• Title/Summary/Keyword: Neutral red

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Cloning and Expression of the Pasteurella haemolytica A1 leukotoxin Gene in Bacillus subtilis (Bacillus subtilis을 이용한 Pasteurella haemolytica A1 leukotoxin 유전자의 cloning 및 발현)

  • Yoo, Han-sang;Maheswaran, Samuel K.
    • Korean Journal of Veterinary Research
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    • v.36 no.3
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    • pp.665-680
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    • 1996
  • Bovine Pneumonic Pasteurellosis는 수송열(輸送熱)로 일반적으로 알려져 있는 질병으로서, 여러가지 요인의 복합적(複合的)인 작용에 의해 발병하는 것으로 알려져 있으나, Pasteurella haemolytica A1이 가장 주요(主要)한 인자(因子)로 밝혀져 있다. P haemolytica A1은 leukotoxin(LKT), lipopolysaccharide(LPS), capsular polysaccharide 등 여러가지의 병원성인자(病原性因子)을 생성한다. 이들 인자중 LKT가 가장 중요한 병원성인자로 밝혀져 있다. 이에 본 실험은 P haemolytical A1의 LKT 유전자를 Bacillus subtilis에서 발현(發現)시킴으로서 LPS에 오염(汚染)되지 않은 LKT을 대량으로 생산할 목적으로 실시되었다. 실험의 첫 단계(段階)로서 pLKT52 plasmid을 Sau3 A1의 제한효소을 이용하여 부분소화(部分消化)시킨 후 이 부분 소화(消化)된 유전자들로부터 3~5kb 크기의 유전자들을 순수분리하여 pUC18와 결합시킨 후 E coli NM522에 형질전환(形質轉換)시켰다. 이때 형질전환된 균주들은 LKT에 대한 단크론 항체인 MAb601을 이용하여 colony blot 법에 의해서 LKT 유전자 보유 및 발현여부(發現與否)을 조사하였다. 이들 양성 clone들은 제한효소분석(制限酵素分析), 염기서열분석(鹽基序列分析) 및 Western blot 등에 의해서 재확인(再確認)하였다. 총 9개의 양성 clone중 위의 방법에 의해서 한 clone을 선택(選擇)하여 lktCA insert를 재분리하여 shuttle vector에 subcloning 하였다. Subcloning된 LKT 유전자들은 shuttle vector의 종류(種類)(pHPS9, p602/20, pHPS9-Sac)와 각기(各其) 다른 종류(種類)의 B subtilis(spoO12A, BR121, WB3O, Raj1105) 숙주내(宿主內)에서 발현정도를 Western blot 법에 의해서 비교(比較)하였다. 이때 최적발현조건(最適發現條件)은 p602/20와 pBL1의 dual plasmid system을 이용하여 B subtilis spoO12A에서 2시간동안 IPTG로 발현을 유도(誘導)하는 것이었다. B subtilis에서 발현된 LKT을 visual 법과 neutral red uptake 법을 이용하여 소 폐포(肺胞) 대식구(大食求)에 대한 biological activity를 확인하였다. 발현된 LKT에 대한 LPS 오염은 LKT을 SDS-PAGE 후 silver stain에 의해서 확인하였다. 본 실험을 통해서 볼 때에 lktCA 유전자를 보유(保有)하고 있는 p602/20는 B subtilis에서 매우 불안정(不安定)하였고, 발현된 LKT는 세균자체(細菌自體)에서 생성되는 protease들에 의해서 파괴(破壞)됨으로서 농도(濃度)가 매우 낮았다. 이러한 문제점들은 다음 단계(段階)의 실험에서 해결되어야할 문제들이다.

