate2, insular, pisiform cortex and Kokchi group in motor1, motor2, sensory1, cingulate2, ectorhinal cortex as compared to arbitrary group. And there was no significant difference between Choksamni & Kokchi group. The optical densities of VIP neurons of Choksamni group were significantly different in all areas except for motor1, auditory cortex and Kokchi group in sensory1, insular, ectorhinal, perirhinal, visual, pisiform cortex as compared to arbitrary group. And there was a significant difference in cingulate1, cingulate2, retrosplenial, auditory corterx between Choksamni & Kokchi group. Conclusion : Our results demonstrated that acupuncture on Choksamni(ST36) & Kokchi(LI11) changes the control activities of the NO system in the cerebral cortex of SHR and according to areas there were significant difference between two groups. In all cerebral cortex areas there were distributed NPY & VIP and there were no significant difference among Choksamni(ST36), Kokchi(LI11) and arbitrary group.

• 대한약리학회지
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• 제24권1호
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• pp.83-92
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• 1988

세포질 내 칼슘 농도의 증가는 다형핵 백혈구의 산화성 대사를 자극하는 주요 인자로 여겨지고 있다. 활성화된 다형핵 백혈구로부터 lysosomal enzyme의 유리는 세포내 cyclic nucleotide농도에 따라 조절될 수 있지만 신경전달물질과 ${\beta}$-아드레날린 또는 무스카린성 수용체의 결합에 따른 그밖의 반응은 아직도 분명하지 않다. 덧붙여, ${\alpha}$-아드레날린성 수용체의 중개에 의한 다형핵 백혈구의 기능은 알려져 있지 않다. Atropine, phentolamine과 propranolol은 활성화된 다형핵 백혈구의 칼슘흡수, superoxide 생성, NADPH oxidase 활성도 그리고 식작용을 억제하였으며, 이에 반하여 carbachol과 isoproterenol은 활성화된 세포의 반응을 약간 더 자극하였다. Carbachol또는 isoproterenol 의하여 항진된 superoxide 생성은 각각 그들의 길항제인 atropine과 propranolol 의하여 억제되었다. 활성화된 다형핵 백혈구의 반응은 chlorpromazine, verapamil과 dantrolene에 의하여 억제되었으나 lithium에 의 하여 약긴 항진되었다. 한편 chlorpromazine과 dibucaine은 NADPH oxidase 활성도에 영향을 주지 않았다. Atropine, phentolamine과 propranolol은 칼슘에 의존적인 식작용을 억제 하였다. 이상의 결과로부터 atropine, phentolamine과 propranolol은 칼슘 유입을 억제하고 자율 신경계의 수용체와 연관이 있는 NADPH oxidase계에 직접 작용함으로써 활성화된 다형핵 백혈구로부터 superoxide 생성을 억제할 것으로 시사되었다.

• 한국식품위생안전성학회지
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• 제12권2호
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• pp.97-101
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• 1997

Effect of allylisothiocyanate on the enzyme activites including malate degydrogenase, isocitrate dehydrogenase, NADPH and acetyl CoA which were related to aflatoxin production of Aspergillus parasticus R-716 were invetigated. The activities of malate dehydrogenase (EC.1.1.1.37), isocitrate dehydrogenase (E.C.1.1.1.42) and NADPH oxidase (E.C.1.6.99.1) indicated relatively high in the 50 ppm allylisothiocyanate-added-culture. In contrast, the activity of acetyl CoA in the 50 ppm allylisothiocyanate-added-culture showed rather lower level through the cultivation.