• Archives of Pharmacal Research
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• 제27권9호
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• pp.961-967
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• 2004

The development of an antibody labeling method with $^{99m}$Tc is important for cancer imaging. Most bifunctional chelate methods for $^{99m}$Tc labeling of antibody incorporate a $^{99m}$Tc chelator through a linkage to lysine residue. In the present study, a novel site-specific $^{99m}$Tc labeling method at carbohydrate side chain in the Fc region of 2 antibodies (T101 and rabbit anti-human serum albumin antibody (RPAb)) using dihydrazinophthalazine (DHZ) which has 2 hydrazino groups was developed. The antibodies were oxidized with sodium periodate to pro-duce aldehyde on the Fc region. Then, one hydrazine group of DHZ was conjugated with an aldehyde group of antibody through the formation of a hydrazone. The other hydrazine group was used for labeling with $^{99m}$Tc. The number of conjugated DHZ was 1.7 per antibody. $^{99m}$Tc labeling efficiency was 46-85％ for T101 and 67∼87％ for RPAb. Indirect labeling with DHZ conjugated antibodies showed higher stability than direct labeling with reduced antibodies. High immunoreactivities were conserved for both indirectly and directly labeled antibodies. A biodistribution study found high blood activity related to directly labeled T1 01 at early time point as well as low liver activity due to indirectly labeled T101 at later time point. However, these findings do not affect practical use. No significantly different biodistribution was observed in the other organs. The research concluded that DHZ can be used as a site-specific bifunctional chelating agent for labeling antibody with $^{99m}$Tc. Moreover, $^{99m}$Tc labeled antibody via DHZ was found to have excellent chemical and biological properties for nuclear medicine imaging.edicine imaging.

• Animal cells and systems
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• 제3권1호
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• pp.73-78
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• 1999

Although there are several methods for the preparation of mitochondrial DNA (mtDNA) from mammalian tissues, most are relatively long ultracentrifugation or manipulations by a small-scale method. We escribed a rapid method for large-scale extraction of mtDNA from human placental and horse liver tissues. The method is based on the preparation and homogenization of tissues, urification of crude mitochondria by differential centrifugations and isolation of mtDNA by alkaline Iysis. It was improved from Pre-existing methods by replacing some steps with simpler ones and discarding many others. This method gives a high yield of pure mtDNA(approximately 1-5mg from one placenta; ca. 400-600 g wet weight), depending on its sources (fresh tissue gave better results than frozen one). The resulting mtDNA indicated that this method can yield mtDNA in sufficient purity and quantity to identify the direct restriction analysis on agarose gel, random-primed labeling as a probe, and end labeling. Therefore, the method is ideal for obtaining good mtDNA samples to conduct routine restriction fragment length polymorphism (RFLP) analyses of natural populations for genetic studies.

• IMMUNE NETWORK
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• 제12권6호
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• pp.223-229
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• 2012

Clinical and preclinical in vivo immune cell imaging approaches have been used to study immune cell proliferation, apoptosis and interaction at the microscopic (intra-vital imaging) and macroscopic (whole-body imaging) level by use of ex vivo or in vivo labeling method. A series of imaging techniques ranging from non-radiation based techniques such as optical imaging, MRI, and ultrasound to radiation based CT/nuclear imaging can be used for in vivo immune cell tracking. These imaging modalities highlight the intrinsic behavior of different immune cell populations in physiological context. Fluorescent, radioactive or paramagnetic probes can be used in direct labeling protocols to monitor the specific cell population. Reporter genes can also be used for genetic, indirect labeling protocols to track the fate of a given cell subpopulation in vivo. In this review, we summarized several methods dealing with dendritic cell, macrophage, and T lymphocyte specifically labeled for different macroscopic whole-body imaging techniques both for the study of their physiological function and in the context of immunotherapy to exploit imaging-derived information and immune-based treatments.