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Influence of Yeoldahanso-tang on the Hypoxic Damage of Cultured Cerebral Neurons from mouse and SK-N-MC cells (열다한소탕(熱多寒少湯)이 저산소성(低酸素性) 대뇌신경세포(大腦神經細胞) 손상에 미치는 영향(影響))

  • Kim, Hyoung-Soon;Bae, Young-Chun;Lee, Sang-Min;Kim, Kyung-Yo;Won, Kyoung-Sook;Sihm, Gyue-Hearn;Park, Su-Jeong
    • Journal of Sasang Constitutional Medicine
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    • v.15 no.1
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    • pp.72-89
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    • 2003
  • To elucidate the neuroprotective effect of Yeoldahanso-tang(YHT) on nerve cells damaged by hypoxia, the cytotoxic effects of exposure to hypoxia were determined by XTT(SODIUM3,3'-{I-[(PHENYLAMINO) CARBONYL]-3,4-TETRAZOLIUM}- BIS (4-METHOXY-6-NITRO) BENZENE SULFONIC ACID HYDRATE), NR(Neutral red), MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and SRB(Sulforhodamin B) asssay. The activity of catalase and SOD(Superoxide dismutase) was measured by spectrophometry, and $TNF-{\alpha}$(Tumor cell necrosis $fector-{\alpha}$) and PKC(Protein kinase C) activity was measured after exposure to hypoxia and treatment of YHTWE. Also the neuroprotective effect of YHTWE was researched for the elucidatioion of neuroprotective mechanism. The results were as follows; 1. Hypoxia decreased cell viability measured by XTT, NR assay when cultured cerebral neurons were exposed to 95% N2/5% CO2 for $2{\sim}26$ minutes in these cultures and YHTWE inhibited the decrease of cell viability. 2. H2O2 treatment decreased cell viability measured by MTT, and SRB assay when cultured cerebral neurons were exposed to 1-80 ${\mu}M$ for 6 hours, but YHTWE inhibited the decrease of cell viability. 3. Hypoxia decreased catalase and SOD activity, and also $TNF-{\alpha}$ and PKC activity in these cultured cerebral neurons, but YHTWE inhibited the decrease of the catalase and SOD activity in these cultures. 4. Hypoxia triggered the apoptosis via caspase activation and internucleosomal DNA fragmentation. Also hypoxia stimulate the release of cytochrome c forom mitochondria. YHTWE inhibited the apoptosis via caspase activation induced by hypoxia. From these results, it can be suggested that brain ischemia model induced hypoxia showed neurotoxicity on cultured mouse cerebral neurons, and the YHTWE has the neuroprotective effect in blocking the neurotoxicity induced by hypoxia in cultured mouse cerebral neurons.

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Histological effect of cyclophosphamide on diethylnitrosamine-induced hepatic tumors in rats (항암제 cyclophosphamide가 diethylnitrosamine에 의한 랫드 간암에 미치는 조직학적 영향)

  • Kwak, Soo-dong;Kang, Chung-boo;Koh, Phil-ok;Kim, Chong-sup
    • Korean Journal of Veterinary Research
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    • v.39 no.3
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    • pp.593-601
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    • 1999
  • This study was designed to evaluate the effect of cyclophosphamide(CY) on diethylnitrosamine(DEN)-induced hepatic tumors in rats. Thirty five male or female Sprague Dawley rats were continiously given water containing 0.01% DEN for 10 weeks and then were given with CY 25mg/rat/day in water for 3, 5, 7 or 9 days. The livers of rats were removed and fixed in 10% buffered neutral formalin. he appearances of positive cells by immunohistochemical methods using proliferating cell nuclear antigen (PCNA) antibody, p53 antibody and apoptotic kit were investigated. The livers of rats given with CY were grossly brilliant, red-brown color, flexible, and thin border, and stainability of the liver cells were restored microscopically, and the vaccuolated and degenerated regions were differentiated from restored regions. These restored findings also were advanced in control group because of no DEN treatment but tended to be less avanced. In immunohistochemistry, positive cells to PCNA antibody appeared more numerous in control groups than that of CY treated groups. Appearance of positive cells in CY-treated group for 7 days and for 9 days were more numerous than those of CY-treated groups for 3 days and for 5 days, respectively. So these findings suggested that CY suppressed cell proliferations and effects of these action were decreased with CY-treated days. The numbers of positive cells to PCNA antibody were more prominent in hepatocellular carcinoma regions and cholangiocarcinoma regions, and then were ranked as order of large liver cell regions and normal liver cell regions. Also the numbers of the positive cells by apoptotic kit tended to be higher in hepatocellular carcinoma regions and cholangiocarcinoma regions but not uniformly in order in all regions and were much less numbers than those of PCNA positive cells. So immunohistochemical methods using PCNA antibody together than using apoptotic kit alone when anti-carcinogen experiments. Rats with positive cells by p53 antibody were 11 of 15 rats(73.4%) in control groups and 12 of 18 rats(66.7%) in CY treated group, respectively. These positive cells appeared focally in early vacuole-occurring regions and were very low in numbers.

