• 한국동물학회지
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• 제22권4호
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• pp.141-152
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• 1979

고양이 백혈병 바이러스에서 reverse transcriptase를 분리하여 생화학적 및 면역학적 연구를 하였다. 분자량은 72,000이고, DNA polymerase와 RNase H의 활성은 0.05-1 mM $M_n^2+$와 50-80 mM KCl에서 가장 좋았다. DNA polymerase와 RNase H는 같은 단백질 분자에 있으며, chymotrypsin 처리로서 RNase H를 쪼개낼 수 있으며, 이 RNase H도 reverse transcriptase의 항체에 의해서 활성이 거의 억제 된다. Reverse transcriptase의 항체 결합위치와 활성을 내는 위치는 다른 것 같다.

• 한국균학회지
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• 제17권4호
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• pp.177-183
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• 1989

1. 느타리버섯 중의 미토콘드리아는 설탕농도 44% 층에서 분리 정제되었다. 2. 파장 변화에 따른 mitochondrial ATP synthase의 활성도는 480nm의 빛이 조사될 때 가장 크게 증가되었다. 3. 최적 파장 480nm의 빛 조사 시간 변화에 따른 활성도는 15분 동안 조사하였을 때 가장 크게 증가하였다. 4. 위의 최적 빛 조사 조건에서 이 효소의 최적 pH는 7.5, 최적 온도는 $56^{\circ}C$였다. 5. 최적 광 조건에서 얻은 이 효소는 $Fe^{3+}$, $Fe^{2+}$ 그리고 $K^{+}$ 이온에 의하여 활성화 되었으나, $Na^{+}$ 이온에 의해서는 억제되었다.

• 대한수의학회지
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• 제12권1호
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• pp.77-84
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• 1972

이 실험(實驗)은 뇌염(腦炎)바이러스(MVE virus)들 acetone, Tween-ether 그리고 Tween-ether-protamine-sulphate 로 처리한 후 그 바이러스가 지니고 있는 적혈구응집력가(赤血球凝集力價), 보체결합력가(補體結合力價) 그리고 감염력가(感染力價)의 변화 여부를 관찰함과 아울러 초원심분리법(超遠心分離法)에 의하여 이 바이러스가 지니고 있는 위의 세 가지 활성물질(活性物質)의 분획(分劃) 가능성(可能性)을 검토한 결과 다음과 같은 결과를 얻었다. 1) Acetone, Tween-ether 그리고 Tween-ether protamine-sulphate 처리에 의하여 MVE virus가 지니는 적혈구응집력가(赤血球凝集力價)는 8~16 배(倍)로 증가 하였다. 2) Acetone 및 Tween-ether 처리로 보체결합력가(補體結合力價)는 4 배(倍)로 증가 되었으나 Tween-ether protamine-sulphate 처리로는 저하되는 경향이 있었다. 3) Tween-ether로 처리하면 감염력가(感染力價)는 완전히 상실 되나 acetone으로 처리하면 $TCID50/log_{10}$ 3이 감퇴 되었다. 4) 위의 화학제의 처리와 관계없이 바이러스의 적혈구응집력가(赤血球凝集力價)는 $37^{\circ}C$ 에서 10 분간 가열(加熱)됨으로써 완전 소실 되었으나 보체결합력가(補體結合力價)는 상승 하였으며 이 활성도(活性度)는 $65^{\circ}C$ 에서 20 분간 까지도 계속 유지되었다. 5) 처리되지 않은 바이러스와 acetone 처리된 바이러스의 보체결합력가(補體結合力價)나 적혈구응집력가(赤血球凝集力價)는 Tween-ether 나 Tween-ether-protamine-sulphate 로 처리한 바이러스의 그것보다 열(熱)에 대해서 더 안정(安定)하였다. 6) 적혈구응집억제반응용(赤血球凝集抑制反應用) 항원제조(抗原製造)에 있어서 Tween-ether 로 처리하거나 acetone 으로 처리하여 만든 두가지 항원(抗原)은 모두 동일(同一)한 역가(力價)를 보였다. 7) 10~60%의 sucrose gradient centrifugation 에서 처리되지 않은 바이러스는 두개의 보체결합(補體結合)피크가 저농도(低濃渡)와 고농도(高濃渡)에서 각각 나타났으며 저농도(低濃渡)에서 얻은 재료는 적혈구응집력(赤血球凝集力)과 감염력(感染力)을 동반하고 있었다. 8) Acetone 으로 처리된 바이러스의 초원심분리(超遠心分離)에서는 위에 말한 두 보체결합(補體結合)피크 중 고농도부(高濃渡部)의 것은 감소 되었으나 다른 하나는 영향을 받지 않았다. 그리고 적혈구응집력가(赤血球凝集力價)가 증가된 반면 감염최고력가(感染最高力價)는 감퇴 되었으며 이 세 가지 활성(活性)은 acetone 처리 후에는 명확히 분획(分劃)되지 않았다. Tween-ether 로 처리된 바이러스는 초원심분리(超遠心分離)에 의하여 고농도부(高濃渡部)에 있는 보체결합(補體結合)피크는 완전 소실된 반면 저농도부(低濃渡部)에 있는 보체결합최고력가(補體結合最高力價)는 영향을 받지 아니 하였으며 이 피크는 감염력(感染力)을 동반하지 않는 적혈구응집소(赤血球凝集素)와 분획(分劃)이 가능 하였다.

