• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1905-1911
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• 2020

Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of ʟ-aspartate-4-semialdehyde to ʟ-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its recognized application in the development of pesticides and is being utilized in the large scale production of ʟ-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for ʟ-homoserine oxidation and found that BsHSD exclusively prefers NADP+ to NAD+ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. By kinetic analysis, Km values for ʟ-homoserine and NADP+ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the Vmax values were 2.72 ± 0.06 μmol/min-1 mg-1 and 2.79 ± 0.11 μmol/min-1 mg-1, respectively. The apparent molecular mass determined with size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. Thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was 54.8℃. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.

• 대한한방부인과학회지
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• 제35권3호
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• pp.1-23
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• 2022

Objectives: The purpose of this study is to evaluate anti-breast cancer and anti-inflammatory effects of Chungganhaewool-tang and Shipyeukmiyeugi-eum. Methods: MDA-MB-231 cells were used to measure cytotoxicity, Reactive oxygen species (ROS) production, protein expression amounts of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xl), Cytochrome C Caspase-3, Caspase-7, Caspase-9, Poly ADP-ribose polymerase (PARP), Nuclear factor erythroid-2-related factor 2 (Nrf2), Heme oxygenase-1 (HO-1) and NAD (P) H Quinone Oxidoreductase 1 (NQO1) to evaluate the anti-breast cancer effects of Chungganhaewool-tang (CHT) and Shipyeukmiyeugi-eum (SYE), and THP-1 cells, differentiated into macrophage and induced inflammation with Lipopolysaccharide (LPS), were used to measure production amounts of ROS, Nitric oxide (NO), and protein expression amounts of Inducible nitric oxide synthase (iNOS), Cyclooxygenase (COX-2), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6) and Tumor necrosis factor-alpha (TNF-α) to evaluate the anti-inflammatory effects of CHT and SYE. Results: CHT and SYE reduced MDA-MB-231 cell counts, increased protein expression of Bax and Cytochrome C, and decreased protein expression of Bcl-2, Bcl-xl. The protein expression amounts of Caspase-3, 7, and 9 decreased, but amounts of the active form, cleaved Caspase-3, 7, and 9, increased. In addition, PARP protein expression decreased, the amount of PARP protein in the cleaved form increased, and the amount of protein expressions of Nrf2 and HO-1 decreased, but NQO1 showed no significant difference. In THP-1 cells CHT and SYE reduced ROS and NO, and reduced protein expressions of iNOS, COX-2, IL-1, and TNF-α, but only SYE groups reduced IL-6. Conclusions: This study suggests that CHT and SYE have potential to be used as treatments for breast cancer.

• Journal of Ginseng Research
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• 제48권4호
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• pp.384-394
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• 2024

Background: Herpes simplex virus type 1 (HSV-1), known to latently infect the host's trigeminal ganglion, can lead to severe herpes encephalitis or asymptomatic infection, potentially contributing to neurodegenerative diseases like Alzheimer's. The virus generates reactive oxygen species (ROS) that significantly impact viral replication and induce chronic inflammation through NF-κB activation. Nuclear factor E2-related factor 2 (Nrf2), an oxidative stress regulator, can prevent and treat HSV-1 infection by activating the passive defense response in the early stages of infection. Methods and results: Our study investigated the antiviral effects of ginsenoside Rg5, an Nrf2 activator, on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection inhibited Nrf2 activity in host cells, induced ROS/NF-κB signaling, and triggered inflammatory cytokines. However, treatment with ginsenoside Rg5 inhibited ROS/NF-κB signaling and reduced inflammatory cytokines through NRF2 induction. Interestingly, the Nrf2 inhibitor ML385 suppressed the expression of NAD(P)H quinone oxidoreductase 1(NQO1) and enhanced the expression of KEAP1 in HSV-1 infected cells. This led to the reversal of VP16 expression inhibition, a protein factor associated with HSV-1 infection, thereby promoting HSV-1 replication. Conclusion: These findings suggest for the first time that ginsenoside Rg5 may serve as an antiviral against HSV-1 infection and could be a novel therapeutic agent for HSV-1-induced neuroinflammation.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.77-83
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• 2024

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuronal cell death and memory impairment. Corticosterone (CORT) is a glucocorticoid hormone produced by the hypothalamic-pituitary-adrenal axis in response to a stressful condition. Excessive stress and high CORT levels are known to cause neurotoxicity and aggravate various diseases, whereas mild stress and low CORT levels exert beneficial actions under pathophysiological conditions. However, the effects of mild stress on AD have not been clearly elucidated yet. In this study, the effects of low (3 and 30 nM) CORT concentration on Aβ_25-35-induced neurotoxicity in SH-SY5Y cells and underlying molecular mechanisms have been investigated. Cytotoxicity caused by Aβ_25-35 was significantly inhibited by the low concentration of CORT treatment in the cells. Furthermore, CORT pretreatment significantly reduced Aβ_25-35-mediated pro-apoptotic signals, such as increased Bim/Bcl-2 ratio and caspase-3 cleavage. Moreover, low concentration of CORT treatment inhibited the Aβ_25-35-induced cyclooxygenase-2 and pro-inflammatory cytokine expressions, including tumor necrosis factor-α and interleukin-1β. Aβ_25-35 resulted in intracellular accumulation of reactive oxygen species and lipid peroxidation, which were effectively reduced by the low CORT concentration. As a molecular mechanism, low CORT concentration activated the nuclear factor-erythroid 2-related factor 2, a redox-sensitive transcription factor mediating cellular defense and upregulating the expression of antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase, glutamylcysteine synthetase, and manganese superoxide dismutase. These findings suggest that low CORT concentration exerts protective actions against Aβ_25-35-induced neurotoxicity and might be used to treat and/or prevent AD.