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Studies on the Natural Pigments (Part I) -Contents and Properties of Gardenia Jasminoides Ellis Pigment- (천연색소(天然色素)에 관(關)한 연구(硏究)제1보(第一報) -치자색소(梔子色索)의 함량(含量) 및 성질(性質)에 관(關)한 연구(硏究)-)

  • Kim, Dong-Yun;Kim, Kwan
    • Applied Biological Chemistry
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    • v.18 no.2
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    • pp.98-101
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    • 1975
  • The components of Gardenia pigment in rind, flesh, fresh fruit and stored fruit, its heat resistance and stability in pH, its dilution and colour-degree were studied. The results are summarized as follows; 1. The components of CROCIN in fresh fruit and stored fruit were 0.0157% and 0.0136% respectively. One year storage of stored fruit resulted in the decrease of 15% of CROCIN. 2. The components of Gardenia pigment in flesh were contained nearly twice as much as in rind. 3. There were traces of ${\beta}-carotene$ and some unknown carotenoid pigments. 4. Gardenia pigment was stable at $100^{\circ}C$ for 60 minutes, but, at $150^{\circ}C$ for 30 minutes, nearly half of them was destructed. 5. Gardenia pigment turned to colurless but was stable in neutral or alkali condition after 10 days. 6. Supercially, dark Gardenia pigment looked yellowish red and thin Gardenia pigment looked yellow: however, each ${\lambda}_{max}$. was all the same.

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Identification of the Volatile Components in Korean Ordinary Kochujang (한국재래식(韓國在來式) 고추장의 향기성분동정(香氣成分同定))

  • Ahn, Cheol-Woo;Kim, Jong-Kyu;Sung, Nack-Kie
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.16 no.3
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    • pp.27-34
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    • 1987
  • The present paper was attempted to obtain the basic date concerning a reasonable preparing method and optimum fermentation conditions of Kochujang (Red pepper paste). To establish the standard quality of Kochujang, the chemical compositions and the volatile components of Kochujang was discussed. The native Kochujang collected from 80 households contained 40.51% of moisture, 6.00% of salts, 3.25 % of crude fat, 10.30 % of crude ash, pH 4.79, 9.28ml of tillable acidity. 19.60% of reducing sugar, 179.51 mg/100g of amino nitrogen and 4.43% of total nitrogen. The volatiles of Kochujang were extracted by a steam-distillation under the reduced pressure and determined by gas chromatography-mass spectrometry (GC-MS). Total volatile components identified in the native Kochujang were 46 components, i. e., 30 components in neutral fraction, 8 components in phenolic fraction and 8 components in acidic fraction, respectively. But no components were detected basic fraction.

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산삼의 배양 및 그 응용에 관한 연구

  • 신미희
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.27 no.2
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    • pp.45-56
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    • 2001
  • Korea mountain ginseng known as oriental miracle drug is an important medicinal plant. The effect of mountain ginseng adventitious roots extract has been described. The valuable root of mountain ginseng contained several kinds of ginsenosides that have been confirmed to have many active functions for the human body. However, the study of mountain ginseng has a limit because the price of wild ginseng is very expensive and rare. The mountain ginseng adventitious roots were derived from mountain ginseng callus that were induced from mountain ginseng roots. Adventitious roots were separated from callus and grown in solid media(Murachige and stoog media). It was cultured in a 20L bioreactor. After culturing for 40days, adventitious roots were harvested. Afterwards the harvested mountain ginseng adventitious roots were dryed and extracted. We examined the effect on melanogenesis of mountain ginseng adventitious roots extract. Here, we report the inhibitory effect of melanin biosynthesis on the adventitious roots extract of In vitro test. Also, we assessed the safety of adventitious roots extract. In vitro, cytotoxicity of adventitious roots extract was assessed in mouse fibroblast using two method: The neutral red uptake assay and the MTT assay. In vivo, the allergic and irritant were Patch teated in 30 patients. Consequently, extract of mountain ginseng adventitious roots have inhibitory effect on melanin biosynthesis in B-16 melanoma cell test, tyrosinase inhibitory test and DOPA auto-oxidation test. There were decreased 86%(0.5% concentration), 45%(1% concentration) and 61%(1% concentration), respectively.

