• Maxillofacial Plastic and Reconstructive Surgery
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• 제28권4호
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• pp.277-286
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• 2006

In the present study, I investigated the effects of N-methyl-D-aspartate (NMDA), arachidonic acid (AA), and Nitric Oxide Synthase Inhibitor (NOS-I), alone or in combination, on the viability of cultured primary normal human oral keratinocytes (NHOK). Specifically, we examined whether AA and NOS-I could protect primary NHOK from glutamate cytotoxicity. The purpose of this study was therefore the preliminary study for the examination of the interaction between these agents and NHOK in order to elucidate the mechanisms by which epithelial growth and regeneration are regulated. NHOK were obtained from gingival tissue of 20 individuals aged 20 to 29, and third passage (P3) cells were used for this study. Cell viability was measured by the MTT assay. NMDA and NNA, a calcium dependent NOS inhibitor, induced an initial increase in cell number, which subsequently decreased by the $7^{th}$ day. Low concentration of AA ($0.5\;{\mu}M$ & $1\;{\mu}M$) induced an increase in cell number while high concentrations of AA ($5\;{\mu}M$ & $10\;{\mu}M$) induced a decrease in cell number. The decrease in cell number induced by NMDA at the $7^{th}$ day was abolished by the addition of low concentrations of AA ($0.5\;{\mu}M$ & $1\;{\mu}M$) or NOS inhibitors. Low concentrations of AA ($0.5\;{\mu}M$ & $1\;{\mu}M$) or NOS inhibitors may protect the NHOK from NMDA induced cytotoxicity. These reactions might be related to the NMDA receptor in the cell and the change of the intracellular calcium ion concentration.

• Asian-Australasian Journal of Animal Sciences
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• 제33권7호
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• pp.1167-1179
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• 2020

Objective: This study was conducted to fathom the underlying mechanisms of nutrition intervention and redox sensitive transcription factors regulated by Antrodia cinnamomea fermented product (FAC) dietary supplementation in broiler chickens. Methods: Four hundreds d-old broilers (41±0.5 g/bird) assigned to 5 groups were examined after consuming control diet, or control diet replaced with 5% wheat bran (WB), 10% WB, 5% FAC, and 10% FAC. Liver mRNA expression of antioxidant, inflammatory and lipid metabolism pathways were analyzed. Prostaglandin E2 (PGE₂) concentration in each group were tested in the chicken peripheral blood mononuclear cells (cPBMCs) of 35-d old broilers to represent the stress level of the chickens. Furthermore, these cells were stimulated with 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH) and lipopolysaccharide (LPS) to evaluate the cell stress tolerance by measuring cell viability and oxidative species. Results: Heme oxygenase-1, glutathione S-transferase, glutamate-cysteine ligase, catalytic subunit, and superoxide dismutase, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) that regulates the above antioxidant genes were all up-regulated significantly in FAC groups. Reactive oxygen species modulator protein 1 and NADPH oxygenase 1 were both rather down-regulated in 10% FAC group as comparison with two WB groups. Despite expressing higher level than control group, birds receiving diet containing FAC had significantly lower expression level in nuclear factor-kappa B (NF-κB) and other genes (inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-1β, nucleotide-binding domain, leucine-richcontaining family, pyrin domain-containing-3, and cyclooxygenase 2) involving in inflammatory pathways. Additionally, except for 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase that showed relatively higher in both groups, the WB, lipoprotein lipase, Acetyl-CoA carboxylase, fatty acid synthase, fatty acid binding protein, fatty acid desaturase 2 and peroxisome proliferator-activated receptor alpha genes were expressed at higher levels in 10% FAC group. In support of above results, promoted Nrf2 and inhibited NF-κB nuclear translocation in chicken liver were found in FAC containing groups. H₂O₂ and NO levels induced by LPS and AAPH in cPBMCs were compromised in FAC containing diet. In 35-d-old birds, PGE₂ production in cPBMCs was also suppressed by the FAC diet. Conclusion: FAC may promote Nrf2 antioxidant pathway and positively regulate lipid metabolism, both are potential inhibitor of NF-κB inflammatory pathway.

