• Plant Biotechnology Reports
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• 제2권2호
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• pp.123-131
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• 2008

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM $CaCl_2$, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20-45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7-11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds.

• Archives of Plastic Surgery
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• 제36권2호
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• pp.135-139
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• 2009

Purpose: The use of an autogenous fat graft has become a common procedure in plastic surgery. However, questions remain concerning on the viability of fat cells and preservation method of aspirated fat. The purpose of this study was to examine the viability of fat cells stored at $-20^{\circ}C$ in the freeze for 1 year after harvest from abdominal liposuction. Methods: Eighteen adults (aged 24 to 65 years old, 16 female and 2 male) were recruited for this study. Harvested aspirated fat tissues were obtained by suction - assisted lipectomy and frozen at $-20^{\circ}C$ commercial refrigerator for one year (average 12.5 months). The viability off at cells in specimens were measured after thawing. The numbers of viable cells were measured on a fluorescence microscope after staining with fluorescein diacetate and propidium iodide. GPDH (Glycerol - 3 - phosphate dehydrogenase) activity was measured. Cell culture was done for 3 weeks. Results: There were no viable cells under the fluorescence microscope, no detectable GPDH activity, and no cultured cells. Conclusion: These findings suggest that aspirated fat after frozen storage for one year at $-20^{\circ}C$ freezer is inadequate to reuse.

• Molecules and Cells
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• 제43권9호
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• pp.813-820
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• 2020

NB4 cell, the human acute promyelocytic leukemia (APL) cell line, was treated with various concentrations of arsenic trioxide (ATO) to induce apoptosis, measured by staining with 7-amino-actinomycin D (7-AAD) by flow cytometry. 2', 7'-dichlorodihydro-fluorescein-diacetate (DCF-DA) and MitoSOX™ Red mitochondrial superoxide indicator were used to detect intracellular and mitochondrial reactive oxygen species (ROS). The steady-state level of SO₂ (Cysteine sulfinic acid, Cys-SO₂H) form for peroxiredoxin 3 (PRX3) was measured by a western blot. To evaluate the effect of sulfiredoxin 1 depletion, NB4 cells were transfected with small interfering RNA and analyzed for their influence on ROS, redox enzymes, and apoptosis. The mitochondrial ROS of NB4 cells significantly increased after ATO treatment. NB4 cell apoptosis after ATO treatment increased in a time-dependent manner. Increased SO₂ form and dimeric PRX3 were observed as a hyperoxidation reaction in NB4 cells post-ATO treatment, in concordance with mitochondrial ROS accumulation. Sulfiredoxin 1 expression is downregulated by small interfering RNA transfection, which potentiated mitochondrial ROS generation and cell growth arrest in ATO-treated NB4 cells. Our results indicate that ATO-induced ROS generation in APL cell mitochondria is attributable to PRX3 hyperoxidation as well as dimerized PRX3 accumulation, subsequently triggering apoptosis. The downregulation of sulfiredoxin 1 could amplify apoptosis in ATO-treated APL cells.

• Osong Public Health and Research Perspectives
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• 제8권6호
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• pp.389-396
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• 2017

Objectives: To circumvent the limitations of the current golden standard method, colony-forming unit (CFU) assay, for viability of Bacille Calmette-$Gu{\acute{e}}rin$ (BCG) vaccines, we developed a new method to rapidly and accurately determine the potency of BCG vaccines. Methods: Based on flow cytometry (FACS) and fluorescein diacetate (FDA) as the most appropriate fluorescent staining reagent, 17 lots of BCG vaccines for percutaneous administration and 5 lots of BCG vaccines for intradermal administration were analyzed in this study. The percentage of viable cells measured by flow cytometry along with the total number of organisms in BCG vaccines, as determined on a cell counter, was used to quantify the number of viable cells. Results: Pearson correlation coefficients of FACS and CFU assays for percutaneous and intradermal BCG vaccines were 0.6962 and 0.7428, respectively, indicating a high correlation. The coefficient of variation value of the FACS assay was less than 7%, which was 11 times lower than that of the CFU assay. Conclusion: This study contributes to the evaluation of new potency test method for FACS-based determination of viable cells in BCG vaccines. Accordingly, quality control of BCG vaccines can be significantly improved.

• The Korean journal of internal medicine
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• 제34권1호
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• pp.146-155
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• 2019

Background/Aims: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. Methods: The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of $NF-{\kappa}B$ was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. Results: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt in HK-2 cells. $NF-{\kappa}B$ promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. Conclusions: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, $NF-{\kappa}B$, and Akt signaling pathway in HK-2 cells.

