• Journal of Microbiology
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• 제42권4호
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• pp.285-291
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• 2004

The soil bacterial community and some inoculated bacteria were monitored to assess the microbial responses to prescribed fire in their microcosm. An acridine orange direct count of the bacteria in the unburned control soil were maintained at a relatively stable level $(2.0\~2.7\times10^9\;cells/g^{-1}{\cdot}soil)$ during the 180 day study period. The number of bacteria in the surface soil was decreased by fire, but was restored after 3 months. Inoculation of some bacteria increased the number of inoculated bacteria sev­eral times and these elevated levels lasted several months. The ratios of eubacteria detected by a flu­orescent in situ hybridization (FISH) method to direct bacterial count were in the range of $60\~80\%$ during the study period, with the exception of some lower values at the beginning, but there were no definite differences between the burned and unburned soils or the inoculated and uninoculated soils. In the unburned control soil, the ratios of $\alpha-,\beta-\;and\;\gamma-subgroups$ of the proteobacteria, Cytophaga-Fla­vobacterium and other eubacteria groups to that of the entire eubacteria were 13.7, 31.7, 17.1, 16.8 and $20.8\%,$ respectively, at time 0. The overall change on the patterns of the ratios of the 5 subgroups of eubacteria in the uninoculated burned and inoculated soils were similar to those of the unburned con­trol soil, with the exception of some minor variations during the initial period. The proportions of each group of eubacteria became similar in the different microcosms after 6 months, which may indicate the recovery of the original soil microbial community structure after fire or the inoculation of some bac­teria. The populations of Azotobacter vinelandii, Bacillus megaterium and Pseudomonas fluorescens, which had been inoculated to enhance the microbial activities, and monitored by FISH method, showed similar changes in the microcosms, and maintained high levels for several months.

• 한국수정란이식학회지
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• 제27권2호
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• pp.107-111
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• 2012

Miniature pig sperm cryopreservation is continually researched in biotechnology for breed conservation and reproduction. It is important to control the temperature at each stage of cryopreservation and cryoprotectant. It is also necessary to find the optimal cryoprotectant concentration and chemical elements of the extender. Recently, many studies have used various cryoprotectant materials, such as dimethyl sulphoxide (DMSO), ethylene glycol (EG), antifreeze protein (AFP), amides, and glycerol. Glycerol is a commonly used cryoprotectant. However, glycerol has critical cytotoxic properties, including osmotic pressure and it can cause irreversible damage to live cells. Therefore, We focused on membrane fluidity modifications can reduce cell damage from freezing and thawing procedures and evaluated on the positive effects of trehalose to the viability, chromatin integrity, and motility of boar sperm. Miniature pig sperm was separated from semen by washing with modified- Modena B (mMB) extender. After centrifugation, the pellet was diluted with the prepared first extender. This experiment was designed to compare the effects that sperm cryopreservation using two different extenders has on sperm chromatin. The control group used the glycerol only and it was compared with the glycerol and glycerol plus trehalose extender. Sperm viability and motility were evaluated using WST1 assays and computer-assisted semen assays (CASA). Chromatin structure was examined using acridine orange staining. For the motility descriptors, trehalose caused a significant (p<0.01) increase in total motility ($57.80{\pm}4.60%$ in glycerol vs. $75.50{\pm}6.14%$ in glycerol + trehalose) and progressive ($51.20{\pm}5.45%$ in glycerol vs. $70.74{\pm}8.06%$ in glycerol + trehalose). A significant (p<0.05) increase in VAP ($42.70{\pm}5.73{\mu}m/s$ vs. $59.65{\pm}9.47{\mu}m/s$), VSL ($23.06{\pm}3.27{\mu}m/s$ vs. $34.60{\pm}6.58{\mu}m/s$), VCL ($75.36{\pm}11.36{\mu}m/s$ vs. $99.55{\pm}12.91{\mu}m/s$), STR ($54.4{\pm}2.19%$ vs. $58.0{\pm}1.63%$), and LIN ($32.2{\pm}2.05%$ vs. $36.0{\pm}2.45%$) were also detected, respectively. The sperm DNA fragmentation index was 48.8% to glycerol only and 30.6% to glycerol plus trehalose. Trehalose added group showed higher percentages of sperm motility, stability of chromatin structure than glycerol only. In this study, we suggest that trehalose is effective in reducing freezing damage to miniature pig sperm and can reduce chromatin damage during cryopreservation.

