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Consideration About the Bacterial Endotoxin Test Showing False Positive Test Result When Performing LAL Test (LAL Test에서 위양성을 나타내는 원인들에 대한 고찰)

  • Hwang, Ki-Young;Cho, Yong-Hyun;Lee, Yong-Suk;Kim, Hyung-Woo;Lee, Hong-Jae;Kim, Hyun-Ju
    • The Korean Journal of Nuclear Medicine Technology
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    • v.13 no.3
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    • pp.156-158
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    • 2009
  • Purpose: Since radiopharmaceuticals are intended for human administration, it is imperative that we should undergo quality control very strictly. Now almost all the PET laboratories have adopted Bacterial Endotoxin Test as the stand quality control method to monitor whether pyrogen is free or not in the product vial containing crude solution. The aim of this study is to find out the reason why false positive result is observed when using commercially available test vial. Materials and Methods: For this experiment, we used commercially available single test kit (Associates of Cape Code. Inc. USA) and we made pH samples by mixing each buffer whose pH ranges are 1.0 to 12.0. Otherwise we made Ethanol samples diluted with distilled water. After making test samples, it added 0.2 mL to the test vial. Assay mixture in the test vial was incubated in a water bath (Chang Shin Co. KOR) for 60 min at $35{\pm}2^{\circ}C$. Results: After incubation period ($60{\pm}1^{\circ}C$), we inverted the test vial about $180^{\circ}$ To know what pH and how many percentage of Ethanol (Fisher Scientific Korea. Ltd) will affect the reaction. With pH buffer, false positive result was observed at pH 1.0 to 5.0 and 7.7 to 12.6 but at pH 5.2 to.7.5, the test results show negative. It's very strange that we couldn't observe negative test result with Tris buffer at pH 8.4, 8.6, 8.8, 9.0. in other case Ethanol, the test result was seen with 5 to 10% Ethanol. But to my surprise we could see very thick gel formation with 100% Ethanol. Conclusions: In this study, we could notice that pH which is too much acidic or alkalic or high concentrated Ethanol would affect Bacterial Endotoxin Test result. As you know, LAL test is sensitive and very reliable method. Therefore, we are needed to elicit the accurate test result as possible as we can.

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Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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Characteristics and Genesis of Terrace Soils in Yeongnam Area -V : Soil Genesis and Classification (영남지역(嶺南地域)에 분포(分布)된 단구지토양(段丘地土壤)의 특성(特性)과 생성연구(生成硏究) -제(第)5보(報) : 토양생성(土壤生成)과 분류(分類))

  • Jung, Yeun-Tae
    • Korean Journal of Soil Science and Fertilizer
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    • v.19 no.4
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    • pp.275-282
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    • 1986
  • A series of studies on the properties of clayey terrace soils distributed at the inland (Yeongcheon) and coastal (Yeongjil) regions in Yeongnam district was carried out. On the base of the facts found and already reported about the macro morphological features as well as on pedological characters in micro scale, physicochemical properties, mineralogical characteristics etc., the present study dealt with soil genesis and tried to classify the soils for reasonable use and managements. 1. Although the both regions belonged to "Mesic" soil temperature regime and "red and yellow earths" areas of "Thornthwaite" pedo-climatic diagram, climatic indices as a soil forming factor indicate that the coastal Yeongil had milder than the inland Yeongcheon. 2. All the terrace soils had developed soil profiles with an "Argrllic B". Upyeong soils in Yeongil region had "Argillans" even in the "II B horizons" that possibly be "Paleo-argillic". 3. The bisequum profiles of Bancheon in Yeongcheon and Upyeong in Yeongil revealed that they were developed on Late Mesozoic shale and on semiconsolidated Tertiary deposits respectively, therefore the overlying clayey terrace deposits were assumed to be originated from the Early Quaternary deposits, Diluvium. 4. To supplement the Soil Taxonomy of USDA, the terrace soils with different degrees of gleyzation were classified as follows; Deogpyeong and Hwadong soils which have less than 50cm of paddified gley horizons (redness less than 0.5) in the upper part of the profiles by artificial surface irrigation, tentatively classified into "Anthrepiaquic Hapludalfs" and the Geugrag soils that have more than 50cm of paddified gley horizons within 1.2m of the profiles, into "Anthr-aquic Ochraqualfs" while the Upyeong soils that had greyish mottles in subsoils by natural ground water remain as an "Aquic Hapludalfs" the same as present. The Bancheon soils with free mottles are into "Typic Hapludalfs" as used at present.