• The Plant Pathology Journal
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• 제39권1호
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• pp.52-61
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• 2023

Fire blight, caused by Erwinia amylovora, is one of the major bacterial disease of apple and pear, causing enormous economic losses worldwide. Several control measures against E. amylovora have been reported till date, however, none of them have proved to be effective significantly against the pathogen. In this study, mechanisms of the copper-based control agents (CBCAs): copper oxychloride (COCHL), copper oxide (COX), copper hydroxide (CHY), copper sulfate basic (CSB), and tribasic copper sulfate (TCS) and their disease severity reduction efficacy against E. amylovora were analyzed. Bis-1,3-dibutylbarbituric acid trimethine oxonol, carboxyl fluorescein diacetate succinimidyl ester, and 5-cyano-2,3-ditolyl tetrazolium chloride staining were used to check the damage of membrane potential, cytoplasmic pH_in, and respiration of CBCAs-treated E. amylovora, respectively. High disturbance in the membrane potential of E. amylovora was found under COX and COCHL treatments. Similarly, higher significant changes in the inner cytoplasmic pH_in were observed under COX, COCHL, and TCS treatment. CHY and COCHL-treated E. amylovora showed a significant reduction in respiration. In vitro bioassay results revealed that CHY, CSB, and TCS at 2,000 ppm reduced the severity of fire blight both in pre- and post-treatment of CBCAs in immature apple fruits and seedlings. Overall, the most effective CBCAs against E. amylovora could be CHY at 2,000 ppm as its showed inhibition mechanisms and disease severity reduction.

• 한국환경농학회지
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• 제27권3호
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• pp.260-266
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• 2008

시설 유기농 포도 재배지에서 공중 질소 고정과 잡초 억제를 목적으로 도입할 수 있는 월동 녹비가 녹비 생육 초기인 봄에 토양 미생물 활성과 다양성에 미치는 효과를 검토하기 위하여 헤어리 베치,자운영,레드 클로버를 녹비로 파종한 후 효소 활성과 토양 미생물 군락의 기질 이용성 및 인지질 지방산 차이에 의한 토양 미생물 다양성을 조사하였다. 탈수소효소 활성과 FDA 수화도에 의한 미생물 활성으로 측정한 토양 미생물 활성은 처리간에 차이가 없었다. Biolog Ecoplate의 평균 색도 변화는 레드 클로버가 대조구에 비하여 높았다. Biolog EcoPlate에 의한 토양 미생물 군락의 기질 이용성은 자운영 처리구가 헤어리 베치구 및 레드 클로버 처리구와 뚜렷이 구분되었다. 그리고 미생물 군락의 기능적 다양성 지수는 헤어리 베치구가 대조구 보다 유의성 있게 높았다. 인지질 지방산 구성에 의한 미생물 군락의 차이를 주요인 분석으로 분석한 결과 자운영이 대조구와 현저히 구분되었고 녹비 처리구 간에는 차이가 없었다.

• 한국토양비료학회지
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• 제40권4호
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• pp.234-241
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• 2007

여러가지의 선발된 미생물로 구성된 미생물비료는 토양 개량과 식물 생장 촉진을 위해서 여러 유기물과 결합하여 이용되기도 한다. 미생물 비료를 미생물 비료 단독으로 그리고 도시 가로수 부산물 퇴비, 가금류 분뇨 부산물, 레드클로버와 귀리의 피복작물 등의 유기물과 같이 토양에 처리하여 토양의 화학적 또는 생물학적 특성에 미치는 효과를 측정하였다. 액체상의 미생물 비료를 2년동안 3회 처리하였다. 미생물 비료 단독으로는 pH, K, 유기물 함량에 영향을 미치지 않았지만, 미생물비료의 처리는 2년 가을 모두 가금류 분뇨 부산물을 처리한 토양의 인산 함량을 증가시켰고, 첫해 가을에 퇴비를 처리한 토양의 칼슘함량을 증가시켰으며, 레드클로버를 처리한 토양의 Ca, Mg, 그리고 양이온교환용량을 감소시켰다. 미생물 비료는 레드클로버가 처리된 토양에서 첫 해 7월에 탈수소효소 활성을 증가시켰다. 미생물 비료는 유기물이 처리되지 않은 토양이나 퇴비가 처리된 토양에서 FDA의 가수분해도를 가끔 증가시켰다. 가금류 분뇨 부산물과 레드 클로버가 처리된 토양의 FDA 가수분해도와 가금류 분뇨 부산물이 처리된 토양의 탈수소효소활성은 미생물 비료의 처리로 감소하였다. 한편, 미생물 비료의 처리는 BIOLOG에 의한 토양 미생물 군락의 생리생태적 다양성에는 영향을 미치지 못했다. 따라서 토양의 미생물학적 특성에 미치는 미생물비료의 효과는 같이 투여되는 유기물의 종류에 따라 다양하다고 할 수 있으며, 탈수소효소의 활성은 레드클로버가 처리된 토양에서, 그리고 FDA 가수분해도는 퇴비와 귀리가 처리된 토양에서 가끔 증가했다.