• 대한수의학회지
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• 제37권2호
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• pp.409-416
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• 1997

Bovine theileriosis caused by Theileria sergenti is the tick-borne intraery- throcytic piroplasmosis, that occurs in most regions of Korea. It results in severe economic losses on a farm caused by anemia, milk production loss, abortion and death. This study was undertaken to confirm the effects of the growth hormone and the insulin-like growth factor-I which are associated in the growth of cattle infected by T sergenti. The blood of one hundred and twenty ten-month Holstein was collected and the prepared blood smear was stained with acridine orange to investigate their parasitemia. And the hematological profiles were observed. According to the value of the hematocrit, they were categorized into four groups : Group 1 was under 20 percent, groups 2 and 3 were from over 21 to under 30 percent and from over 31 to under 35 percent and group 4 was over 36 percent. As the value of the hematocrit decreased, parasitemia(%) in erythrocytes was observed to increase(Y=-1.064X + 30.537, r=0.660). The amounts of the growth hormone and the insulin-like growth factor-I in the serum were measured by the radioimmunoassay. The growth hormone in serum of the group 1, group 2, group 3 and group 4 were observed as $0.238{\pm}0.043nmol/l$, $0.21{\pm}0.024nmol/l$, $0.366{\pm}0.035nmol/l$ and $0.646{\pm}0.223nmol/l$, respectively. The quantitative of the insulin-like growth factor-I in the same groups were observed also as $209.686{\pm}18.94ng/ml$, $250.9{\pm}12.609ng/ml$, $279.3{\pm}8.883ng/ml$ and $365.9{\pm}22.45ng/ml$, respectively. It can be concluded that the growth hormone and the insulin-like growth factor-I were observed to decrease in severe anemia due to theileriosis.

• Korean Journal of Acupuncture
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• 제26권1호
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• pp.79-84
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• 2009

Objective : In 1960's Bonghan Kim's team found BongHan(BH) ducts which were presumed as acupuncture meridians and BH corpuscles. They asserted Bonghan theory and SanAl theory which was involved in cell division and cell restoration. However, many other experiments which had been operated to demonstrate and find the existence of BH ducts had failed because of the secret of blue stain drugs. During the last several years, BongHan theory has been revived through experimental researches to find the anatomical structures of BH ducts and corpuscles by Soh's Biomedical Physics Lab. Soh's research team used the staining with Janus Green B, Alcian blue, nanoparticles and Acridine Orange. We used DAPI staining to find the existence of BH ducts and the corpuscles and to observe nuclear arrangement. Methods : We used japan white rabbits as experimental animals. BH ducts and corpuscles were stained with DAPI. The nucleus configuration in BH ducts stained with DAPI were observed with microscope. Results : In this study, we found thread like structures in silver white color distinguished from the blood vessels, nerves and lymph vessels. These thread like vessels in the linear duct shape were connected to same colored mass in the ball shape. Thread like structures we found could be separated easily from the surrounding other organ mass. The nuclei of the thread like structure in DAPI staining, are about 10${\sim}$20${\mu}m$ length, in rod shape and linear arrangement. Conclusion : We concluded that the thread like structure we found was same vessel reported by Soh's research team, BongHan ducts and corpuscle.

• Biomolecules & Therapeutics
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• 제28권2호
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• pp.202-210
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• 2020

Fluoxetine is used widely as an antidepressant for the treatment of cancer-related depression, but has been reported to also have anti-cancer activity. In this study, we investigated the cytotoxicity of fluoxetine to human gastric adenocarcinoma cells; as shown by the MTT assay, fluoxetine induced cell death. Subsequently, cells were treated with 10 or 20 µM fluoxetine for 24 h and analyzed. Apoptosis was confirmed by the increased number of early apoptotic cells, shown by Annexin V- propidium iodide staining. Nuclear condensation was visualized by DAPI staining. A significant increase in the expression of cleaved PARP was observed by western blotting. The pan-caspase inhibitor Z-VAD-FMK was used to detect the extent of caspase-dependent cell death. The induction of autophagy was determined by the formation of acidic vesicular organelles (AVOs), which was visualized by acridine orange staining, and the increased expression of autophagy markers, such as LC3B, Beclin 1, and p62/SQSTM 1, observed by western blotting. The expression of upstream proteins, such as p-Akt and p-mTOR, were decreased. Autophagic degradation was evaluated by using bafilomycin, an inhibitor of late-stage autophagy. Bafilomycin did not significantly enhance LC3B expression induced by fluoxetine, which suggested autophagic degradation was impaired. In addition, the co-administration of the autophagy inhibitor 3-methyladenine and fluoxetine significantly increased fluoxetine-induced apoptosis, with decreased p-Akt and markedly increased death receptor 4 and 5 expression. Our results suggested that fluoxetine simultaneously induced both protective autophagy and apoptosis and that the inhibition of autophagy enhanced fluoxetine-induced apoptosis through increased death receptor expression.

