• 한국균학회지
• /
• 제32권2호
• /
• pp.112-118
• /
• 2004

한국산 흰구름버섯(Trametes hirsuta S1)로부터 배지 내로 분비된 laccase를 ultrafiltration과 anion exchange chromatography, adsorption chromatography를 이용하여 분리 정제하고 정제된 효소의 특성을 조사하였다. Laccase는 균주의 일차 대사 과정에서 주로 생산되는 세포의 페놀 산화효소였다. 흰구름버섯을 기본 배지에서 배양하였을 때 생장은 배양 6일까지 급속히 이루어졌고, laccase의 활성은 배양 5일에 최대활성을 나타냈으며 배양액에서 LiP와 VAO의 활성은 측정되지 않았다. Laccase의 생산에 미치는 유도원의 영향을 조사하기 위하여 배양 중인 흰구름버섯에 몇몇 유도원을 첨가한 결과, 2,5-xylidine은 대조구에 비하여 laccase의 생산을 약4배 증가 시켰다. 정제된 laccase는 SDS 젤 전기영동에서 대략 66 kDa의 분자량을 가지는 단일 폴리펩타이드(single polypeptide)였고, 탄수화물 함량은 12%였다. 정제된 laccase의 $K_m$과 $V_{max}$를 ABTS[2,2-azino-bis(3-ethylbenzthiazo line-6-sulfonic acid)]를 기질로 사용하여 조사한 결과 각각 $51.2\;{\mu}M$과 $56.8\;{\mu}mole{\cdot}min^1{\cdot}mg^{-1}$로 측정되었다. Laccase 활성의 최적 pH는 3.0이며, 이 효소는 $50^{\circ}C$ 미만에서 1시간 동안 처리하였을 때 안정적이었고 $70^{\circ}C$에서 20분간 처리하였을 때 효소의 활성이 반감되었다. Laccase의 분광학적 특성을 조사한 결과 구리를 포함하는 단백질로 나타났다. 일반적으로 알려진 laccase의 기질들에 대한 특이성을 조사한 결과, 5 mM ABTS에서 가장 높은 활성을 나타내었으며 tyrosine에서는 laccase의 활성이 나타나지 않았다. 저해제의 영향을 조사한 결과, 일반적으로 구리를 포함하는 단백질의 저해제인 $NaN_3$, TGA, DDC를 일정 농도로 처리한 실험구에서는 효소의 활성이 완전하게 억제되었으며, EDTA 처리구에서는 효소의 활성이 억제되지 않았다. 한국산 흰구름버섯 S1 균주로부터 생산되는 laccase의 N-말단의 아미노산의 서열은 Coriolus hirsutus의 laccase와 100%의 상동성을 나타냈고, T. versicolor의 laccase I과는 68%의 상동성을 나타냈다.

• 한국버섯학회지
• /
• 제20권2호
• /
• pp.86-91
• /
• 2022

큰느타리(Pleurotus eryngii)는 azo계 염료인 CR, heterocyclic계 염료인 MB, triphenyl methane계 염료인 CV와 MG를 고체와 액체 배양 상태에서 모두 분해할 수 있었으며 고체 배양 조건에서는 CR을, 액체 배양 상태에서는 MG을 가장 효과적으로 분해했다. MB는 고체와 액체 배양 조건에서 모두 가정 적게 분해되었다. 4 종류의 염료 모두 균사의 생장을 저해했으나 MG의 영향이 가장 크게 나타났다. 활성을 측정한 두 종류의 리그닌 분해 효소 중 모두 laccase의 활성이 훨씬 높았으며 MnP 역시 활성은 낮았으나 laccsse와 비슷한 경향을 보였다. 따라서 큰느타리는 주로 laccase를 사용하여 방향족 합성염료를 분해하는 것으로 추정된다. CR, MB, CV는 laccase의 활성과 염료 분해 정도가 양의 상관관계를 보인 반면 MG의 경우 높은 효소 활성을 나타낸 후 염료의 분해가 진행되었다. 또한 MG가 모두 분해되었을 때의 laccase 활성(0.07 U/ml)이 다른 염료가 가장 많이 분해되었을 때의 활성(0.10 U/ml)보다 낮았는데 이는 큰느타리의 균사체가 MG를 분해하는 기작이 CR, MB 및 CR 등의 염료를 분해하는 기작과 차이를 보인다는 것을 알 수 있었다. 본 연구에서는 큰느타리의 방향족 합성염료 분해 능력에 대해 밝힘으로써 앞으로 국내산 버섯균주를 이용한 친환경적인 합성염료 처리 기술 개발에 도움을 줄 것으로 기대된다.