• Journal of Periodontal and Implant Science
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• 제44권6호
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• pp.266-273
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• 2014

Purpose: We previously reported that human serum significantly reduces the invasion of various oral bacterial species into gingival epithelial cells in vitro. The aims of the present study were to characterize the serum component(s) responsible for the inhibition of bacterial invasion of epithelial cells and to examine their effect on periodontitis induced in mice. Methods: Immortalized human gingival epithelial (HOK-16B) cells were infected with various 5- (and 6-) carboxy-fluorescein diacetate succinimidyl ester-labeled oral bacteria, including Fusobacterium nucleatum, Provetella intermedia, Porphyromonas gingivalis, and Treponiema denticola, in the absence or presence of three major serum components (human serum albumin [HSA], pooled human IgG [phIgG] and ${\alpha}1$-antitrypsin). Bacterial adhesion and invasion were determined by flow cytometry. The levels of intracellular reactive oxygen species (ROS) and activation of small GTPases were examined. Experimental periodontitis was induced by oral inoculation of P. gingivalis and T. denticola in Balb/c mice. Results: HSA and phIgG, but not ${\alpha}1$-antitrypsin, efficiently inhibited the invasion of various oral bacterial species into HOK-16B cells. HSA but not phIgG decreased the adhesion of F. nucleatum onto host cells and the levels of intracellular ROS in HOK-16B cells. N-acetyl-cysteine (NAC), a ROS scavenger, decreased both the levels of intracellular ROS and invasion of F. nucleatum into HOK-16B cells, confirming the role of ROS in bacterial invasion. Infection with F. nucleatum activated Rac1, a regulator of actin cytoskeleton dynamics. Not only HSA and NAC but also phIgG decreased the F. nucleatum-induced activation of Rac1. Furthermore, both HSA plus phIgG and NAC significantly reduced the alveolar bone loss in the experimental periodontitis induced by P. gingivalis and T. denticola in mice. Conclusions: NAC and the serum components HSA and phIgG, which inhibit bacterial invasion of oral epithelial cells in vitro, can successfully prevent experimental periodontitis.

• 한국토양비료학회지
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• 제45권3호
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• pp.435-443
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• 2012

본 연구는 논과 목초지에서 관리방법에 따른 토양탄소의 변화에 대해 알아보았다. 논에서는 총탄소 농도의 변화에 비하여 입자상 탄소농도의 변화가 IRG 처리에 따라 더 민감하게 반응하였으며, 이에 입자상 탄소농도는 관리방법 변화에 따른 조기지시자로 활용될 수 있음을 시사하였다. 반면, 초지에서는 입자상 탄소농도의 변화가 총탄소 농도 변화에 비해 오히려 더 민감하지 않게 반응하였는데, 이는 본 연구방법에 따라 분류한 입자상 탄소가 초지와 같은 생태계에서는 의미 있는 탄소부분이 아닐 수 있음을 시사한다. 토양탄소저장을 살펴보았을 때 논의 경우는 겨울동안 IRG 목초작물을 재배하는 관리를 3년 이상 지속한 서천 및 장흥에서 유의한 증가가 있었다. 토양탄소의 유의한 증가는 농도뿐만 아니라 용적밀도를 고려하여 동일 부피 토양을 비교한 경우 및 동일 질량 토양을 비교한 경우 모두 유의하게 관찰되었는데, 이는 IRG 재배에 따른 토양탄소 저장의 증가가 단순히 표토에 축적된 식물체 유기물에 의한 일시적 농도의 증가가 아닌 토심 0-15 cm 깊이의 토양탄소의 증가였음을 시사한다. 목초지에서는 목초지 조성이후 3년, 5년, 7년, 10년이 지남에 따라 표토 (0-5 cm)에서의 탄소농도 증가가 현저하였다. 반면 5-15 cm 깊이의 토양에서는 이러한 증가경향이 뚜렷하지 않거나 오히려 감소하기도 하였다. 이 결과 토양내 저장되어 있는 탄소량을 용적밀도를 고려하고 동일 질량을 비교한 경우에 시간에 따른 뚜렷한 증가경향이 흐릿해짐을 알 수 있었다. 이는 초지 조성시기가 오래됨에 따라 토양의 용적밀도가 높아짐으로 인해 용적밀도 증가에 따른 토양질량의 증가분을 보정하였을 때 나타나는 결과로써, 초지조성연도가 오래될수록 저장된 탄소의 양이 선형적으로 증가할 것이라는 기존의 통념을 수정할 필요가 있음을 시사한다. IPCC (2006)에서 제안한 지침에 따르면, 토양탄소는 20년 동안 직선적인 증가를 한다는 가정을 하였는데, 본 연구의 결과에서 특정 관리방법 착수시점에서 초기 5-7년 동안토양탄소는 빠르게 증가하고, 증가속도는 그 이후에 느려지고 있음을 보여준다. 이는 지속적 토양탄소 저장 증가를 위해서는 적절한 토양관리 및 유기물 관리를 해야 한다는 점을 의미하기도 한다. 한편, 토양 내 FDA 활성도에 비추어본 미생물 활성도는 논의 경우는 IRG 처리구에서 대조구에 비해 유의하게 낮게 나타났으며 초지의 경우는 조성연도별 유의한 차이가 없었다. 이는 FDA 활성도가 토양의 관리방법별 차이에 따른 토양 질의 변화를 표현하기에는 민감하거나 변별력이 높은 지시자는 아님을 의미하며, 미생물 생체량이나 토양호흡량 등과 함께 측정하여 보조적인 자료로 활용하는 것이 좋다고 판단된다.