• 한국식품영양과학회지
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• 제26권4호
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• pp.743-750
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• 1997

세포막에서의 지방산 이동은 매우 빠르게 일어나므로 방사성 원소를 사용해서는 여러가지 단점이 있고, 정확한 이동속도 측정에도 어려움이 많았다 최근에 개발된 FRET assay는 형광성 물질과 형광성 물질을 상쇄시키는 quencher를 사용한 실험방법 이다. 이는 공명 에너지 이동의 원리를 이용한 것으로 형광광도계, stopped-flow장치를 사용하여 소수성 물질 이동을 직접 컴퓨터 모니터로 측정하는 방법으로 기존방법의 단점을 보완하였다. Donor막에는 형광성 표지를 붙인 지방산이 들어 있고 acceptor막에는 형광을 흡수하는 물질이 들어 있어서 형광성 지방산이 donor에서 acceptor로 이동하면 형광도가 감소하며, 시간에 따른 형광도 감소를 측정하여 지방산 이동속도를 측정하는 방법이다. 형광성 표지를 이용하여 소수성 물질 이동에 사용되는 또 다른 방법은 self-Quenching assay이다. 형광 물질의 농도가 높아지면 서로 형광을 흡수하는 성질을 이용한 방법으로 주로 micelle에서의 물질 이동에 많이 쓰인다. Donor micelle에는 높은 농도의 형광성 지방산이 들어 있고 acceptor micelle에는 형광성 지방산이 들어 있지 않을 때 형광성 지방산이 donor에서 acceptor로 이동하면 형광도가 증가하게 되고 시간에 따른 형광도 증가를 측정하는 방법이다.

• 공업화학
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• 제28권2호
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• pp.177-185
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• 2017

본 연구에서는 스테아르산(SA), 올레산(OA), 리놀레산(LA) 등의 지방산이 지질 소포체 막과의 상호 작용에 미치는 영향에 관하여 살펴보았다. 이를 위하여 지방산 종류 및 농도 변화에 따른 리포좀 평균 입자 크기 및 제타 전위, 리포좀 막의 deformability, fluorescence anisotropy ratio 등을 측정하고 TEM 관찰을 통하여 지방산 첨가가 리포좀 막의 유동성 변화에 미치는 역할에 관하여 살펴보았다. 기본적으로 SA, OA, LA 등의 지방산 첨가는 동일한 경향을 나타내었다. 즉, 지방산을 첨가함에 따라 리포좀이 보다 치밀한 패킹을 갖게 되어서 리포좀의 크기는 감소하고 제타 전위 값은 증가하였으나, 지방산의 과도한 첨가는 리포좀에서 다형(polymorphic) 구조를 가지는 지질 입자 응집체로의 전이를 일으켰다. SA, OA 및 LA 지방산 시스템에서의 최소 리포좀 크기와 가장 치밀한 리포좀 패킹은 레시틴 대비 지방산의 몰 비율이 각각 0.70, 0.50, 0.25인 조건에서 관찰되었으며, 리포좀 막의 deformability와 fluorescence anisotropy ratio 측정에 의한 리포좀 막의 유동성 측정 결과는 TEM 및 입자 크기 측정 결과와 일치함을 알 수 있었다.

• Bulletin of the Korean Chemical Society
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• 제35권12호
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• pp.3509-3513
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• 2014

Phospholipase D (PLD) catalyzed the generation of phosphatidic acid (PA) from phosphatidylcholine (PC) at the outer layer of the vesicles prepared through layer by layer via a double emulsion technique. The generation induced a curvature change in the vesicles, which eventually led them to fuse each other. The ratio of two-fatty-acid-tail ethanolamine (PE) to one-fatty-acid-tail ethanolamine (PE) was found to acquire the condition where the mixed-phospholipid vesicles were stable identically with pure two-fatty-acid-tail PC. The effect of the outer-layer mixture on the PLD-induced vesicle fusion was investigated using the fluorescence intensity change. 8-Aminonaph-thalene-1,3,6-trisulfonic acid disodium salt (ANTS) and p-Xylene-bis(N-pyridinium bromide) (DPX) were encapsulated in the vesicles, respectively, for the quantification of the fusion. The fluorescence scale was calibrated with the fluorescence of a 1/1 mixture of ANTS and DPX vesicles in NaCl buffer taken as 100% fluorescence (0% fusion) and the vesicles containing both ANTS and DPX as 0% fluorescence (100% fusion), considering the leakage into the medium studied directly in a separate experiment using vesicles containing both ANTS and DPX. The fusion data for each composition were acquired with the subtraction of the leakage from the quenching. From the monitoring, the vesicle fusion caused by the PLD reaction seems dominantly to occur rather than the vesicle lysis, because the composition effect on the fusion was observed identically with that on the change in the vesicle structure. Furthermore, the diameter measurements also support the fusion dominancy.

