• Fisheries and Aquatic Sciences
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• 제21권6호
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• pp.16.1-16.7
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• 2018

Background: Matrix metalloproteinases (MMPs) are linked with several complications such as metastasis of cancer progression, oxidative stress, and hepatic fibrosis. Brown seaweeds are being extensively studied for their bioactive molecule content against cancer progression. In this context, Sargassum horneri was reported to possess various bioactivities including antiviral, antimicrobial, and anti-inflammatory partly due to its phenolic compound content. Methods: In this study, potential of S. horneri was evaluated through anti-MMP effect in HT1080 fibrosarcoma cells. S. horneri crude extract was fractionated with organic solvents, namely, water ($H_2O$), n-buthanol (n-BuOH), 85% aqueous methanol (85% aq. MeOH), and n-hexane. The non-toxicity of fraction samples (Sargassum horneri solvent-partitioned extracts (SHEs)) was confirmed by cell-viability assay. SHEs were tested for their ability to inhibit MMP enzymatic activity through gelatin digestion evaluation and cell migration assay. Expressions of MMP-2 and MMP-9 and tissue inhibitors of MMP (TIMPs) were evaluated by reverse transcription and Western blotting. Results: All fractions inhibited the enzymatic activities of MMP-2 and MMP-9 according to gelatin zymography. Except $H_2O$ fraction, fractions hindered the cell migration significantly. All tested fractions suppressed both mRNA and protein levels of MMP-2, MMP-9, TIMP-1, and TIMP-2. Conclusion: Overall, current results suggested that S. horneri has potential to be a good source for anti-MMP agents, and further investigations are underway for better understanding of the action mechanism and isolation and elucidation of the bioactive molecules.

• Ocean and Polar Research
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• 제40권4호
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• pp.249-257
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• 2018

Matrix metalloproteinases (MMPs) are associated with the invasion and metastasis of malignant tumors composed of cancer cells in an increased state of expression. This study evaluates the inhibitory effect of Carex pumila on MMP-2 and MMP-9 activity in phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 human fibrosarcoma cells using gelatin zymography, MMPs enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. C. pumila was extracted twice with dichloromethane ($CH_2Cl_2$) and methanol (MeOH). Treatment with $CH_2Cl_2$ extract and MeOH extract in PMA-stimulated HT-1080 cells effectively reduced the production of MMP-2 and 9. Also, the combined crude extracts ($CH_2Cl_2$ and MeOH) significantly inhibited the enzymatic activities and the expression of MMP-2 and MMP-9 in mRNA and protein levels. The combined crude extracts were partitioned between $CH_2Cl_2$ and water. The organic layer was further fractionated with n-hexane, 85% aqueous methanol (85% aq.MeOH) and the aqueous layer was separated into n-butanol and water, successively. Of the fractions, 85% aq.MeOH fraction showed the highest inhibitory activity of MMP-2 and MMP-9 in gelatin zymography and MMP ELISA kit. Furthermore, 85% aq.MeOH fraction most significantly suppressed cell migration. In RT-PCR and Western blot assay, n-butanol and 85% aq.MeOH fractions exerted the greatest inhibition on mRNA and protein expression of MMP-2 and MMP-9, respectively. As a result, C. pumila can be used as a good anti-invasive agent source.

• 생명과학회지
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• 제28권8호
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• pp.923-930
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• 2018

본 연구는 미생물을 이용하여 갈색거저리를 발효시킨 분말을 water, ethanol, methanol 용매별로 추출하여 다양한 실험을 행하였다. 균주는 3종의 유산균 Lactobacillus plantarum JBMI F3 (F3), Lactobacillus plantarum JBMI F5 (F5), Lactobacullus gasseri Ba9 (Ba9), 1종의 곰팡이 Aspergillus kawachii KCCM 32819 (Ak), 1종의 효모 Saccharomyces cerevisiae KACC 93023 (Sc), 1종의 바실러스 Bacillus subtilis KACC 91157 (Bs) 총 6종의 균주를 사용하였다. 각 균주를 이용한 발효 후 갈색거저리의 효능을 알아보기 위해 DPPH assay, 총 phenolic compound 및 Flavonoid 함량, Cu 환원력, Fibrinolytic activity, 효소-기질 활성 분석을 진행하였다. 그 결과, DPPH assay 결과, Water 추출물이 전반적으로 높은 항산화능을 보였고, 그 중 Bs 균주 발효 추출물이 우수한 라디컬 소거능을 보였다. 총 phenolic compound 및 Flavonoid의 경우, phenolic compound는 Ak발효군이 가장 함량이 많았고, Flavonoid는 Bs발효군이 가장 높았다. 환원력은 ethanol - Bs발효군에서 높은 수치를 확인하였고, Fibrinolytic activity 및 전분분해능은 water - Bs발효군에서 우수한 효과를 나타냈다. 위와 같은 결과를 토대로 발효 갈색거저리분말은, 기존 갈색거저리 분말보다 항산화능 및 생리활성 측면에서 효능이 상당부분 향상됨을 알 수 있었다. 그러므로 발효거저리분말은 식품산업전반에서 높은 이용가치를 증명하는 기초 연구자료로 활용될 것이다.

