• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.146-152
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• 2010

Streptomyces clavuligerus NRRL3585 produces a clinically important $\beta$-lactamase inhibitor, clavulanic acid (CA). In order to increase the production of CA, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (gap) was deleted in S. clavuligerus NRRL3585 to overcome the limited glyceraldehyde-3-phosphate pool; the replicative and integrative expressions of ccaR (specific regulator of the CA biosynthetic operon) and claR (Lys-type transcriptional activator) genes were transformed together into a deletion mutant to improve clavulanic acid production. We constructed two recombinant plasmids to enhance the production of CA in the gap1 deletion mutant of S. clavuligerus NRRL3585: pHN11 was constructed for overexpression of ccaR-claR, whereas pHN12 was constructed for their chromosomal integration. Both pHN11 and pHN12 transformants enhanced the production of CA by 2.59-fold and 5.85-fold, respectively, compared with the gap1 deletion mutant. For further enhancement of CA, we fed the pHN11 and pHN12 transformants ornithine and glycerol. Compared with the gap1 deletion mutant, ornithine increased CA production by 3.24- and 6.51-fold in the pHN11 and pHN12 transformants, respectively, glycerol increased CA by 2.96- and 6.21-fold, respectively, and ornithine and glycerol together increased CA by 3.72- and 7.02-fold, respectively.

• Molecules and Cells
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• v.28 no.6
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• pp.575-581
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• 2009

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the biosynthesis of polyamines, which are essential for cell growth, differentiation, and proliferation. This report presents the characterization of an ODC-encoding cDNA (SlitODC) isolated from a moth species, the tobacco cutworm, Spodoptera litura (Lepidoptera); its expression in a polyamine-deficient strain of yeast, S. cerevisiae; and the recovery in polyamine levels and proliferation rate with the introduction of the insect enzyme. SlitODC encodes 448 amino acid residues, 4 amino acids longer than B. mori ODC that has 71% identity, and has a longer C-terminus, consistent with B. mori ODC, than the reported dipteran enzymes. The null mutant yeast strain in the ODC gene, SPE1, showed remarkably depleted polyamine levels; in putrescine, spermidine, and spermine, the levels were > 7, > 1, and > 4%, respectively, of the levels in the wild-type strain. This consequently caused a significant arrest in cell proliferation of > 4% of the wild-type strain in polyamine-free media. The transformed strain, with the substituted SlitODC for the deleted endogenous ODC, grew and proliferated rapidly at even a higher rate than the wild-type strain. Furthermore, its polyamine content was significantly higher than even that in the wild-type strain as well as the spe1-null mutant, particularly with a very continuously enhanced putrescine level, reflecting no inhibition mechanism operating in the putrescine synthesis step by any corresponding insect ODC antizymes to SlitODC in this yeast system.

• Journal of Nutrition and Health
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• v.12 no.3
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• pp.1-8
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• 1979

단백질을 함유하지 않은 식이를 5일간 먹인 쥐에게 동량으로 혼합된 필수 아미노산 혼합물(amino acid mixture)을 투여 하여 ornithine $\delta$-transamiaase(OT)를 유도(induction)한 군과 또한 amino acid mixture를 먹인 후 $6{\sim}9 \;1/2$시간 후에 glucose를 동시에 먹인 군에서 OT의 유도 기전을 보기 위하여 방사성 동위원소인 L-methionine-$CH_{3}-C^{14}$ 또는 L-Phenylalanine-$H^{3}$(uniformly labelled) 을 사용하여 유도전 OT를 pulse label하여 준비된 항체로 각각 유리 분리시켜서 방사능을 측정하였다. Amino acid mixture만을 투여한 군에서는zero time에 비해 OT활성은 6시간 후에 1.5배, 12시간 후에는 약 3배, 18시간 후에는 13배가 증가되었다. 또한 OT에 incorporate된 표지 아미노산의 방사능은 6시간 후에 1.5배, 12시간 후에 약 3배가 증가 되었을을 보였다. 그러나 이 기간동안 간장의 total soluble protein에 incorporate된 표지 아미노산의 방사능은 거의 증가하지 않았다. 그러므로, amino acid mixture에 의한 OT의 증가는 고유하게 유도되었으며 이때의 관성의 증가는 OT 단백질의 분해률의 감소에 의한 것이 아니라 순수하게 OT 단백질의 생합성에 의찬 증거라고 볼 수 있다. amino acid mixture를 투여한지 6시간 후에 glucose 용액을 동시에 먹인 쥐에서는 OT 활성은 증가를 하였으나 amino acid mixture만을 먹인 군보다는 약간 감소를 보였다. 또한 glucosedp이 투여된 후에는 OT에 표지 아미노산이 더 이상 incorporate되지는 않아 방사능의 증가는 보이지 않았으나 glucose가 투석될 당시보다 더 이상 감소되지는 않았다. 그러므로 glucose에 의한 유도에 대한 억제 (repression)는 단백질의 분해률의 증가에서 온 것이라기 보다는 OT 단백질의 생합성을 억제하여서 $OT_{1}$활성을 감소하지 않았는가 본다.