• 한국동물학회지
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• 제33권1호
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• pp.35-44
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• 1990

생쥐 악하선으로부터 분리한 전 RNA를 poly(U)-sepharose chromatography와 sucrose linear density gradient centrifugation 방법으로 레닌 mRNA를 분리하여 in vitro translation과 immunoprecipitation에 의하여 레닌 mRNA를 확인하였다. 레닌 mRNA로부터 레닌 cDNA를 합성하여 EcoRI inker를 이용하여 pUC19에 삽입시켰고, Taq, polymerase를 이용한 PCR방법으로 합성한 레닌 cDNA는 pUC19의 Hinalll 부의에 삽입하여 재조합 plamid를 각각 작성하였다. 이것을 JM103에 형질전환시켜 레닌 유전자 발현을 유도하여 45,000의 분자량을 갖는 레닌을 얻었으며 이 레닌 단백은 토끼으 혈압을 850115mmHg에서 115-140mmHg로 증가시키는 생리 활성을 나타냈다. 토끼의 신장 DNA를 EMBL3 phage에 삽입시켜 genomic library를 작성한 후, 레닌 cDNA로부터 합성한 probe로 plaque hybridization시켜 genomic 유전자를 갖는 재조합 phage를 분리하였다.

• 한국동물생명공학회지
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• 제36권2호
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• pp.91-98
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• 2021

The objective of this study was to establish a selection process for high quality sperm in bovine semen using sperm separation by magnetic activation (MACS). For this, semen from 21 Nellore bulls was collected using an artificial vagina. To guarantee the presence of pathologies in the ejaculate, animals previously declassified in four consecutive spermiogram were used. Semen was analyzed in five statuses: (1) fresh semen (fresh); (2) density gradient centrifugation (DGC), percoll column; (3) non-apoptotic fraction after separation by MACS (MAC); (4) apoptotic fraction from the separation (MACPOOR); and (5) MAC followed by DGC (MACDGC). Using a computerized analysis system (CASA), motility was measured. The sperm morphology was evaluated by phase contrast, and the supravital test was completed with eosin/nigrosin staining. For DGC, 20 × 10⁶ cells were used in a gradient of 90% and 45% percoll. MACS used 10 × 10⁶ cells with 20 μL of nanoparticles attached to annexin V, and filtered through the MiniMACS magnetic separation column. Membrane integrity was assessed with SYBR-14/IP and mitochondrial potential with JC-1 by flow cytometry. Processing sperm by MACDGC, was more effective in obtaining samples with high quality sperm, verified by the total of abnormalities in the samples: 35.04 ± 2.29%, 21.50 ± 1.47%, 17.30 ± 1.10%, 30.68 ± 1.94% and 10.50 ± 1.46%, respectively for fresh, DGC, MAC, MACPOOR, and MACDGC. The subpopulation of non-apoptotic sperm had a high number of live cells (82.65%), membrane integrity (56.60%) and mitochondrial potential (83.98%) (p < 0.05). These findings suggest that this nanotechnological method, that uses nanoparticles, is efficient in the production of high-quality semen samples for assisted reproduction procedures in cattle.