• 한국미생물·생명공학회지
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• 제28권2호
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• pp.117-123
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• 2000

P. phosphoreum의 생존과 생체발광도는 온도에 의해 많은 영향을 받는다. 냉동 저장한 세포의 경우 glycerol의 보호 작용으로 세포농도와 생균수는 측정기간 동안 일정하게 유지된 반면 생체발광도는 glycerol 첨가 직후 급속히 감소하였으며, 저장 이후에도 감소된 생체발광도가 활성화되지 못하였다. 최적 생육온도인 $20^{\circ}C$의 경우 저장 초기 세포가 성장함에 따라 세포수의 증가를 보였으나 일정 시간 이후 세포 분해 현상으로 인하여 생균수 및 세포 집락수의 감소를 나타내었으며, 생체발광도는 저장 3일 이후 소멸되었다. 이와는 대조적으로 $4^{\circ}C$에 저장한 세포의 생체발광도는 저장 10일 동안 지속되어 가장 높은 생체발광 유지도를 나타내었으나 장기간 저온 저장으로 인하여 세포가 VBNC 상태에 돌입됨에 따라 총균수와 생균수는 일정한 반면 저장 10일 이후 세포 집락수의 급격한 감소를 나타내었으며, 저장 20일 이후 간균에서 구균으로 세포 형태상의 변화를 나타내었다. 이에 따라 세포 저장 시 접종원의 농도를 달리하여 VBNC 상태와 생체발광도의 관련성을 조사한 결과 VBNC 세포가 증가할수록 생체발광도의 감소를 나타내었다. 따라서 VBNC 세포를 감소시키기 위하여 세포를 고정화하여 저장한 결과 별도의 활성제 없이 실온에서 다시 활성화되어 고정화하지 않은 세포에 비해 2.3배 높은 생체발광유지도를 나타내었으며, 저온저장에 따른 platebility 소실과 세포 응축현상이 나타나지 않았다. 이러한 결과는 세포의 고정화 방법을 이용하여 $4^{\circ}C$에서도 세포의 생존 및 생체발광 유지도를 향상시킬 수 있으며, 동결 건조법의 단점을 보완해 줄 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제27권1호
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• pp.197-205
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• 2017

Exposure of Jurkat T cell clone (J/Neo cells) to acacetin (5,7-dihydroxy-4'-methoxyflavone), which is present in barnyard millet (Echinochloa esculenta (A. Braun)) grains, caused cytotoxicity, enhancement of apoptotic $sub-G_1$ rate, Bak activation, loss of mitochondrial membrane potential (${\Delta}{\Psi}m$), activation of caspase-9 and caspase-3, degradation of poly(ADP-ribose) polymerase, and FITC-Annexin V-stainable phosphatidylserine exposure on the external surface of the cytoplasmic membrane without accompanying necrosis. These apoptotic responses were abrogated in Jurkat T cell clone (J/Bcl-xL) overexpressing Bcl-xL. Under the same conditions, cellular autophagic responses, including suppression of the Akt-mTOR pathway and p62/SQSTM1 down-regulation, were commonly detected in J/Neo and J/Bcl-xL cells; however, formation of acridine orange-stainable acidic vascular organelles, LC3-I/II conversion, and Beclin-1 phosphorylation (Ser-15) were detected only in J/Neo cells. Correspondingly, concomitant treatment with the autophagy inhibitor (3-methyladenine or LY294002) appeared to enhance acacetin-induced apoptotic responses, such as Bak activation, ${\Delta}{\Psi}m$ loss, activation of caspase-9 and caspase-3, and apoptotic $sub-G_1$ accumulation. This indicated that acacetin could induce apoptosis and cytoprotective autophagy in Jurkat T cells simultaneously. Together, these results demonstrate that acacetin induces not only apoptotic cell death via activation of Bak, loss of ${\Delta}{\Psi}m$, and activation of the mitochondrial caspase cascade, but also cytoprotective autophagy resulting from suppression of the Akt-mTOR pathway. Furthermore, pharmacologic inhibition of the autophagy pathway augments the activation of Bak and resultant mitochondrial damage-mediated apoptosis in Jurkat T cells.

• Journal of Korean Neurosurgical Society
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• 제63권5호
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• pp.566-578
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• 2020