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Effects of Organic Matter Applications on General Components and Essential Oils in Codonopsis lanceolata $T_{RAUTV}$ (유기물(有機物) 종류(種類)에 따른 더덕 근(根)의 일반성분(一般成分)과 정유성분(精油成分) 변화(變化))

  • Lee, Seong-Phil;Kim, Sang-Kuk;Choi, Boo-Sull;Lee, Sang-Chul;Yeo, Soo-Kab
    • Korean Journal of Medicinal Crop Science
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    • v.6 no.1
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    • pp.21-27
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    • 1998
  • This experiment was conducted to increase aromatics in roots of Condonopsis lanceolata by applications of organic matters. Fresh root wt. was increased by conifer/moss application to 79.1g per plant. Crude protein content was also higher at rice straw application than native soil application and crude saponin content was increased by conifer/moss application, but contents of crude fat, fiber and ash were not different in all treatments. Although contents of K, Ca, and Mg were increased by rice straw application, Fe, Mn, Zn, Na and Cu were not significantly different in all treatments, The highest free amino acid was arginine, it was increased by the application of fallen leaves and the highest yield (0,008%) of essential oils was obtained by conifer/moss application. As a result, to produce C. lanceolata plant showing higher quality and aromatic essential oils, it was considered that the most effective organic matter showing high yield and higher aromatic constituents was conifer/moss application of over 3M/T per 10a.

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Processing and Property of Olive Flounder Paralichthys olivaceus Steak (넙치(Paralichthys olivaceus)스테이크제품의 제조 및 품질특성)

  • Yoon, Moon-Joo;Kwon, Soon-Jae;Lee, Jae-Dong;Park, Si-Young;Kong, Cheong-Sik;Joo, Jong-Chan;Kim, Jeong-Gyun
    • Journal of Fisheries and Marine Sciences Education
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    • v.27 no.1
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    • pp.98-107
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    • 2015
  • Olive flounder contains rich amount of lysine which is required for children's growth. Moreover, it is good foodstuffs for elderly, convalescent and diabetics because of low lipid content and high digestibility. This study was investigated for the purpose of obtaining basic data which can be applied to the processing of olive flounder steak. Olive flounder 100 g were chopped, mixed with vegetable (onion 20%, celery 10%, carrot 15%, garlic 1% of chopped olive flounder meat) and ingredient (bread crumbs 20 g, onion 15 g, celery 10 g, egg 1 ea, tarragon 1/2 t, blanc sauce 20 g, fresh cream 20 mL, salt and pepper pinch). Mixed dough was molded into steak shape ($12{\times}7cm$) and was processed by two types of products, Steak-1 {Roasting for 2 minutes in a frying pan wrapped with olive oil and then vacuum packaging in polyethylene film ($20{\times}30{\times}0.05mm$), and then storage at $-20^{\circ}C$ for 7 days, next thawed and warmed by microwave for 2 minutes} and Steak-2 {vacuum-packaging in polyethylene film ($20{\times}30{\times}0.05mm$), and then storage at $-20^{\circ}C$ for 7 days, after thawed, roasted during 2 minutes in a frying pan wrapped with olive oil}. The factors such as pH, TBA value, amino-N, free amino acid, chemical composition, color value (L, a, b), texture profile, sensory evaluation and viable bacterial count of the olive flounder steak (Steak-1, Steak-2) were measured. From the result of sensory evaluation, Steak-2 showed a bit more high scores than Steak-1 but it was difficult to distinguish significant difference (color, odor, taste, texture and acceptance) between Steak-1 and Steak-2 products.