• 생태와환경
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• 제56권1호
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• pp.36-44
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• 2023

자연생태계에서 환경유전자 (eDNA)는 세포의 내부(intracellular)와 외부(extracellular) 형태로 존재할 수 있다. 유해남조류를 대상으로 할 때, 세포 외부 eDNA는 남조류의 흔적, 세포 내부 eDNA는 남조류의 발생 잠재성을 의미한다. 하지만 기존의 퇴적물 eDNA 분석법인 silica bead를 이용한 파쇄법으로는 존재 형태를 구분할 수 없기 때문에 실질적인 유해남조류 발생 잠재성을 파악하기 어렵다. 본 연구는 기존의 파쇄법의 한계를 극복하고자 퇴적물 내 세포 내 eDNA를 선택적으로 분석할 수 있는 퇴적물 전처리 방법인 밀도구배 원심분리법(Ludox method)의 적용성을 분석하였다. 그 결과, 기존의 파쇄법은 퇴적물을 그대로 사용하여 eDNA를 추출하기 때문에 eDNA에서 증폭한 mic 유전자가 세포 내 존재하는지 혹은 세포 외 DNA로만 존재하는지 알 수 없었다. 하지만 Ludox method는 여과 및 밀도 구배를 통해 퇴적물의 세포 내 eDNA를 농축하므로 남조류 세포 내부에 존재하는 mic 유전자만을 증폭할 수 있었다. 결론적으로 Ludox method는 충분한 세포내부 유전자 농도를 확보하고 세포 내부와 외부의 eDNA를 명확히 구분함으로써 보다 정확하고 세밀한 잠재성 분석이 가능하였다. 이는 퇴적물 유해남조류의 유전자 활성을 확인할 수 있는 eRNA 분석과 차세대염기서열분석(next generation sequencing; NGS)을 이용한 meta-barcoding에 Ludox method를 활용함으로써 보다 현실적인 잠재성을 분석할 수 있을 것으로 판단된다.

• Journal of Periodontal and Implant Science
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• 제38권4호
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• pp.639-644
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• 2008

Purpose: The aim of this study was to compare the number of live and dead bacteria attached to, or within, the stratified squamous epithelium lining the tissue side of the gingival sulcus. Materials and Methods: A total of 50 patients was examined and classified into healthy or diseased sites according to inflammatory status of the gingival tissue. The surface of stratified squamous epithelium was removed by gentle scraping of the gingival sulcus with curettes. The cells were processed in the laboratory by density-gradient centrifugation to separate the epithelial cells from the loose bacteria and debris. The LIVE/$DEAD^{(R)}$ $BacLight^{TM}$ Bacterial Viability Kit was applied and the specimens were observed by an epifluorescent microscope and the number of bacteria was counted. Results: Live and dead bacteria were stained to green and red, irrespectively. Generally, the number of total bacteria in the diseased sites was significantly higher than in the healthy sites. The mean number of detected bacteria in the diseased sites was $58.6{\pm}36.0$ (red bacteria $10.4{\pm}9.2$ / green bacteria $48.2{\pm}30.5$), while it was $1.5{\pm}1.7$ in the healthy sites (red bacteria $0.1{\pm}0.3$ / green bacteria $1.4{\pm}1.5$). The percentage of red bacteria was $17.5{\pm}11.2%$ in the diseased sites and $2.0{\pm}5.8%$ in the healthy sites. Conclusion: The total number of bacteria in the diseased sites was significantly higher than that of the healthy sites. The ratio and the number of red bacteria were also significantly higher in the diseased sites.

• BMB Reports
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• 제29권3호
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• pp.253-260
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• 1996