• The Korean Journal of Physiology and Pharmacology
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• 제1권6호
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• pp.673-679
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• 1997

It has been generally accepted that glutamate mediates the ischemic brain damage, excitotoxicity, and induces release of neurotransmitters, including norepinephrine(NE), in ischemic milieu. In the present study, the role of nitric oxide(NO) in the ischemia-induced $[^3H]norepinephrine([^3H]NE)$ release from cortex slices of the rat was examined. Ischemia, deprivation of oxygen and glucose from $Mg^{2+}-free$ artificial cerebrospinal fluid, induced significant release of $[^3H]NE$ from cortex slices. This ischemia-induced $[^3H]NE$ release was significantly attenuated by glutamatergic neurotransmission modifiers. $N^G-nitro-L-arginine$ methyl ester(L-NAME), $N^G-monomethyl-L-arginine$ (L-NMMA) or 7-nitroindazole, nitric oxide synthase inhibitors attenuated the ischemia-evoked $[^3H]NE$ release. Hemoglobin, a NO chelator, and 5, 5- dimethyl-L-pyrroline-N-oxide(DMPO), an electron spin trap, inhibited $[^3H]NE$ release dose-dependently. Ischemia-evoked $[^3H]NE$ release was inhibited by methylene blue, a soluble guanylate cyclase inhibitor, and potentiated by 8-bromo-cGMP, a cell permeable cGMP analog, zaprinast, a cGMP phosphodiesterase inhibitor, and S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide generator. These results suggest that the ischemia-evoked $[^3H]NE$ release is mediated by NMDA receptors, and activation of NO system is involved.

• Nutrition Research and Practice
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• 제14권2호
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• pp.95-101
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• 2020

BACKGROUND/OBJECTIVES: Folate plays a critical role in DNA synthesis and methylation. Intracellular folate homeostasis is maintained by the enzymes folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH). FPGS adds glutamate residues to folate upon its entry into the cell through a process known as polyglutamylation to enhance folate retention in the cell and to maintain a steady supply of utilizable folate derivatives for folate-dependent enzyme reactions. Thereafter, GGH catalyzes the hydrolysis of polyglutamylated folate into monoglutamylated folate, which can subsequently be exported from the cell. The objective of this review is to summarize the scientific evidence available on the effects of intracellular folate homeostasis-associated enzymes on cancer chemotherapy. METHODS: This review discusses the effects of FPGS and GGH on chemosensitivity to cancer chemotherapeutic agents such as antifolates, such as methotrexate, and 5-fluorouracil. RESULTS AND DISCUSSION: Polyglutamylated (anti)folates are better substrates for intracellular folate-dependent enzymes and retained for longer within cells. In addition to polyglutamylation of (anti)folates, FPGS and GGH modulate intracellular folate concentrations, which are an important determinant of chemosensitivity of cancer cells toward chemotherapeutic agents. Therefore, FPGS and GGH affect chemosensitivity to antifolates and 5-fluorouracil by altering intracellular retention status of antifolates and folate cofactors such as 5,10-methylenetetrahydrofolate, subsequently influencing the cytotoxic effects of 5-fluorouracil, respectively. Generally, high FPGS and/or low GGH activity is associated with increased chemosensitivity of cancer cells to methotrexate and 5-fluorouracil, while low FPGS and/or high GGH activity seems to correspond to resistance to these drugs. Further preclinical and clinical studies elucidating the pharmocogenetic ramifications of these enzyme-induced changes are warranted to provide a framework for developing rational, effective, safe, and customized chemotherapeutic practices.

• Journal of Ginseng Research
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• 제40권2호
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• pp.113-120
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• 2016

Background: The present study aimed to compare the relative abundance of proteins and amino acid metabolites to explore the mechanisms underlying the difference between wild and cultivated ginseng (Panax ginseng Meyer) at the amino acid level. Methods: Two-dimensional polyacrylamide gel electrophoresis and isobaric tags for relative and absolute quantitation were used to identify the differential abundance of proteins between wild and cultivated ginseng. Total amino acids in wild and cultivated ginseng were compared using an automated amino acid analyzer. The activities of amino acid metabolism-related enzymes and the contents of intermediate metabolites between wild and cultivated ginseng were measured using enzyme-linked immunosorbent assay and spectrophotometric methods. Results: Our results showed that the contents of 14 types of amino acids were higher in wild ginseng compared with cultivated ginseng. The amino acid metabolism-related enzymes and their derivatives, such as glutamate decarboxylase and S-adenosylmethionine, all had high levels of accumulation in wild ginseng. The accumulation of sulfur amino acid synthesis-related proteins, such as methionine synthase, was also higher in wild ginseng. In addition, glycolysis and tricarboxylic acid cycle-related enzymes as well as their intermediates had high levels of accumulation in wild ginseng. Conclusion: This study elucidates the differences in amino acids between wild and cultivated ginseng. These results will provide a reference for further studies on the medicinal functions of wild ginseng.