• 생명과학회지
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• 제18권2호
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• pp.264-272
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• 2008

풀망둑(Acanthogobius hasta) 조직의 젖산탈수소효소(EC 1.1.1.27 Lactate dehydrogenase, LDH) 동위 효소의 특성을 생화학적, 면역화학적 및 역학적 방법에 의해 연구하였다. 풀망둑 골격근과 눈 조직의 젖산탈수소효소 활성이 65.30과 53.25 units였고, 심장과 간 조직에서는 낮게 나타났다. 골격근 조직의 LDH/CS는 22.29로 가장 높고, LDH 특이활성도는 뇌 56.45, 눈 38.04 및 골격근 11.04 units/mg였다. 각 조직에 대해 $A_4,\;B_4$, eye-specific $C_4$, 및 liver-specific $C_4$에 대한 항혈청으로 면역 침강 반응시킨 후 Polyacrylamide gel 전기영동 하였다. 골격근, 뇌 및 눈 조직에서 $A_4$ 동위효소가 우세하게 확인되었고, 심장에서는 $A_4$ 동위효소가 확인되었다. 또한 양극의 eye-specific t와 음극의 liver-specific $C_4$가 한 종에서 함께 발현되었으며, 눈 조직의 eye-specific $C_4$는 활성이 크고 간 조직의 liver-Specific $C_4$의 활성은 낮게 나타났다. 결과 $A_4$와 $C_4,\;A_4$와 $B_4$ 및 eye-specific $C_4$와 liver-specific $C_4$의 분자구조의 일부가 서로 유사하지만 $B_4$와 $C_4$의 구조는 서로 다른 것으로 나타났으므로 하부단위체 A는 보존적이고 하부단위체 B는 하부단위체 A보다 빠르게 진화된 것으로 사료된다. 골격근 조직의 LDH $A_4$ 동위효소는 affinity chromatography에서 $NAD^+$를 함유한 buffer를 유입한 후 용출된 분획에서 정제되었고, eye-specific $C_4$ 동위효소는 $A_4$ 분획에 이어 용출되었으므로 eye-specific $C_4$가 $A_4$의 분자 구조와 유사한 것으로 보인다. 그리고 LDH에 대한 피루브산의 저해 실험 결과 35.22-43.47% 의 활성이 남았고, $Km_{pyr}$은 0.080-0.098 mM이고 골격근과 눈조직의 Vmax은 153.85와 35.09 units였다. 또한 $B_4$ 동위효소가 열에 대해 가장 안정하였고 $C_4$는 $A_4$보다 안정하였으며, 최적 pH는 6.5로 나타났다. 본 실험 결과 풀망둑은 저 산소 환경조건에 적응되어져 조직들의 동위효소들이 $A_4$와 $B_4$ 동위효소 사이의 특성을 나타냈고, 골격근과 눈 조직에서 피루브산에 대한 LDH의 친화력이 상당히 크므로 LDH가 혐기적 조건에서 효율적으로 기능을 하는 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제24권6호
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• pp.748-755
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• 2014

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

• BMB Reports
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• 제49권11호
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• pp.617-622
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• 2016

Oxidative stress is closely associated with various diseases and is considered to be a major factor in ischemia. NAD(P)H: quinone oxidoreductase 1 (NQO1) protein is a known antioxidant protein that plays a protective role in various cells against oxidative stress. We therefore investigated the effects of cell permeable Tat-NQO1 protein on hippocampal HT-22 cells, and in an animal ischemia model. The Tat-NQO1 protein transduced into HT-22 cells, and significantly inhibited against hydrogen peroxide ($H_2O_2$)-induced cell death and cellular toxicities. Tat-NQO1 protein inhibited the Akt and mitogen activated protein kinases (MAPK) activation as well as caspase-3 expression levels, in $H_2O_2$ exposed HT-22 cells. Moreover, Tat-NQO1 protein transduced into the CA1 region of the hippocampus of the animal brain and drastically protected against ischemic injury. Our results indicate that Tat-NQO1 protein exerts protection against neuronal cell death induced by oxidative stress, suggesting that Tat-NQO1 protein may potentially provide a therapeutic agent for neuronal diseases.