• BMB Reports
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• v.39 no.6
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• pp.730-736
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• 2006

To evaluated the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) on cell cycle distribution in colorectal cancer cell and investigated underlying regulatory responses, human colorectal cancer cells HT-29 were cultured in RPMI 1640 medium and infected with Ad-ODC-AdoMetDCas. Cell cycle progression was detected by flow cytometry analysis. The expression levels of cell cycle regulated proteins were measured by Western blot analysis. The mRNA level of cyclin D1 was measured by RT-PCR. And a luciferase reporter plasmid of cyclin D1 promoter was constructed to observe the effect of Ad-ODC-AdoMetDCas on cyclin D1 promoter activity. The results showed that recombinant adenovirus Ad-ODC-AdoMetDCas significantly induced $G_1$ arrest, decreased levels of cyclin D1 protein and mRNA and suppressed the promoter activity. Ad-ODC-AdoMetDCas also inhibited nuclear translocation of $\beta$-catenin. In conclusion, downregulation of ODC and AdoMetDC mediated by Ad-ODC-AdoMetDCas transfection induces $G_1$ arrest in HT-29 cells and the arrest was associated with suppression of cyclin D1 expression and inhibition of $\beta$-catenin nuclear translocation. As a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of colorectal cancers.

• Preventive Nutrition and Food Science
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• v.20 no.4
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• pp.238-245
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• 2015

A non-protein amino acid, L-ornithine (Orn), has been shown to stimulate the urea cycle and tissue protein synthesis in the liver. The purpose of the current study was to assess whether Orn affects the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway, which is involved in protein synthesis. Primary cultured cells isolated from Wistar rat liver were incubated in an amino acid-free medium, followed by addition of Orn for 3 h. The cell lysate was subjected to immunoblotting to evaluate the phosphorylation of downstream targets of mTORC1, including p70S6K, S6, and 4EBP1. To assess the involvement of mTORC1 for the effect of Orn, the cells were pretreated with the mTOR inhibitor rapamycin before the addition of Orn and the cell lysate was subjected to immunoblotting. We next examined whether the effects of Orn were exerted in vivo. Orn was orally administered to 18 h food-deprived rats, the blood and the livers were collected at 1 and 3 h after administration for immunoblotting. Orn treatment for primary cultured cells for 3 h enhanced the phosphorylation of p70S6K, S6, and 4EBP1. In addition, rapamycin blocked the effects of Orn completely (p70S6K and S6) or partially (4EBP1). The oral administration of Orn to the rat also augmented the phosphorylation of mTORC1 downstream targets notably in S6 at 1 h. Our findings demonstrate that Orn has the potential to induce the phosphorylation of downstream targets of mTORC1 in the rat liver. This may be mediated by the augmentation of mTORC1 activity.

• The Korean Journal of Food And Nutrition
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• v.26 no.4
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• pp.945-951
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• 2013

Changes in fermentation properties and ornithine levels of Baechu Kimchi by storage conditions were investigated. After making and fermenting Kimchi at $15^{\circ}C$ for 32 hr (S1), 36 hr (S2), 40 hr (S3), 44 hr (S4), and 48 hr (S5) during the first 10 days of storage. The Kimchi samples are subsequently stored in the -$1^{\circ}C$ Kimchi refrigerator for up to 60 days. Changes in the pH values and lactic acid contents of S4 and S5 samples are slightly bigger than the S1, S2 and S3 samples which have no significance differences. According to lactic acid bacteria (LAB) number, all samples show the largest augmentation according to the number of Lactobacilli during the first 20 days of storage. After 20 days of storage, the S4 and S5 samples show larger accumulations of LAB than S1, S2 and S3 samples. The Weissella genus is predominated at the 40 day of storage in the S5 sample. Ornithine levels are increased up to 170mg per 100 g during the storage period of 40~50 days in the S5 sample. However, the increase of ornithine levels in S1, S2 and S3 samples is smaller than those of the S4 and S5 samples. These results indicate that the conditions of Kimchi fermentation, which is 48 hr at $15^{\circ}C$ before storage, is proved to be the most superior for ornithine levels within the Kimchi refrigerator.