• Molecular & Cellular Toxicology
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• 제1권2호
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• pp.92-98
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• 2005

DNA microarray analysis of gene expression in toxicogenomics typically requires relatively large amounts of total RNA. This limits the use of DNA microarray when the sample available is small. To confront this limitation, different methods of linear RNA amplification that generate antisense RNA (aRNA) have been optimized for microarray use. The target preparation strategy using amplified RNA in DNA microarray protocol can be divided into direct-incorporation labeling which resulted in cDNA targets (Cy-dye labeled cDNA from aRNA) and indirect-labeling which resulted in cRNA targets (i.e. Cy-dye labeled aRNA), respectively. However, despite the common use of amplified targets (cDNA or cRNA) from aRNAs, no systemic assessment for the use of amplified targets and bias in terms of hybridization performance has been reported. In this investigation, we have compared the hybridization performance of cRNA targets with cDNA targets from aRNA on a 10 K cDNA microarrays. Under optimized hybridization conditions, we found that 43% of outliers from cDNA technique and 86% from the outlier genes were reproducibly detected by both targets hybridization onto cDNA microarray. This suggests that the cRNA labeling method may have a reduced capacity for detecting the differential gene expression when compared to the cDNA target preparation. However, further validation of this discordant result should be pursued to determine which techniques possesses better accuracy in identifying truly differential genes.

• Applied Microscopy
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• 제51권
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• pp.2.1-2.7
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• 2021

Synaptic vesicles, which are endogenous to neurotransmitters, are involved in exocytosis by active potentials and release neurotransmitters. Synaptic vesicles used in neurotransmitter release are reused via endocytosis to maintain a pool of synaptic vesicles. Synaptic vesicles show different types of exo- and endocytosis depending on animal species, type of nerve cell, and electrical activity. To accurately understand the dynamics of synaptic vesicles, direct observation of synaptic vesicles is required; however, it was difficult to observe synaptic vesicles of size 40-50 nm in living neurons. The exo-and endocytosis of synaptic vesicles was confirmed by labeling the vesicles with a fluorescent agent and measuring the changes in fluorescence intensity. To date, various methods of labeling synaptic vesicles have been proposed, and each method has its own characteristics, strength, and drawbacks. In this study, we introduce methods that can measure presynaptic activity and describe the characteristics of each technique.

• 대한핵의학회지
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• 제26권2호
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• pp.380-391
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• 1992

This study was designed to evaluate a direct method of $^{99m}Tc$ labeling using $\beta-mercaptoethanol$ as a reducing agent, and to investigate whether $^{99m}Tc$ labeled specific monoclonal antibody against carcinoembryonic antigen (CEA-92) can be used for the scintigraphic localization of human colon cancer xenograft. Purified CEA-92 IgG was fragmented into F $(ab')_2$ and then labeled with $^{99m}Tc$ by transchelation method using glucarate as a chelator. Labeling efficiency, immunological reactivity and in vitro stability of $^{99m}Tc$ CEA-92 F $(ab')_2$ were measured and then injected intravenously into nude mice bearing human colon cancer (SNU-C4). Scintigrams were obtained at 24 hour after injection. Then nude mice were sacrificed and the radioactivity was measured Labeling efficiency of injected $^{99m}Tc$ CEA-92 F $(ab')_2$, immunoreative fraction and in vitro stability at 24 hour of injected $^{99m}Tc$ CEA-92 F $(ab')_2$ was 45.2%, 32.8% and 57.4%, respectively. At 24 hour after injection, % ID/g in kidney (46.77) showed high uptake, but %ID/g in tumor (1.65) was significantly higher than spleen (0.69), muscle (0.16), intestine (0.45), stomach (0.75), heart (0.48) and blood (0.45). There was no significant difference between tumor and liver (1.81). Tumor contrast as quantitated by tumor to blood ratio of $^{99m}Tc$ CEA-92 F $(ab')_2$ was increased significantly (p<0.005) until 24 hours (3.70), and there was no statistical differece from tumor to blood ratio of I-131 CEA-92 F $(ab')_2$. The scintigram demonstrated localization of radioactivity over transplanted tumor, but significant background radioactivity was also noted over kidney and abdomen. It is concluded that CEA-92 F $(ab')_2$ can be labeled with $^{99m}Tc$ by a direct transchelation method using $\beta-mercaptoethanol$ as a reducing agent and $^{99m}Tc$ labeled CEA-92 F $(ab')_2$ can be used for the scintigraphic localization of human colon cancer xenograft in nude mice model.