Objective : Radiation is known to induce autophagy in malignant glioma cells whether it is cytocidal or cytoprotective. Dexamethasone is frequently used to reduce tumor-associated brain edema, especially during radiation therapy. The purpose of the study was to determine whether and how dexamethasone affects autophagy in irradiated malignant glioma cells and to identify possible intervening molecular pathways. Methods : We prepared p53 mutant U373 and LN229 glioma cell lines, which varied by phosphatase and tensin homolog (PTEN) mutational status and were used to make U373 stable transfected cells expressing GFP-LC3 protein. After performing cell survival assay after irradiation, the IC₅₀ radiation dose was determined. Dexamethasone dose (10 μM) was determined from the literature and added to the glioma cells 24 hours before the irradiation. The effect of adding dexamethasone was evaluated by cell survival assay or clonogenic assay and cell cycle analysis. Measurement of autophagy was visualized by western blot of LC3-I/LC3-II and quantified by the GFP-LC3 punctuated pattern under fluorescence microscopy and acridine orange staining for acidic vesicle organelles by flow cytometry. Results : Dexamethasone increased cell survival in both U373 and LN229 cells after irradiation. It interfered with autophagy after irradiation differently depending on the PTEN mutational status : the autophagy decreased in U373 (PTEN-mutated) cells but increased in LN229 (PTEN wild-type) cells. Inhibition of protein kinase B (AKT) phosphorylation after irradiation by LY294002 reversed the dexamethasone-induced decrease of autophagy and cell death in U373 cells but provoked no effect on both autophagy and cell survival in LN229 cells. After ATG5 knockdown, radiation-induced autophagy decreased and the effect of dexamethasone also diminished in both cell lines. The diminished autophagy resulted in a partial reversal of dexamethasone protection from cell death after irradiation in U373 cells; however, no significant change was observed in surviving fraction LN229 cells. Conclusion : Dexamethasone increased cell survival in p53 mutated malignant glioma cells and increased autophagy in PTEN-mutant malignant glioma cell but not in PTEN-wildtype cell. The difference of autophagy response could be mediated though the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling pathway.

• Journal of Gastric Cancer
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• 제6권3호
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• pp.132-138
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• 2006

목적: 도파민은 중추신경전달물질이지만 위장관에서 도파민수용체와 결합하여 점막상피세포 증식, 상피세포의 보호, 위암 세포증식과 관련이 있는 것으로 알려져 있다. 본 연구에서는 위암에서 기원한 세포주를 이용하여 도파민과 각각의 도파민 수용체가 위암 세포 증식과 억제에 작용하는 역할에 대해 알아보았다. 대상 및 방법: 위암세포기원에서 각각 유래한 세포주인 SNU601과 KCU-C2를 이용하여 RNA 추출 후 RT-PCR 시행 후 도파민수용체 D1, D2L과 D2S 각각에 대한 primer로 PCR을 시행하여 수용체 유전자의 상대적인 발현정도를 측정하였다. 도파민과 Dl 수용체의 대항제인 SCH 23390과 D2 수용체 대항제인 raclopride를 사용하여 약물처리에 따른 위암세포주에서 세포 증식에 대한 분석을 하였다. 결과: KCU-C2 세포주에서 D1과 D2L과 D2S 유전자 mRNA의 상대적인 발현정도는 모두 높은 발현을 보였지만, SNU 601 세포주에는 mRNA의 발현이 모두 낮은 수준이었으며, 특히 D2L mRNA는 발현되고 있지 않았다. 약물처리에 따른 위암세포주에서 세포증식에 대한 분석에서는 D1과 D2S 수용체를 통한 도파민의 신호는 세포의 증식을 억제하였고 D2L 수용체를 통한 도파민의 신호는 세포의 증식을 유도하였다. 결론: 본 연구를 통해 도파민이 위암의 세포증식과 억제에 관여하며, 도파민의 이러한 효과는 도파민의 신호가 어느 수용체를 통해 전달되었느냐에 따라 위암세포의 증식과 억제가 이루어짐을 알 수 있었다.

• 미생물학회지
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• 제44권1호
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• pp.43-48
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• 2008

정도관리는 분석 결과의 신뢰성을 높이기 위해 필수적인 과정이며, 이를 위해서는 일정한 농도의 표준시료가 필요하다. 본 연구에서는 정도관리의 정략분석을 위해 실온에서도 일정시간 개체수가 유지되는 미생물 정도관리용 표준시료를 제작하였다. 대장균으로 조제한 미생물 시료에 세포 분열을 억제하기 위해 nalidixic acid ($100\;{\mu}g/ml$)와 cephalexin ($50\;{\mu}g/ml$)을 첨가하였다. 이후 12시간 간격으로 acridine orange로 염색 후 현미경으로 계수하는 방법과 한천 배지에 배양하여 집락을 계수하는 평판집락계수법으로 개체수 변화를 측정하였다. 현미경 계수 결과 초기간이 $3.7{\times}10^5\;cells/ml$이었을 때 대장균의 개체수는 48시간 동안 $3.5{\sim}4.2{\times}10^5\;cells/ml$의 범위를 보였고, 평판집락법 결과 초기값이 $1.2{\times}10^4\;CFU/ml$ 이었을 경우에는 $1.0{\sim}1.2{\times}10^4\;CFU/ml$로 조사되어 냉장 및 냉동의 필요 없이 실온에서도 48시간 동안 개체수가 비교적 안정적으로 유지되는 미생물 표준시료를 제작할 수 있었다. 따라서 본 연구에서 제작된 표준시료는 현재 상업적으로 판매되고 있는 표준균주 제품과 함께 정도관리의 정략분석을 위해 사용될 수 있을 것이다.