• Journal of Mushroom
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• v.15 no.1
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• pp.38-44
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• 2017

Sparassis latifolia (formerly S. crispa) is used in food and nutraceuticals or dietary supplements, as rich in flavor compounds and ${\beta}-glucan$. Some previous studies have reported the effects of mushroom on brain function, including its neuroprotective effect. Thus, for this mushroom to be used as an effective nutraceutical for brain function, it would be desirable for it to contain other compounds such as ${\gamma}-aminobutyric$ acid (GABA) in addition to ${\beta}-glucan$. In this study, the enhancement of growth and GABA production in the mycelium of medicinal and edible mushroom S. latifolia was investigated. Amino acids were added externally as the main source of nutrition, and the effects of amino acids were investigated using liquid medium, specifically amino acid-free potato dextrose broth (PDB). The amino acids added were L-glutamic acid (named PDBG medium) and L-ornithine (named PDBO medium). The growth of mycelia was determined to be $0.9{\pm}0.00g/L$, $2.2{\pm}0.16g/L$, and $1.93{\pm}0.34g/L$ PDBG respectively. The GABA content was $21.3{\pm}0.9mg/100g$ in PDB medium, and it in PDBG 1.4% medium, at $115.4{\pm}30.2mg/100g$. However, the PDBO medium was not effective in increasing the GABA content of mycelia. Amino acids had little effect on the ${\beta}-glucan$ content of mycelia. The ${\beta}-glucan$ content was $39.7{\pm}1.4mg/100mg$, $34.4{\pm}0.2mg/100mg$, and $35.2{\pm}9.2mg/100mg$ in PDB, PDBG 1.8% and PDBO 1.4% media, respectively. Addition of glutamic acid and ornithine positively affected the growth of S. latifolia mycelia, and glutamic acid positively affected GABA production; no degradation of GABA was observed with addition of glutamic acid.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.15 no.3
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• pp.165-170
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• 2015

Ornithine transcarbamylase (OTC) deficiency is a rare X-linked genetic disorder of urea synthesis in newborns. It is the most common urea cycle disorder and leads to elevated levels of ammonia in the blood. Excessive ammonia can cause various symptoms, including vomiting, lethargy, and coma. Boys have a more serious form of OTC deficiency than girls. If not treated immediately, severe OTC deficiency can lead to neurologic abnormalities, hyperammonemic coma, and death. Because late-onset OTC deficiency, which is more common in girls, presents mild symptoms, it is easy to miss diagnosis and prompt treatment. We describe an 11-month-old girl who presented periodic vomiting, intermittent lethargy, and seizure. She was diagnosed with OTC deficiency by elevated serum ammonia and urine orotic acid levels. Genetic analysis of the OTC gene revealed a missense mutation in exon 5 (c.418G>C). We reported an experience of exact diagnosis and successful treatment of late-onset OTC deficiency in our patient.

• Applied Biological Chemistry
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• v.40 no.3
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• pp.189-195
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• 1997

The structural basis of Iysine specificity of Achromobacter protease I (API) was investigated by means of site-directed mutagenesis. The precursor protein in which Glu190, one of the two candidates for determining Iysine specificity, was substituted by glutamine, aspartic acid or leucine was processed autocatalytically to attaln full pretense activity with lysine specificity. The substitution of the other candidate, Asp225, for asparagine or leucine produced no mature active forms of pro-API. The precursor protein of the mutant D225E slowly matured autocatalytically. The lysylendopeptidase activity of the mature D225E was 0.25% of that of native API, and this reduced activity is mainly due to a decrease in the affinity of the enzyme for lysine. These results suggest that Asp225 plays a critical rol in restricted substrate specificity as a lysylendopeptidase. However, D225E exhibited no measurable activity for synthetic ornithine substrate. Since the hydroxyl group of Ser194 in this mutant retained essentially the same reactivity to DFP or PMSF as that in native API, it can be noted that a methylene unit longer side chain of residue 225 is not compensated by a methylene unit shorter side chain at subsite P1 in the bound substrate.

• Journal of Life Science
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• v.18 no.3
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• pp.381-386
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• 2008

Deep sea water was tested for cancer chemopreventive activity by measuring the activities of ${\beta}-$ naphthoflavone $({\beta}-NF)-induced$ cytochrome P 450 1A2 (CYP 1A2), quinone reductase (QR) and glutathione-S-transferase (GST), glutathione (GSH) levels, and ornithine decarboxylase (ODC) activity. The in vitro incubation of rat liver microsome with deep sea water (a hardness range of $100{\sim}1,000$) showed a hardness-dependent inhibition of CYP 1A2 activity. QR and GST activities were induced about $1.1{\sim}1.2$ fold with the treatment of deep sea water in murine hepatoma Hepa 1clc7 cells. In addition GSH levels were increased $1.3{\sim}1.4$ fold in a hardness range of $100{\sim}1,000$. The deep sea water showed 20.3 and 35.0% inhibition of 12-O- tetradecanoylphorbol-13-a-cetate (TPA)-induced ODC activity at hardness 800 and 1,000, respectively. Therefore, deep sea water is worth further investigation with respect to cancer chemoprevention or therapy.