• 대한의생명과학회지
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• 제16권4호
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• pp.279-285
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• 2010

Endogenous cannabinoids (endocannabinoids) display various pharmacological effects including pain control, anti-inflammation, and neuroprotection. The synthesis and release of endocannabinoids are regulated under both physiological and pathological conditions. The main degrading enzyme of endocannabinoid is fatty acid amide hydrolase (FAAH). Therefore we have developed the fluorescence-based assay system for FAAH. We established stable CosM6 cell lines expressing human FAAH. We also synthesized 2-oxo-2H-chromen-7-yl decanoate (DAEC) as a fluorogenic substrate for FAAH. When crude membrane extracts stably expressing FAAH was incubated with DAEC at $25^{\circ}C$, FAAH reacted specifically to DAEC and catalyzes the hydrolysis of DAEC into decanoic acid and highly fluorescent coumarin. Furthermore, the serin hydrolase inhibitor, phenylmethanesulfonylfluoride, inhibited the coumarin release to the reaction buffer in concentration dependent manner. This assay system is suitable for high-throughput screening since this system has simple experimental procedure and measurement method.

• Journal of Photoscience
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• 제7권2호
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• pp.39-44
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• 2000

To examine the chilling tolerance lipids, we compared the chilling susceptibility of photosystem II of wild type tobacco plants with that of transgenic tobacco plants, in which the sensitivity to chilling had been enhanced by genetic modification of fatty acid unsaturation of chloroplast membrane lipids. The transgenic tobacco plants were found to contain reduced levels of unsaturated membrane fatty acids by being tansformed with cDNA for glycerol-3-phosphate acyltransferase from squash. For the purpose of studying on the functional integrity of photosystem II during low-temperature photoinhibition, the photochemical efficiency was measured as the ration of the maximun fluorescence of chlorophyll (Fv/Fm) of photosystem II. In parallel with an investigation on the transgenic plants, susceptibility of chilling-resistant species, such as spinah and pea, and of chilling-sensitive ones, such as squash and sweet potato, to low-temperature photoinhibition was also compared in terms of room temperature-induced chlorophyll fluorescence from photosystem II. When leaf disks from the two genotypes of tobacco plants were exposed to light at 5$^{\circ}C$, the transgenic plants showed more rapid decline in photochemical activity of photosysytme II than wild-type plants. When they were pretreated with lincomycin, an inhibitor of chloroplast-encoded protein synthesis, the extent of photoinhibition was even more accelerated. More impottantly, they showed a comparable extent of photoinhibition in the presence of lincomycin, making a clear contrast to the discrepancy observed in the discrepancy observed in the absence of lincomycin. Restoration of Fv/Fm during recovery from low-temperature photoinhibition occurred more slowly in the transgenic tobacco plants than the wild-type. These findings are discussed in relation to fatty acid unsaturation of membrane phosphatidylglycerol. It appears that the ability of plants to rapidly regenerate the active photosystem II complex from might explain, in part, why chilling-resistant plants can toleratlow-temperature photoinhibition.

• International Journal of Oral Biology
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• 제33권2호
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• pp.65-70
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• 2008

Oral spirochetes are anaerobes known as one of causative agents for periodontal diseases. In this study, we investigated the possibility of utilizing fluorescent fatty acids for labeling oral spirochetes. Bacterial labeling was standardized with three different lengths of fluorescent fatty acids: 5-octadecanoylaminfluorescein (OAF), 5-dodecanoylamin-fluorescein (DAF), and 5-hexadecanoylaminfluorescein (HAF). Among these fatty acids, OAF showed the best labeling activity. Treponema denticola ATCC 35405 was totally saturated to the maximum when incubated with OAF $1\;{\mu}g/ml$ for 1 hour. Treponema vincentii LA-1 also increased in fluorescence in proportion to incubation time length and the concentration. In conclusion, these findings showed the possibility that the fluorescent fatty acid can be used for labeling oral spirochetes.

• International Journal of Oral Biology
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• 제35권3호
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• pp.107-111
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• 2010

Treponema denticola is a gram-negative anaerobe that can cause periodontal disease. The adhesion of this bacterium to host tissues is considered to be the primary event in the colonization and infection of a host. Fibrinogen is generally found in damaged tissues resulting from periodontitis. The binding ability of T. denticola to fibrinogen may therefore be an important virulence factor in inducing periodontal diseases. It has been reported recently that oral spirochetes can be labeled with fluorescent fatty acids and we speculated that this labeling method could be used in an oral spirochete binding assay. The binding of several different strains of T. denticola to immobilized human fibrinogen was therefore tested using the fluorescent fatty acid labeling method. In the case of immobilized fibrinogen, the T. denticola ATCC 35405 strain showed saturable binding to immobilized fibrinogen. Indeed, all four different T. denticola strains tested in this experiment, T. denticola ATCC 35405, T. denticola ATCC 33520, T. denticola ATCC 35404 and T. denticola OTK showed binding to fibrinogen. The fluorescent fatty acid labeling method thus shows utility in binding assays for T. denticola, different strains of which can generally bind to immobilized fibrinogen.