• Journal of Microbiology and Biotechnology
• /
• 제18권4호
• /
• pp.767-772
• /
• 2008

Biodegradation of endocrine-disrupting phthalates [diethyl phthalate (DEP), dimethyl phthalate (DMP), butylbenzyl phthalate (BBP)] was investigated with 10 white rot fungi isolated in Korea. When the fungal mycelia were added together with 100 mg/l of phthalate into yeast extract-malt extract-glucose (YMG) medium, Pleurotus ostreatus, Irpex lacteus, Polyporus brumalis, Merulius tremellosus, Trametes versicolor, and T. versicolor MrP1 and MrP13 (transformant of the Mn-repressed peroxidase gene of T. versicolor) could remove almost all of the 3 kinds of phthalates within 12 days of incubation. When the phthalates were added to 5-day pregrown fungal cultures, most fungi except I. lacteus showed the increased removal of the phthalates compared with those of the non-pregrown cultures. In both culture conditions, p. ostreatus showed the highest degradation rates for the 3 phthalates tested. BBP was degraded with the highest rates among the 3 phthalates by all fungal strains. Only 14.9% of 100 mg/I BBP was degraded by the supernatant of P. ostreatus culture in YMG medium in 4 days of incubation, but the washed or homogenized mycelium of P. ostreatus could remove 100% of BBP within 2 days even in distilled water, indicating that the initial BBP biodegradation by P. ostreatus may be attributed to mycelium-associated enzymes rather than extracellular enzymes. The biodegradation rate of BBP by the immobilized cells of P. ostreatus was almost same as that in the suspended culture. The estrogenic activity of 100 mg/I DMP decreased during biodegradation by P. ostreatus.

• The Korean Journal of Physiology and Pharmacology
• /
• 제15권6호
• /
• pp.363-369
• /
• 2011

The present study elucidated the effect of the selective inducible nitric oxide synthase (iNOS) inhibitor $N^6$-(1-iminoethyl)-L-lysine (L-NIL) on monosodium urate (MSU) crystal-induced inflammation and edema in mice feet. L-NIL (5 or 10 mg/kg/day) was administered intraperitoneally 4 h before injection of MSU (4 mg) into the soles of mice hindlimb feet. Twenty-four hours after MSU injection, foot thickness was increased by 160% and L-NIL pretreatment reduced food pad swelling in a dose dependent manner. Pretreatment of 10 mg/kg/day L-NIL significantly suppressed the foot pad swelling by MSU. Plasma level of nitric oxide (NO) metabolites and gene expression and protein level of iNOS in feet were increased by MSU, which was suppressed by L-NIL pretreatment. Similar pattern of change was observed in nitrotyrosine level. MSU increased the gene expression of tumor necrosis factor (TNF)-${\alpha}$ and interleukin (IL)-$1{\beta}$ and L-NIL pretreatment suppressed MSU-induced cytokines expression. The mRNA levels of superoxide dismutase and glutathione peroxidase1 were increased by MSU and L-NIL pretreatment normalized the gene expression. Phosphorylation of extracellular signal-regulated kinase 1/2 and p38 was increased by MSU, which was suppressed by L-NIL pretreatment. The mRNA levels of iNOS, TNF-${\alpha}$, and IL-$1{\beta}$ were increased by MSU in human dermal fibroblasts, C2C12 myoblasts, and human fetal osteoblasts in vitro, which was attenuated by L-NIL in a dose dependent manner. This study shows that L-NIL inhibits MSU-induced inflammation and edema in mice feet suggesting that iNOS might be involved in MSU-induced inflammation.

