• Biomolecules & Therapeutics
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• 제21권2호
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• pp.126-131
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• 2013

Neuropathic pain is a chronic pain disorder caused by nervous system lesions as a direct consequence of a lesion or by disease of the portions of the nervous system that normally signal pain. The spinal nerve ligation (SNL) model in rats that reflect some components of clinical pain have played a crucial role in the understanding of neuropathic pain. To investigate the direct effects of gabapentin on differential gene expression in cultured dorsal root ganglion (DRG) cells of SNL model rats, we performed a differential display reverse transcription-polymerase chain reaction analysis with random priming approach using annealing control primer. Genes encoding metallothionein 1a, transforming growth factor-${\beta}1$ and palmitoyl-protein thioesterase-2 were up-regulated in gabapentin-treated DRG cells of SNL model rats. The functional roles of these differentially expressed genes were previously suggested as neuroprotective genes. Further study of these genes is expected to reveal potential targets of gabapentin.

• Toxicological Research
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• 제26권1호
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• pp.15-19
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• 2010

Mesenchymal stem cells (MSCs) have greater potential for immediate clinical and toxicological applications, due to their ability to self-renew, proliferate, and differentiate into a variety of cell types. To identify novel candidate genes that were specifically expressed during transdifferentiation of human MSCs to neuronal cells, we performed a differential expression analysis with random priming approach using annealing control primer-based differential display reverse transcription-polymerase chain reaction approach. We identified genes for acyl-CoA thioesterase, tissue inhibitor of metalloproteinases-1, brain glycogen phosphorylase, ubiquitin C-terminal hydrolase and aldehyde reductase were up-regualted, whereas genes for transgelin and heparan sulfate proteoglycan were down-regulated in MSC-derived neurons. These differentially expressed genes may have potential role in regulation of neurogenesis. This study could be applied to environmental toxicology in the field of testing the toxicity of a chemical or a physical agent.

• International Journal of Industrial Entomology and Biomaterials
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• 제7권2호
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• pp.139-144
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• 2003

Over 350 million people worldwide are chronic carriers of hepatitis B virus (HBV). Chronic viral infections of the liver can progress to cirrhosis, which may ultimately lead to hepatic failure or the development of hepatocellular carcinoma. There are two antiviral drugs on the market approved for clinical management of chronic HBV infections; interferon-alpha and the nucleoside analog lamivudine. However, they showed adverse side-effects. In the rational drug design for such therapies we would like to utilize antiviral drugs that inhibit the HBV replication in the liver. Investigation of natural extracts of silkworm exhibiting antiviral potential was held in the functional HBV polymerase activity and the release of virion particle in the HepG2.2.15 cell lines. HBV-producing transgenic mouse fed with silkworm DNJ molecule was shown as an inhibitor of serum HBV particles. We could represent this DNJ molecule as an antiviral potential complementing conventional therapies after preclinical tests against WHBV-infected animal model, woodchuck.

• IMMUNE NETWORK
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• 제11권1호
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• pp.79-94
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• 2011

Background: Dendritic cell (DC)-based vaccines are currently being evaluated as a novel strategy for tumor vaccination and immunotherapy. However, inducing long-term regression in established tumor-implanted mice is difficult. Here, we show that deoxypohophyllotoxin (DPT) induces maturation and activation of bone marrow-derived DCs via Toll-like receptor (TLR) 4 activation of MAPK and NF-${\kappa}B$. Methods: The phenotypic and functional maturation of DPT-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. DPT-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity and for tumor regression against melanoma. Results: DPT promoted the activation of $CD8^+$ T cells and the Th1 immune response by inducing IL-12 production in DCs. In a B16F10 melanoma-implanted mouse model, we demonstrated that DPT-treated DCs (DPT-DCs) enhance immune priming and regression of an established tumor in vivo. Furthermore, migration of DPT-DCs to the draining lymph nodes was induced via CCR7 upregulation. Mice that received DPT-DCs displayed enhanced antitumor therapeutic efficacy, which was associated with increased IFN-${\gamma}$ production and induction of cytotoxic T lymphocyte activity. Conclusion: These findings strongly suggest that the adjuvant effect of DPT in DC vaccination is associated with the polarization of T effector cells toward a Th1 phenotype and provides a potential therapeutic antitumor immunity.