• Journal of Korean Neurosurgical Society
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• 제30권9호
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• pp.1059-1064
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• 2001

Objective : 6R-Tetrahydrobiopterin(BH4) is a cofactor for the aromatic amino acid hydroxylases which is essential for the biosynthesis of catecholamines and serotonin. It also acts as a cofactor for nitric oxide synthase, and stimulates the release of some neurotransmitters such as dopamine, serotonin, acetylcholine and glutamate. Recently, it has been reported that BH4 could induce cellular proliferation and enhance neuronal survival. This study was performed to investigate the antioxidative effect of BH4 on the various oxidative insults in mouse cerebral cortical cell cultures. Methods : Iron ion(FeCl2), zinc ion(ZnCl2), sodium nitroprusside(SNP) and buthionine sulfoximine(BSO, a glutathione depletor) were used as oxidants. Cell death was assessed by measurement of lactate dehydrogenase efflux to bathing media at the end of exposure. Result : All 4 oxidants induced neuronal cell death associated with cell body swelling, which was markedly inhibited by trolox($100{\mu}M$), a vitamin E analog. BH4($10-100{\mu}M$) markedly inhibited the neuronal cell death induced by all 4 oxidants($20{\mu}M\;Cu^{2+}$, $20{\mu}M\;Zn^{2+}$, $1{\mu}M$ SNP or 1mM BSO). However, BH4 failed to inhibit the neuronal cell death induced by 24hr exposure to $20{\mu}M$ NMDA. Conculsion : These results suggest that BH4 has antioxidative action independently of any actions of enzyme cofactor.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.789-796
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• 2000

In order to analyze the effects of tktA, $aroF^{FBR}$, and aroL expression in a tryptophan-producing Escherichia coli, a series of plasmids carrying the genes were constructed. Introduction of tktA, $aroF^{FBR}$, and aroL into the E. coli strain resulted in approximately 10-20 fold increase in the activities of transketolase, the feedback inhibition-resistant 3-deoxy-D-arabinoheptulsonate-7-phosphate synthase, and shikimate kinase. Expression of $aroF^{FBR}$ in the aroB mutant strain of E. coli resulted in the accumulation of 10 mM of 3-deoxy-D-arabinoheptulsonate-7-phosphate (DAHP) in the medium. Simultaneous expression of tktA and $aroF^{FBR}$ in the strain further increased the amount of excreted DAHP to 20 mM. In contrast, the mutant strain which has no gene introduced accumulated 0.5 mM of DAHP. However, the expression of tktA and $aroF^{FBR}$ in a tryptophan-producing E. coli strain did not lead to the increased production of tryptophan, but instead, a significant amount of shikimate, which is an intermediate in the tryptophan biosynthetic pathway, was excreted to the growth medium. Despite the fact that additional expression of shikimate kinase in the strain could possibly remove 90% of excreted shikimate to 0.1 mM, the amount of tryptophan produced was still unchanged. Removing shikimate using a cloned aroL gene caused the excretion of glutamate, which suggests disturbed central carbon metabolism. However, when cultivated in a complex medium, the strain expressing tktA, $aroF^{FBR}$, and aroL produced more tryptophan than the parental strain. These data indicate that additional rate-limiting steps are present in the tryptophan biosynthetic pathway, and the carbon flow to the terminal pathway is strictly regulated. Expressing tktA in E. coli cells appeared to impose a great metabolic burden to the cells as evidenced by retarded cell growth in the defined medium. Recombinant E. coli strains harboring plasmids which carry the tktA gene showed a tendency to segregate their plasmids almost completely within 24h.

• 생명과학회지
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• 제16권1호
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• pp.101-107
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• 2006

상황버섯균사체를 이용한 발효주 (FMLP)가 인체간암세포와 횐쥐 간에서 염증관련단백질의 발현과 간 손상관련효소에 미치는 영향에 대해 조사하였다. 에탄올, FLMP를 인체 간암세포인 $HepG_2$세포에 24시간 처리한 경우, 대조군에 비해 에탄올을 처리한 세포는 농도가 증가할수록 세포의 형태 변화가 심한 반면 FLMP를 처리한 세포는 알코올의 농도가 증가해도 세포의 형태 변화를 관찰할 수 없었다. $HepG_2$세포의 염증 관련 단백질 발현을 보기 위해 Western blot과 RT-PCR을 한 결과 COX-1, COX-2, iNOS, $TNF-\alpha$의 발현이 에탄올에 의해 현저하게 유도되었으나, FLMP는 $HepG_2$세포에서 mRNA와 단백질의 발현이 에탄올에 비해 억제되었다. 흰쥐를 이용한 동물실험에서 간 기능의 지표가 되는 혈청내 GOT와 GPT양 등이 에탄을 투여 그룹에서 현저히 증가했으나 FLMP를 투여한 그룹에서 에탄을 투여 그룹에 비해 낮게 나타났다. 면역조직화학적 분석에서 에탄올을 투여한 쥐의 간세포에서 iNOS와 COX-2의 발현이 현저히 증가하나 FLMP를 투여한 쥐의 간세포에서 미약한 면역반응이 관찰되었다. 이상의 결과로 보아 FLMP는 동일한 농도의 에탄올에 비하여 간 손상의 가능성이 적은 것으로 사료되며 이 자료들은 상황버섯에 대한 연구와 다른 기능성 주류에 대한 기초 자료가 될 것이다.