• The Plant Pathology Journal
• /
• 제29권4호
• /
• pp.374-385
• /
• 2013

Environmental stresses induce several plant pathogenic bacteria into a viable but nonculturable (VBNC) state, but the basis for VBNC is largely uncharacterized. We investigated the physiology and morphology of the copper-induced VBNC state in the plant pathogen Ralstonia solanacearum in liquid microcosm. Supplementation of $200{\mu}M$ copper sulfate to the liquid microcosm completely suppressed bacterial colony formation on culture media; however, LIVE/DEAD BacLight bacterial viability staining showed that the bacterial cells maintained viability, and that the viable cells contain higher level of DNA. Based on electron microscopic observations, the bacterial cells in the VBNC state were unchanged in size, but heavily aggregated and surrounded by an unknown extracellular material. Cellular ribosome contents, however, were less, resulting in a reduction of the total RNA in VBNC cells. Proteome comparison and reverse transcription PCR analysis showed that the Dps protein production was up-regulated at the transcriptional level and that 2 catalases/peroxidases were present at lower level in VBNC cells. Cell aggregation and elevated levels of Dps protein are typical oxidative stress responses. $H_2O_2$ levels also increased in VBNC cells, which could result if catalase/peroxidase levels are reduced. Some of phenotypic changes in VBNC cells of R. solanacearum could be an oxidative stress response due to $H_2O_2$ accumulation. This report is the first of the distinct phenotypic changes in cells of R. solanacearum in the VBNC state.

• The Korean Journal of Physiology and Pharmacology
• /
• 제3권1호
• /
• pp.19-27
• /
• 1999

Apoptosis has been implicated in the pathophysiological mechanisms of various neurodegenerative diseases. In a variety of cell types, oxidative stress has been demonstrated to play an important role in the apoptotic cell death. However, the exact mechanism of oxidative stress-induced apoptosis in neuronal cells is not known. In this study, we induced oxidative stress in IMR-32 human neuroblastoma cells with tert- butylhydroperoxide (TBHP), which was confirmed by significantly reduced glutathione content and glutathione reductase activity, and increased glutathione peroxidase activity. TBHP induced decrease in cell viability and increase in DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. TBHP also induced a sustained increase in intracellular $Ca^{2+}$ concentration, which was completely prevented either by EGTA, an extracellular $Ca^{2+}$ chelator or by flufenamic acid (FA), a non-selective cation channel (NSCC) blocker. These results indicate that the TBHP-induced intracellular $Ca^{2+}$ increase may be due to $Ca^{2+}$ influx through the activation of NSCCs. In addition, treatment with either an intracellular $Ca^{2+}$ chelator (BAPTA/AM) or FA significantly suppressed the TBHP-induced apoptosis. Moreover, TBHP increased the expression of p53 gene but decreased c-myc gene expression. Taken together, these results suggest that the oxidative stress-induced apoptosis in neuronal cells may be mediated through the activation of intracellular $Ca^{2+}$ signals and altered expression of p53 and c-myc.

• Journal of Veterinary Science
• /
• 제22권4호
• /
• pp.54.1-54.13
• /
• 2021

Background: Hypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase. Objectives: This study aims to investigate the correlation between melatonin and hypoxia induction in cardiomyocytes differentiation. Methods: Mouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated. Results: Under hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects. Conclusions: This study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway.

• The Korean Journal of Physiology and Pharmacology
• /
• 제23권2호
• /
• pp.113-120
• /
• 2019

Mannosylerythritol lipids (MELs) are glycolipids and have several pharmacological efficacies. MELs also show skin-moisturizing efficacy through a yet-unknown underlying mechanism. Aquaporin-3 (AQP3) is a membrane protein that contributes to the water homeostasis of the epidermis, and decreased AQP3 expression following ultraviolet (UV)-irradiation of the skin is associated with reduced skin moisture. No previous study has examined whether the skin-moisturizing effect of MELs might act through the modulation of AQP3 expression. Here, we report for the first time that MELs ameliorate the UVA-induced downregulation of AQP3 in cultured human epidermal keratinocytes (HaCaT keratinocytes). Our results revealed that UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels, but that MEL treatment significantly ameliorated these effects. Our mitogen-activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. To explore a possible mechanism, we tested whether MELs could regulate the expression of peroxidase proliferator-activated receptor gamma ($PPAR-{\gamma}$), which acts as a potent transcription factor for AQP3 expression. Interestingly, UVA irradiation significantly inhibited the mRNA expression of $PPAR-{\gamma}$ in HaCaT keratinocytes, whereas a JNK inhibitor and MELs significantly rescued this effect. Taken together, these findings suggest that MELs ameliorate UVA-induced AQP3 downregulation in HaCaT keratinocytes by suppressing JNK activation to block the decrease of $PPAR-{\gamma}$. Collectively, our findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