• 대한의생명과학회지
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• 제18권4호
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• pp.438-444
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• 2012

N-acylethanolamines (NAEs) including endocannabinoids, anadamide, are long chain fatty acid ethanolamines and express ubiquitously in animal and plant tissues. NAEs have several pharmacological effects including anti-inflammatory, analgesic and anorexic effects. The levels of NAEs in tissues are strictly regulated by synthesizing and hydrolyzing enzymes because NAEs are not stored in the cell but rather made on demand. NAEs are hydrolyzed to free fatty acids and ethanolamines by fatty acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase (NAAA). Here, we suggest the fluorescence-based assay system for NAAA. We developed N-(4-methy-2-oxo-2H-chromen-7-yl)palmitamide (PAAC) as a fluorogenic substrate for NAAA and we also generated NAAA stably expressing COSM6 cell line. When extracts of cells expressing NAAA were incubated with PAAC, NAAA specifically hydrolyzed PAAC to palmitic acids and fluorogenic dye, coumarin. Release of coumarin was monitored by using fluorometer. NAAA hydrolyzed PAAC with an apparent Km of $20.05{\mu}M$ and Vmax of 32.18 pmol/mg protein/min. This assay system can be used to develop inhibitors or activators of NAAA.

• Asian-Australasian Journal of Animal Sciences
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• 제32권11호
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• pp.1789-1800
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• 2019

Objective: Although alveolar macrophages play a key role in the respiratory immunity of livestock, studies on the mechanism of differentiation and survival of alveolar macrophages are lacking. Therefore, we undertook to investigate changes in the lipid metabolism and survival rate, using 3D4/31 macrophages and Dudleya brittonii which has been used as a traditional asthma treatment. Methods: 3D4/31 macrophages were used as the in vitro porcine alveolar macrophages model. The cells were activated by exposure to phorbol 12-myristate 13-acetate (PMA). Dudleya brittonii extraction was performed with distilled water. For evaluating the cell survival rate, we performed the water-soluble tetrazolium salt cell viability assay and growth curve analysis. To confirm cell death, cell cycle and intracellular reactive oxygen species (ROS) levels were measured using flow cytometric analysis by applying fluorescence dye dichlorofluorescein diacetate and propidium iodide. Furthermore, we also evaluated cellular lipid accumulation with oil red O staining, and fatty acid synthesis related genes expression levels using quantitative polymerase chain reaction (qPCR) with SYBR green dye. Glycolysis, fatty acid oxidation, and tricarboxylic acid (TCA) cycle related gene expression levels were measured using qPCR after exposure to Dudleya brittonii extract (DB) for 12 h. Results: The ROS production and cell death were induced by PMA treatment, and exposure to DB reduced the PMA induced downregulation of cell survival. The PMA and DB treatments upregulated the lipid accumulation, with corresponding increase in the acetyl-CoA carboxylase alpha, fatty acid synthase mRNA expressions. DB-PMA co-treatment reduced the glycolysis genes expression, but increased the expressions of fatty acid oxidation and TCA cycle genes. Conclusion: This study provides new insights and directions for further research relating to the immunity of porcine respiratory system, by employing a model based on alveolar macrophages and natural materials.

• Journal of Photoscience
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• 제5권1호
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• pp.1-10
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• 1998

The susceptibility of chilling-resistant spinach plants. and of chilling-sensitive squash plants to photoinhibition was compared in terms of the activity of photosystem II, in relation to the deuce of fatty acid unsaturation of chloroplast membrane lipids. From thylakoid membranes of the plants. monogalactosyl diacylgtycerol, digalactosyl diacylglycerol. sulfoquinovosyt diacylglycerol, and phosphatidylglycerol were seperated as major lipid classes. It was found that the content of cis-unsaturated fatty acids of phosphatidylglycerol was greater by 32% in spinach than that in squash. When leaf disks were exposed to light at 5$\circ$C, 15$\circ$C and 25$\circ$C, photochemical efficiency of photosystem II. measured as the ratio of the variable to the maximum fluorescence of chlorophyll, declined markedly in squash plants, as compared to spinach plants. When leaf disks were exposed to strong light in the presence of lincomycin, an inhibitor of protein synthesis in chloroplasts, photoinhibition was accelerated in the two types of plants. Moreover, lincomycin treatment abolished the differences in the degree of susceptibility to strong light, which had been observed between the two types of plants. When the extent of photoinhibition of photosystem II-mediated electron transport was compared in thylakoid membranes isolated from the two types of plants, there were no differences in the degree of inactivation of photosystem II activity. However, when intact leaf disks were exposed to strong light either at 10$\circ$C or at 25$\circ$C, and then were allowed to recover either at 17$\circ$C or at 25$\circ$C in dim light. chilling-resistant plants such as spinach and pea showed marked recovery from photoinhibition, in contrast to chilling-sensitive plants, such as squash and sweet potato. whose recovery was strongly dependent on the temperature. These findings are discussed in relation to the unsaturation of fatty acids in membrane phosphatidylglycerol. It appears that fatty acid unsaturation of membrane lipids accelerates the recovery of photosystem H from photoinhibition, without affecting the photo-induced inactivation process of photosystem II associated with photoinhibition.