• IMMUNE NETWORK
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• 제10권6호
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• pp.188-197
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• 2010

Background: Lichen-derived glucans have been known to stimulate the functions of immune cells. However, immunostimulatory activity of glucan obtained from edible lichen, Umbilicaria esculenta, has not been reported. Thus we evaluated the phenotype and functional maturation of dendritic cells (DCs) following treatment of extracted glucan (PUE). Methods: The phenotypic and functional maturation of PUE-treated DCs was assessed by flow cytometric analysis and cytokine production, respectively. PUE-treated DCs was also used for mixed leukocyte reaction to evaluate T cell-priming capacity. Finally we detected the activation of MAPK and NF-${\kappa}B$ by immunoblot. Results: Phenotypic maturation of DCs was shown by the elevated expressions of CD40, CD80, CD86, and MHC class I/II molecules. Functional activation of DCs was proved by increased cytokine production of IL-12, IL-$1{\beta}$, TNF-${\alpha}$, and IFN-${\alpha}/{\beta}$, decreased endocytosis, and enhanced proliferation of allogenic T cells. Polymyxin B, specific inhibitor of lipopolysaccharide (LPS), did not affect PUE activity, which suggested that PUE was free of LPS contamination. As a mechanism of action, PUE increased phosphorylation of ERK, JNK, and p38 MAPKs, and enhanced nuclear translocation of NF-${\kappa}B$ p50/p65 in DCs. Conclusion: These results indicate that PUE induced DC maturation via MAPK and NF-${\kappa}B$ signaling pathways.

• Clinical and Experimental Vaccine Research
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• 제12권1호
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• pp.47-59
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• 2023

Purpose: The development and study of hepatitis C virus (HCV) vaccine candidates' individualized responses are of great importance. Here we report on an HCV DNA vaccine candidate based on selected envelope (E1/E2) epitopes. Besides, we assessed its expression and processing in human peripheral blood mononuclear cells (PBMCs) and in vivo cellular response in mice. Materials and Methods: HCV E1/E2 DNA construct (EC) was designed. The antigen expression of EC was assayed in PBMCs of five HCV-uninfected donors via a real-time quantitative polymerase chain reaction. Serum samples from 20 HCV antibody-positive patients were used to detect each individual PBMCs expressed antigens via enzyme-linked immunosorbent assay. Two groups, five Swiss albino mice each, were immunized with the EC or a control construct. The absolute count of lymph nodes' CD4⁺ and CD8⁺ T-lymphocytes was assessed. Results: Donors' PBMCs showed different levels of EC expression, ranging between 0.83-2.61-fold in four donors, while donor-3 showed 34.53-fold expression. The antigens expressed in PBMCs were significantly reactive to the 20 HCV antibody repertoire (all p=0.0001). All showed comparable reactivity except for donor-3 showing the lowest reactivity level. The absolute count % of the CD4⁺ T-cell significantly increased in four of the five EC-immunized mice compared to the control group (p=0.03). No significant difference in CD8⁺ T-cells % was observed (p=0.89). Conclusion: The inter-individual variation in antigen expression and processing dominance was evident, showing independence in individuals' antigen expression and reactivity levels to antibodies. The described vaccine candidate might result in a promising natural immune response with a possibility of CD4⁺ T-cell early priming.