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Effect of 840 nm Light-Emitting Diode(LED) Irradiation on Monosodium Iodoacetate-Induced Osteoarthritis in Rats (흰쥐의 MIA 유발 무릎 뼈관절염에 대한 840 nm LED의 효과)

  • Jekal, Seung-Joo;Kwon, Pil-Seung;Kim, Jin-Kyung;Lee, Jae-Hyoung
    • Journal of the Korean Society of Physical Medicine
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    • v.9 no.2
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    • pp.151-159
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    • 2014
  • PURPOSE: The purpose of this study was to evaluate whether light-emitting diodes (LED) irradiation could be effective in a noninvasive, therapeutic device for the treatment of osteoarthritis(OA). METHODS: Twenty-four male Sprague-Dawley rats were divided into four groups: Vehicle control (saline); monosodium iodoacetate-injection (MIA); LED irradiation after MIA injection (MIA-LED); indomethacin-treatment after MIA injection (MIA-IMT). OA was induced by intra-articular injection of 3 mg MIA through the patellar ligament of the right knee. Vehicle control rats were injected with an equivalent volume of saline. The LED was irradiated for 15 min/day for a week after 7 days of MIA treatment. To compare with the effect of LED irradiation, the indomethacin was administrated 20 mg/kg twice a week orally after 7 days of MIA treatment. Knee joints were removed and fixed overnight in 10% neutral buffered formalin and decalcified by EDTA for 2 week before being embedded in paraffin. The assessment of OA induction were monitored by knee movement and radiographic finding. Histologic analysis were performed following staining with hematoxylin and eosin, safranin O-fast green, or toluidine blue, picrosirius red, and histologic changes were scored according to a modified Mankin system. Apoptotic cell in tissue sections was detected using TUNEL method. RESULTS: Radiographic examination could not show the differences between the MIA-treated and the MIA-LED-treated rats. In the histologic analysis, however, LED irradiation prevented cartilage damage and subchondral bone destruction, and significantly reduced mononuclear inflammatory cell infiltration and pannus formation. LED irradiation also reduced apoptosis of cartilage cells, but it prevented apoptosis of infiltrated inflammatory cells in synovium. In addition, LED irradiation showed an increase of collagen production in the meniscus. CONCLUSION: These results suggest that the 840 nm LED irradiation would be a suitable non-thermal phototherapy for the treatment of OA, as a cartilage protection and anti-inflammatory modality.

Radioanalytical and Spectroscopic Characterizations of Hydroxo- and Oxalato-Am(III) Complexes (방사분석과 분광학을 이용한 Am(III) 가수분해와 옥살레이트 착물 화학종 연구)

  • Kim, Hee-Kyung;Cho, Hye-Ryun;Jung, Euo Chang;Cha, Wansik
    • Journal of Nuclear Fuel Cycle and Waste Technology(JNFCWT)
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    • v.16 no.4
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    • pp.397-410
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    • 2018
  • When considering the long-term safety assessment of spent-nuclear fuel management, americium is one of the most radio-toxic actinides. Although spectroscopic methods are widely used for the study of actinide chemistry, application of those methods to americium chemistry has been limited. Herein, we purified $^{241}Am$ to obtain a highly pure stock solution required for spectroscopic studies. Quantitative and qualitative analyses of purified $^{241}Am$ were carried out using liquid scintillation counting, and gamma and alpha radiation spectrometry. Highly sensitive absorption spectrometry coupled with a liquid waveguide capillary cell and time-resolved laser fluorescence spectroscopy were employed for the study of Am(III) hydrolysis and oxalate (Ox) complexation. $Am^{3+}$ ions under acidic conditions exhibit maximum absorbance at 503 nm, with a molar absorption coefficient of $424{\pm}8cm^{-1}{\cdot}M^{-1}$. $Am(OH)_3(s)$ colloidal particles formed under near neutral pH conditions were identified by monitoring the absorbance at around 506-507 nm. The formation of ${Am(Ox)_3}^{3-}$ was detected by red-shifts of the absorption and luminescence spectra of 4 and 5 nm, respectively. In addition, considerable enhancements of the luminescence intensities were observed. The luminescence lifetime of ${Am(Ox)_3}^{3-}$ increased from 23 to 56 ns, which indicates that approximately six water molecules are replaced by carboxylate ligands in the inner-sphere of the Am(III). These results suggest that ${Am(Ox)_3}^{3-}$ is formed through the bidentate coordination of the oxalate ligands.