• Journal of Oral Medicine and Pain
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• 제34권3호
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• pp.227-235
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• 2009

타액의 보호작용은 주로 타액 당단백질의 생물학적, 물리적, 구조적 성질 및 유동학적 성질과 관련이 있다. 그러므로 이상적인 타액 대체제의 개발을 위해서는 인체 타액의 생물학적 성질 뿐만 아니라 유동학적 특성을 이해하여야 한다. 본 연구의 목적은 타액 대체제가 인체 타액에 존재하는 효소의 활성에 미치는 영향을 파악하고 다양한 타액 대체제의 점도와 인체 타액의 점도를 비교하기 위해서 시행되었다. Moi-Stir, Stoppers4, MouthKote, Saliva Orthana 및 서울대학교치과병원 타액 대체제(SNU)를 사용하였으며, lysozyme 활성은 turbidimetric 법으로, peroxidase 활성은 NbsSCN 법으로, $\alpha$-amylase 활성은 maltotriose와 결합된 2-chloro-p-nitrophenol를 사용하여 시행하였다. 타액 대체제의 pH를 측정하였으며 cone-and-plate 형태의 점도계를 이용하여 다양한 전단율에서 점도를 측정하였다. 본 연구에 사용된 다양한 타액 대체제는 타액 효소 활성에 각기 다른 영향을 미쳤다. Stoppers4는 hen egg-white lysozyme, bovine lactoperoxidase (bLP) 및 $\alpha$-amylase 활성을 증가시켰고, Saliva Orthana와 SNU는 bLP 활성은 저해하였으며 $\alpha$-amylase 활성은 증가시켰다. MouthKote는 $\alpha$-amylase 활성을 저해하였으며, Moi-Stir는 bLP와 $\alpha$-amylase 활성을 저해하였다. 타액 대체제의 pH는 타액 대체제의 종류에 따라 매우 달랐다. Stoppers4, MouthKote 및 Saliva Orthana는 낮은 전단율에서는 인체 타액보다 낮은 점도를 높은 전단율에서는 인체 타액보다 높은 점도를 나타내었다. Moi-Stir와 SNU는 인체 타액보다 매우 높은 점도를 나타내었다. 결론적으로 본 연구결과는 각각의 타액 대체제는 각기 다른 생물학적 기능과 유동학적 특성을 가지고 있음을 알 수 있다. 타액 대체제의 사용은 사용하는 타액 대체제의 종류에 따라 타액 효소 활성에 각기 다른 영향을 미치고 궁극적으로는 구강건강에 다른 영향을 미칠 수 있을 것이다.

• Natural Product Sciences
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• 제17권1호
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• pp.26-32
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• 2011

Novel tyrosinase inhibitors are important for pigmentation in the skin. Following extraction of tyrosinase inhibitors from edible vegetables or fruits, we found that the Prunus persica flesh extract (PPFE) exhibited potential inhibitory activity for melanogenesis. PPFE showed tyrosinase inhibitory activity in an enzymatic assay and PPFE also significantly inhibited the melanin formation in cultured mouse melan-a cells. Moreover, real-time RT-PCR analysis revealed that the inhibition of melanin production by PPFE was closely related to marked suppression of mRNA expression of tyrosinase and tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2) in melan-a cells. Further investigation found that the modulation of tyrosinase expression by PPFE was associated with the transcriptional regulation of the microphthalmia-associated transcription factor (MITF). PPFE inhibited the promoter activity of MITF and suppressed MITF mRNA expression in melan-a cells. These results indicate that PPFE down-regulates melanogenesis-associated gene expression through MITF-mediated transcriptional regulation and these events might be related to the hypopigmentary effects of PPFE.