• 대한핵의학회지
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• 제33권1호
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• pp.84-93
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• 1999

목적: 주기율표상에서 7족에 위치하며, 유사한 화학적 특성을 지닌 $^{99m}Tc$와 $^{188}Re$의 항체에 표지하는 조건의 차이점과 앞서 표지된 항체의 핵종에 따른 거동의 차이를 비교 실험해 보고자 하였다. 대상 및 방법: mercaptoethanol을 사용하여 non-specific human IgG의 disulfide 결합을 환원시켜, -SH 기의 발생을 유도하였다. 앞서 처리된 IgG을 stannous ion 을 사용하여 $^{99m}Tc$과 $^{188}Re$을 환원하여 $^{99m}Tc$-IgG과 $^{188}Re$-IgG을 제작하였다. HPLC를 사용하여 $^{99m}Tc$-IgG은 1시간, 4시간. 6시간, 24시간마다 $^{188}Re$-IgG은 1시간, 4시간, 16시간, 24시간마다 시간경과에 따른 안정도 변화를 확인하였다. 결과: 한 분자당 약 2개의 -SH기를 유도 면역 활성이 최대한 유지되는 환원된 IgG를 얻을 수 있었다. 각각 90%와 95% 이상의 높은 표지반응수율로 $^{99m}Tc$-IgG과 $^{188}Re$-IgG을 얻었다. $^{99m}Tc$-IgG의 %peak area를 1시간, 4시간, 6시간, 24시간마다 측정한 값은 각각 91%, 83%, 78%, 7%이었다. $^{188}Re$-IgG의 경우 1시간, 4시간, 16시간, 24시간에서 94%, 80%, 47%, 42%이었다. $^{99m}Tc$-IgG을 정맥주사 후 4시간 후의 %ID/g는 장기별로 신장, 혈액, 위, 농양($9.42{\pm}0.68,\;1.43{\pm}0.24,\;0.86{\pm}0.18,\;0.72{\pm}0.10$)순이었고, 24시간에서는 신장, 농양, 위, 혈액($7.61{\pm}1.58,\;0.57{\pm}0.07,\;0.37{\pm}0.09,\;0.281{\pm}0.09$)의 순이었다. $^{188}Re$-IgG의 경우 4시간에서 신장, 혈액, 농양, 위($3.92{\pm}0.62,\;1.32{\pm}0.08,\;0.88{\pm}0.01,\;0.26{\pm}0.06$)의 순이였고, 24시간에서 신장, 농양, 혈액, 위($4{\pm}0.26,\;0.37{\pm}0.04,\;0.29{\pm}0.01,\;0.13{\pm}0.03$)의 순이었다. 결론: $^{99m}Tc$-IgG과 $^{188}Re$-IgG의 표지항체는 직접표지 법을 사용하여 효과적으로 제조되었다. 그러나, 직접표지법을 사용한 $^{99m}Tc$-IgG과 $^{188}Re$-IgG의 안정도는 시간경과에 따라 불안정함을 보여 안정성을 증가시키기 위한 항체표지 방법들에 대한 연구가 필요할 것으로 생각되었다.

• 한국정보통신학회논문지
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• 제5권7호
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• pp.1257-1262
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• 2001

최근에 경제적인 대안으로 IP over WDM 네트워크가 주목을 받고 있다. IP over WDM은 광대역 수용 능력, 네트워크 확장성 외에 여러 장점을 가지고 있다. IP 기반에서, "lambda-labeling"은 IP over WDM에 직접적으로 적용할 수 있다. 본 논문에서는 현재 IETF측 중심으로 연구중인 MPLambdaS 개념을 기초로 하여 광 인터넷의 진화를 고려한 구조적인 접근 방법을 연구한다. MPLS 도메인과 MPLamdaS 도메인간에 연동을 고려하여 전자적인 다수의 LSP를 광 LSP로 모으는 레이블 스택 개념을 이용한 람다 LSP 터널링 기술을 적용하여 전자적인 MPLS 와 광 MPLamdaS 간의 연동 방안을 제안하고 이를 지원하는 Edge Optical LSR의 구조를 제안한다. 구조를 제안한다.