Characterization of Cellular Growth, CO2 Assimilation and Neutral Lipid Production for 4 Different Algal Species (미세조류 4종의 성장, CO2 동화 및 지질 생성 특성)

  • Shin, Chae Yoon;Noh, Young Jin;Jeong, So-Yeon;Kim, Tae Gwan
    • Microbiology and Biotechnology Letters
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    • v.48 no.4
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    • pp.547-555
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    • 2020
  • Microalgae are a promising resource in energy and food production as they are cost-effective for biomass production and accumulate valuable biological resources. In this study, CO2 assimilation, biomass, and lipid production of 4 microalgal species (Chlorella vulgaris, Mychonastes homosphaera, Coelastrella sp., and Coelastrella vacuolata) were characterized at different CO2 concentrations ranging from 1% to 9%. Microscopic observation indicated that C. vulgaris was the smallest, followed by M. homosphaera, C. vacuolata, and Coelastrella sp. in order of size. C. vulgaris grew and consumed CO2 more rapidly than any other species. C. vulgaris exhibited a linear increase in CO2 assimilation (up to 9.62 mmol·day-1·l-1) as initial biomass increased, while the others did not (up to about 3 mmol·day-1·l-1). C. vulgaris, Coelastrella sp., and C. vacuolata showed a linear increase in the specific CO2 assimilation rate with CO2 concentration, whereas M. homosphaera did not. Moreover, C. vulgaris had a greater CO2 assimilation rate compared to those of the other species (14.6 vs. ≤ 11.9 mmol·day-1·l-1). Nile-red lipid analysis showed that lipid production per volume increased linearly with CO2 concentration in all species. However, C. vulgaris increased lipid production to 18 mg·l-1, compared to the 12 mg·l-1 produced by the other species. Thus, C. vulgaris exhibited higher biomass and lipid production rates with greater CO2 assimilation capacity than any other species.

Female Vaginitis Active Ingredients of 16 Kinds Natural Herbal Medicine Extracts and Dyeing Effect on Cotton Fabric (16종 천연물 추출물의 여성질염 활성성분 및 면포 염착 효과)

  • Hyun Kyoung Kim;Yungi Lee;Subin Choi;DO Wan Kim
    • The Journal of the Convergence on Culture Technology
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    • v.9 no.1
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    • pp.725-733
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    • 2023
  • The evaluation of functional active ingredients and dyeing effect on cotton fabric using 16 types of natural products water extracts and 50% ethanol extracts, there were differences in color intensity, but 6 types of red, 5 types of yellow, 2 types of orange, and 3 types of green were observed. In general, the color of the 50% ethanol extract was high and clear. As a result of dyeing by immersing cotton gauze in water extracts and 50% ethanol extracts of these natural products for 2 days, the dyeing effect was generally good, and it was observed that there was little fading decrease in color even after drying at room temperature and opening and storing. The pH of the water extracts of 16 natural products ranged from neutral to slightly acidic, with no side effects on the skin. For DPPH free radical inhibition antioxidant effect, the effect of 50% ethanol extract for each sample was better than that of water extract. As for the polyphenol content, the content of the 50% ethanol extract tended to be higher than that of the water extract. Therefore, the purpose of this study was to investigate the correlation between the antioxidant activity of 16 natural extracts and the prevention of female vaginitis.