• Animal Bioscience
• /
• 제36권2호
• /
• pp.284-294
• /
• 2023

Objective: This study investigated the effects of in ovo feeding of γ-aminobutyric acid (GABA) and embryonic thermal manipulation (ETM) on growth performance, organ indices, plasma biochemical parameters, hepatic antioxidant levels, and expression of lipid metabolism-related genes in broilers. Methods: Two hundred and fifty eggs were assigned to one of four treatments: control eggs incubated under standard conditions (CON); eggs that received an in ovo injection of 10% GABA on day 17.5 of incubation (G10); thermally manipulated eggs between days 10 and 18 of incubation at 39.6°C for 6 h daily (TM); and eggs that received both treatments during incubation (G10+TM). After 28 days of rearing, five birds per treatment were selected for blood and organ sampling. Results: No differences were found in hatchability or growth parameters among different treatment groups. Hepatic gene expression of catalase (CAT) and glutathione peroxidase 1 (GPx1) was upregulated (p = 0.046 and p = 0.006, respectively) in the G10+TM group, while that of nuclear factor erythroid 2-related factor 2 (NRF2) was upregulated (p = 0.039) in the G10 group. In addition, the relative gene expression of NADPH oxidase 1 (NOX1) was significantly lower (p = 0.007) in all treatment groups than that in the CON group. Hepatic fatty acid synthase (FAS) levels and average daily feed intake (ADFI) of last week showed a positive correlation (r = 0.50, p = 0.038). In contrast, the relative gene expression of the extracellular fatty acid-binding protein (EXFAB) and peroxisome proliferator-activated receptor-γ (PPAR-γ) were positively correlated (r = 0.48, p = 0.042 and r = 0.50, p = 0.031) with the overall ADFI of birds. Conclusion: Taken together, the results of this study suggest that the combination of in ovo feeding of GABA and ETM can enhance hepatic antioxidant function in broilers.

• Journal of Animal Science and Technology
• /
• 제50권2호
• /
• pp.185-198
• /
• 2008

급성기 반응 중인 육계 병아리에서 미역 2.0% 사료가 영양소 대사, 항산화 효소활성 및 세포성 면역에 미치는 영향이 조사 되었다. 갓 부화한 숫 육계 병아리(Ross)는 4주간의 실험 사육기간에 미역 0.0%(기초) 및 2.0% 사료를 급여하고, 8, 10, 및 12일령에 Salmonella typhymurium lipopolysaccharide(LPS)를 복강내 주입하여 급성기 반응을 활성화 하였다. 실험사육 전기간을 통한 생산성은 사료 중 미역 함량이나 급성기 반응 또는 그 경험의 영향이 없었다. 대조(정상) 병아리에 비하여, 급성기 반응은 미역 2.0% 사료에서 일당 증체량에 영향이 없었으나, 혈장 과산화물 분해효소, 세포외액 SOD(EcSOD) (p<0.05), 적혈구 MnSOD와 CuZnSOD 활성을 높이고, 그리고 사료 중 미역 함량과 관계없이 대사체중(kg0.75) 당 NB와 ME이용량를 낮추고 TNF-α와 ovotransferrin 활성을 높였다. 미역 0.0% 사료에 비하여, 미역 2.0% 사료는, 급성기 반응과 관계없이 kg0.75 당 NB를 증가시켰고, 급성기 반응 중에는 kg0.75당 UAN 배설에 영향이 없었으나, 혈장 과산화물 농도와 적혈구 세포액의 과산화물 분해효소와 MnSOD(p<0.05) 활성을 낮추고 혈장 과산화물 분해효소와 EcSOD 활성은 높이고(p<0.05), 4주령 병아리에서 LPS 자극 PBMC에서 분비한 IL-1 활성을 증가시켰다.(주 단어:미역, LPS, 단백질과 에너지대사, 항산화계, 친염증성 사이토카인, PBMC, 육계 병아리)