• 대한핵의학회지
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• 제30권4호
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• pp.541-547
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• 1996

우리는 국내에서 개발한 항 NCA-95 단일클론항체의 면역학적 특성을 규명하고 $^{99m}Tc$ 표지법을 연구하여 골수면역 신티그라피법을 개발한 바 있다. 그러나 $^{99m}Tc$ 단일클론항체의 표지효율이 낮고, 과정이 복잡하여 시간이 오래 걸리는 단점이 있어 임상적용에 어려움이 있었다. 본 연구에서는 $^{99m}Tc$ 표지 단일클론항체의 표지과정을 개선하여 시간을 단축하고 표지효율을 높이는 연구를 실시하였다. 티올기와 아민 및 하이드록시 그룹을 가진 착화제(chelator)와 $^{99m}Tc$은 안정한 복합체를 형성하므로, 이러한 화학적 성질을 이용하여 항체를 환원시켜 티올기를 생성하고 여기에 약한 착화제와 결합한 $^{99m}Tc$을 가하는 transchelation법을 연구하였다. 본 연구에서는 방법 1. ${\beta}$-mercaptoethanol로 항체를 환원하고 환원제의 존재하에 $^{99m}Tc$-gluconate 를 이용한 표지법, 방법 2. 항체의 환원후 ${\beta}$-mercaptoethanol을 제거하고 $^{99m}Tc$-gluconate를 사용한 표지법, 방법 3. 환원제를 제거한 항체에 MDP를 먼저 가하고 여기에 $^{99m}Tc$을 가하는 표지법을 시도하여 보았다. 고정상으로는 ITLC-SG를, 이동상으로는 생리식염수와 아세톤을 사용하여 표지효율을 구하였다. 방법 1은 표지효율이 40-70%이나 좋은 골수영상을 얻었고, 방법 2의 경우 표지효율은 70-80%로 개선되었으나 혈액풀의 방사능이 높았다. MDP를 착화제로 이용한 방법 3은 표지효율이 $92.4{\pm}5.9%$(n=15) 이고 면역반응성은 약 60%였으며 골수면역 신티그라피에서도 좋은 영상을 얻을 수 있었다. 때로는 $^{99m}Tc$- MDP 방사능이 높아 뼈에 흡수되는 방사능이 증가하는 단점이 있지만 PD-10 컬럼을 이용하여 분리가 가능하였다. 환원된 항체를 냉동보관하여 사용하였을 경우에도 안정하여 냉동보관 47일 까지 90%이상의 표지효율을 보였다. 본 연구 결과 $^{99m}Tc$ 표지 항 NCA-95 단일클론항체의 표지법의 개선으로 더 쉽게 임상에 이용할 수 있게 될 것으로 생각된다.

• Preventive Nutrition and Food Science
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• 제10권1호
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• pp.40-45
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• 2005

Age-related changes in bone metabolism are well established by biochemical markers of bone matrix in serum and urine, but analysis of the residual bone matrix, which is still turning over, has not been investigated. In the present study, we measured in vivo rates of bone protein synthesis using a precursor-product method based on the exchange of ²H from ²H₂O into amino acids. Four percent ²H₂O was administered to mice in drinking water after intraperitonial (i.p) bolus injection of 99.9% ²H₂O. Mice were divided into the two groups: growing young mice were administered 4% ²H₂O for 12 weeks after an i.p bolus injection at 5 week of age, whereas weight stable adult mice started drinking 4% ²H₂O 8 weeks later than the growing group and continued 4% ²H₂O drinking for 8 weeks. Mass isotopomer abundance in alanine from bone protein was analyzed by gas chromatography/mass spectrometry. Body ²H₂O enrichments were in the range of 1.88-2.41% over the labeling period. The fractional synthesis rates (ks) of bone protein were 2.000±0.071%/d for growing mice and 0.243±0.014%/d for adult mice. These results demonstrate that the bone protein synthesis rate decreases with age and present direct evidence of age-related changes in bone protein synthesis.