• 미생물학회지
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• 제12권4호
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• pp.159-179
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• 1974

In order to investigate morphological and cultural characteristics of strains in species, Flammulina velutipes, the author collected isolates of Flammultina velutipes at 49 locations in Korea and cultivated these isolates on the various kinds of solid media. After investigating the cultural characteristics, appeared on the various media, he obtained the following results : 1. The variation of colors in the fruit bodies is connected with the variation of climatic environments(composite effects between mean temperature in January and number of rain days of 1mm and over precipitation). The author, therfore, can find out the trend that brown type is distributed in the midland climatic region and yellow type in the southern climatic regoin. 2. Two types can be classified into several strains respectively : the strain of abundant or insufficient productivity, and strain of selectivity or non-selectivity of media. 3. According to the results of mutual comparison of soluble mycelial proteins by disc electrophoresis using polyacrylamide gels, each type has special common protein fractions(brown type : band located at 26..5mm position from surface of gel, yellow type : band located at 24.5mm position from surface of gel), and each strain has special protein fractions too. Therefore this phenomenon seems to support the results obtained by the above-stated morphological and cultural studies. 4. In the adaptability of strains to the temperature, every strain has the nature of growing in lower temperature(the optimum temperature of 20.deg.C to 25.deg.C) except that YI-1 strain has the optimum temperatue of $25^{\circ}C$-26^{\circ}C. And mycelial growth of every strain is discontinued at $35^{\circ}C.$ 5. In the adaptability of strains to the H-ion concentration, every strain has wide adaptable range of H-ion concentration, and has optimum range of pH 5.5 to 6.6 in mycelial growth excepting YA01, BI-2 and YI-1. 6. In the utility of carbon sources, the mycelial growth of every strain is very poor on the media containing xylose(average diameter of mycelial growth : 18mm), and most strains utilize favorably sucrose(39mm), maltose(37mm) and dextrose(35mm) in mycelial growth. In the utility of nitrogen sources, every strain utilizes favorably organic nitrogens(36 mm)more than inorganic nitrogens (25 mm), and utilizes fully peptone nad asparagine in organic nitrogens. Especially BA-1, BIK-2 and YA-1 strains grow vigorously on each media containing various carbon and nitrogen sources. 7. The characteristic tests of the number of days required for mycelial growth, the number of days requried for sprout of young bodies, the length of stipe and the number of fruit bodies formation seem to be useful methods in the early selection of the strain of the abundant productivity.

• 한국미생물·생명공학회지
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• 제41권4호
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• pp.433-441
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• 2013

본 연구에서는 Endlicheria anomala (Nees) Mez 메탄올 추출물(EAME)의 항산화, 항염증 및 미백 생리활성을 in vitro assay 및 cell culture model system을 이용하여 분석하였다. EAME의 항산화능을 분석한 결과 DPPH, $H_2O_2$로 유도한 ROS, LPS로 유도한 NO 등 다양한 산화적 스트레스원을 효과적으로 소거하였다. 대표적인 항산화 효소들로 천연물에 의한 항산화능 활성에 의해 주로 발현이 유도되는 세효소인 HO-1, TrxR1, NQO1 및 그 전사 인자인 Nrf2의 단백질 발현에 미치는 영향을 분석한 결과 시료 처리 농도의 증가에 따라 세 효소 및 Nrf2의 발현이 유의적으로 증가됨을 보였다. 또한 EAME는 in vitro DOPA oxidation을 강하게 저해하여 tyrosinase inhibitor로서 작용할 가능성을 시사하였고 이에 B16F10 melanocyte를 이용하여 미백 효능을 분석한 결과 유의적인 melanin 생성억제능 및 tyrosinase 효소 활성 억제능을 보였으며 이는 tyrosinase, TRP-1, TRP-2 등 melanin 생성의 핵심 작용 효소들의 단백질 발현 저해를 통해 일어나는 것으로 나타났다. 이러한 결과를 통해 EAME가 높은 항산화능과 항염증 활성, 그리고 미백 활성을 보유함을 처음으로 밝혔으며 향후 기능성 식품 및 피부 미용 소재로서유용하게 활용될 수 있을 것으로 판단된다.

• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1215-1219
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• 2016

Background: Worldwide, breast cancer is the most common cancer among women and is a leading cause of cancer death. In the present study, we investigated the NQO1 C609T genotypic and allelic distribution in north Indian breast cancer patients. Materials and Methods: The genotypic distribution of the NQ01 C609T polymorphism was assessed in 100 invasive ductal carcinoma (IDC) breast cancer patients and 100 healthy controls using allele specific PCR (AS-PCR). Results: A lower frequency of the CC genotype was found in breast cancer patients (24%) than in the controls. On the other hand, TT genotype frequency was also found to be higher in female healthy controls (32%) than the female breast cancer patients (20%). The frequencies of all three genotypes CC, CT, TT in patients were 24%, 56% and 20% and in healthy controls 50%, 22% and 32% respectively. We did not find any significant correlation between the NQO1 C609T polymorphism and age group, grading, menopausal status and distant metastasis. A less significant association was found between the NQ01 C609T polymorphism and the stage of breast cancer (X2=5.931, P=0.05). Conclusions: The present study shows a strong association between NQO1 C609T polymorphism with the breast cancer risk in the north Indian breast cancer patients so that possible use as a risk factor should be further expel.