• 환경생물
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• 제35권4호
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• pp.648-653
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• 2017

The aim of this study was to select various fungal strains indigenous to Korea that have the potential to produce cellulases, including filter paper activity (FPase), $endo-{\beta}$-1,4-glucanase (EG), and ${\beta}-glucosidase$ (BGL). Among the 25 species of Ascomycetes and the 32 species of Basidiomycetes tested in this study, the Bjerkandera adusta KUC10565, Heterobasidion orientale KUC10556, Hyphoderma praetermissum KUC10609, and Trichoderma harzianum KUC1716 all exhibited remarkably high FPase activity. In addition, the T. harzianum KUC1716 showed high levels of EG and BGL activity. This strain has been selected for further study because of their enzymatic potential.

• BMB Reports
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• 제56권11호
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• pp.618-623
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• 2023

Most cancer cells utilize glucose at a high rate to produce energy and precursors for the biosynthesis of macromolecules such as lipids, proteins, and nucleic acids. This phenomenon is called the Warburg effect or aerobic glycolysis- this distinct characteristic is an attractive target for developing anticancer drugs. Here, we found that Phosphofructokinase-1 (PFK-1) is a substrate of the Protein Phosphatase 4 catalytic subunit (PP4C)/PP4 regulatory subunit 1 (PP4R1) complex by using immunoprecipitation and in vitro assay. While manipulation of PP4C/PP4R1 does not have a critical impact on PFK-1 expression, the absence of the PP4C/PP4R1 complex increases PFK-1 activity. Although PP4C depletion or overexpression does not cause a dramatic change in the overall glycolytic rate, PP4R1 depletion induces a considerable increase in both basal and compensatory glycolytic rates, as well as the oxygen consumption rate, indicating oxidative phosphorylation. Collectively, the PP4C/PP4R1 complex regulates PFK-1 activity by reversing its phosphorylation and is a promising candidate for treating glycolytic disorders and cancers. Targeting PP4R1 could be a more efficient and safer strategy to avoid pleiotropic effects than targeting PP4C directly.

• BMB Reports
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• 제40권5호
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.652-655
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• 2005

This paper describes the development of an enzymatic assay system for the identification of inhibitors of isocitrate lyase (ICL), one of the key enzymes of the glyoxylate cycle that is considered as a new target for antifungal drugs. A 1.6 kb DNA fragment encoding the isocitrate lyase from Candida albicans ATCC10231 was amplified by PCR, cloned into a vector providing His-Patch-thioredoxin-tag at the N-terminus, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The molecular mass of the purified ICL was approximately 62 kDa, as determined by SDS-PAGE, and the enzyme activity was directly proportional to incubation time and enzyme concentration. The effects of itaconate-related compounds on ICL activity were also investigated. Among them, itaconic acid, 3-nitropropionate, and oxalate had strong inhibitory activities with $IC_{50}$ values of 5.8, 5.4 and $8.6\;{mu}g/ml$, respectively. These inhibitors also exhibited antifungal activity on YPD agar media containing acetate as a sole carbon source, albeit at high concentration. The results indicate that the C. albicans ICL may be a regulatory enzyme playing a crucial role in fungal growth and is a prime target for antifungal agents.

• 한국미생물·생명공학회지
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• 제41권4호
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• pp.456-461
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• 2013

단일가닥 DNA와 이중가닥 DNA의 흡착 율의 차이를 이용하여, 본 연구는 산화 그래핀에 흡착된 단일 가닥 DNA을 사용하여 Klenow fragment의 효소 활성을 검출하기 위하여 실시간 형광에세이 방법을 사용했다. 실험 결과에 의하면, 산화그래핀에 흡착된 형광표지 ssDNA는 형광이 ��칭(quenching) 되지만, cDNA 첨가에 의해서 흡착된 단일가닥 DNA가 유리되었다. Klenow fragment의 활성을 측정하기 위해서 형광표지 틀(template) DNA, 산화그래핀과 시발체(primer)가 존재할 때, 고분자 반응이 진행됨에 따라 ��칭된 형광세기가 증가하였다. 그리고 겔 전기영동 실험은 산화 그래핀에서 DNA 합성과 hybridization 반응을 확인하였다.