An enzyme that hydrolyzes flavin-adenine dinucleotide (FAD) was found to be present in rat liver lysosomal membrane prepared from Triton WR-1339 filled lysosomes (tritosomes) purified by flotation on sucrose. This FAD phosphohydrolase (FADase) exhibited optimal activity at pH 8.5 and had an apparent Km of approximately 3.3 mM. The activity was decreased 50~70% by dialysis against EDTA and this was restored by $Zn^{2+}$, $Mg^{+2}$, $Hg^{+2}$, and $Ca^{+2}$ ions inhibited the enzyme, but $F^-$ and molybdate had no effect. The enzyme was also inhibited by p-chloromercuribenzoate (pCMB), reduced glutathione and other thiols, cyanide, and ascorbate. The presence of ATP, ADP, AMP. ${\alpha}-{\beta}-methylene$ ATP, AMP-p-nitrophenyl phosphate (PNP), GMP, and coenzyme A (CoA) decreased the activity on FAD, but pyrimidine nucleotides, adenosine, adenine, or $NAD^+$ were without effect. Phosphate stimulated the activity slightly. FAD phosphohydrolase activity was separated from ATPase and inorganic pyrophosphatase activities by solubilization with detergents and polyacrylamide gel electrophoresis and by linear sucrose density gradient centrifugation suggesting that the enzyme is different from ATPase, inorganic pyrophosphatase, and soluble lysosomal FAD pyrophosphatase. Paper chromatography showed that FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP which were further hydrolyzed to riboflavin and AMP by phosphatases known to be present in lysosomal membranes. Incubation of the intact Iysosomes with pronase showed that the active site of FAD phosphohydrolase must be oriented to the cytosol. The FAD hydrolyzing activity was detected in Golgi, microsome, and plasma membrane, but not in mitochondria or soluble lysosomal preparations.

• Archives of Plastic Surgery
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• 제35권3호
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• pp.229-234
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• 2008

Purpose: There are some reports presenting that peripheral blood contain circulating hematopoietic cells as well as, in significantly smaller quantities, mesenchymal stem cells. The purposes of this study is to isolate and characterize circulating mesenchymal progenitor cells with osteogenic potential from human peripheral blood. Methods: Human buffycoat containing mononuclear cells was harvested from peripheral blood of normal persons and isolated using a density gradient centrifugation and serially subcultured in osteogenic media for 1-4 weeks. The proliferation capability, phase-contrast microscopy, transmission electron microscopy, immunophenotype FACS analysis, Alizarin red staining and RT-PCR assays for osteogenic differentiation potential were performed. Results: The phenotype of cultured cells changed from small round or cuboidal cells at passage 1 into large spindle-shaped fibroblastic morphology cells at passage 4. Surface marker expressed CD14, but did not express CD34, CD80, CD83. Strong positive staining was observed for Alizarin reds in osteogenic medium on day 14, Using RT-PCR, the mRNA levels of bone- specific genes, such as ALP, c-bfa-1 and osteocalcin were detected. Conclusion: A new subset of peripheral blood derived progenitor cells described here has the ability to proliferate and differentiate into osteogenic cell lineages in vitro, and to be candidate for regenerative therapy.

• Journal of Korean Neurosurgical Society
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• 제56권5호
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• pp.375-382
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• 2014

Objective : To assess the effect of bone marrow mononuclear cells (BMMNCs) transplantation in the expression of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) in spinal cord injury (SCI) in rats. Methods : BMMNCs were isolated from tibia and femur by a density gradient centrifugation. After establishment of acute transection SCI, rats were divided into experiment (BMMNCs), experiment control (0.1 M PBS infused) and sham surgery groups (laminectomy without any SCI). Locomotor function was assessed weekly for 5 weeks post-injury using BBB locomotor score and urinary bladder function daily for 4 weeks post-injury. Activity of NF-${\kappa}B$ in spinal cord was assessed by immunohistochemistry and reverse transcriptase polymerase chain reaction. Results : At each time point post-injury, sham surgery group had significantly higher Basso, Beattie, Bresnahan locomotor and urinary bladder function scores than experiment and experiment control group (p<0.05). At subsequent time interval there were gradual improvement in both experiment and experiment control group, but experiment group had higher score in comparison to experiment control group (p<0.05). Comparisons were also made for expression of activated NF-${\kappa}B$ positive cells and level of NF-${\kappa}B$ messenger RNA in spinal cord at various time points between the groups. Activated NF-${\kappa}B$ immunoreactivity and level of NF-${\kappa}B$ mRNA expression were significantly higher in control group in comparison to experiment and sham surgery group (p<0.05). Conclusion : BMMNCs transplantation attenuates the expression of NF-${\kappa}B$ in injured spinal cord tissue and thus helps in recovery of neurological function in rat models with SCI.