• Radiation Oncology Journal
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• 제16권2호
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• pp.107-114
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• 1998

목적 : 혈관수축이완작용 및 혈액점도저하의 약리작용을 가져 말초혈관의 혈류를 증가시키는 것으로 알려진 은행잎 추출물인 Ginkgo biloba extract(GBE) 투여후 방사선 조사시 마우스 종양에서 방사선 효과가 증강됨이 확인되었다. 즉 저산소세포 분획이 감소되었으며 따라서 암조직의 혈류증가가 간접적으로 증명되었다. 방사선효과 증강제인 경우 암조직에 대한 효과가 정상조직에 대한 효과보다 더 커야한다는 것은 필수적이다. 이에 저자들은 GBE 투여후 방사선조사시 마우스 정상조직 급성 반응의 증가여부 및 그 정도를 확인하기 위하여 본 실험을 시행하였다. 대상 및 방법 : 방사선에 대한 급성 피부 반응의 측정 및 공장 재생 소낭선 측정을 위해서 C3H 마우스를 방사선 단독 조사군과 GBE 투여후 방사선조사군으로 나누었다. GBE는 방사선조사 24시간 전과 1시간 전에 각각 복강 내에 2회 주사하였다. 급성 피부 반응 측정 실험에서는 30-50Gy가 마우스 우측 하지에 조사되었고, 공장 재생 소낭선 측정 실험에서는 11-14Gy가 마우스 전신에 조사되었다. 결과 : 방사선에 의한 급성 피부반응 점수 2.0 이상에 해당하는 $RD_{50}$는 방사선 단독조사군에서 44.2Gy(40.6-48.2Gy)이었고, GBE 투여후 방사선 조사시에는 44.4Gy(41.6-47.4Gy)로서 GBE에 의한 영향이 없었다. 방사선 단독 조사군 및 GBE 투여후 방사선조사군의 각 방사선량에 따른 마우스 공장 재생 소낭선의 수는 차이를 보이지 않았다(p=0.57-0.94). 평균치사선량($D_0$)은 방사선 단독조사시 1.80Gy(1.57-2.09Gy), G8E 투여후 방사선조사를 병용시 1.88Gy(1.65-2.18Gy)로서 GBE에 의한 영향이 없었다. 결론 : C3H 마우스에서 방사선에 의한 급성 정상조직 손상은 GBE에 의하여 전혀 증가되지 않는 것으로 판단되며 이미 증명된 종양세포에 대한 치료효과의 증강과 더불어 방사선치료시 GBE를 병용함으로써 치료적 이득을 얻을 수 있을 것으로 예상된다.

• IMMUNE NETWORK
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• 제3권4호
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• pp.302-309
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• 2003

Background: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. Methods: We have used CHO-$dhfr^-$ cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. Results: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. Conclusion: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection.

• IMMUNE NETWORK
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• 제3권2호
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• pp.126-135
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• 2003

Background: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the $65^{th}$ residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. Methods: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains $57{\sim}80^{th}$ amino acid residues of TP domain. After isolation of mRNA of heavy-variable region ($V_H$) and light-chain variable region ($V_L$) from the spleen of the immunized mouse, DNA of $V_H$ and $V_L$ were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. scFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. Results: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. Conclusion: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

• 한국토양비료학회지
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• 제16권1호
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• pp.20-27
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• 1983

제주도(濟州道) 화산회토양유기물(火山灰土壌有機物)의 화학성(化学性), 유기물(有機物) 성상(性狀) 및 부식산(腐植酸)의 광학적(光学的) 특성(特性)을 알고져 수종(数種)의 화산회토양(火山灰土壌)을 공시(供試)하여 실내시험(室內試験)한 결과(結果)를 요약(要約)하면 다음과 같다. 가. 토양(土壌)의 화학적(化学的) 성질(性質) 1) 화산회토양(火山灰土壌)은 비화산회토양(非火山灰土壌)에 비(比)하여 유기물(有機物)(4~27%), 유효규산(有効珪酸)(291~884ppm), 활성(活性) 알루미늄(150~478ppm) 및 활성철함량(活性鉄含量)(0.77~0.86%)이 많고 반대(反対)로 유효인산(有効燐酸)(4~15ppm) 함량(含量)이 현저(顕著)히 낮았다. 2) 가리(加里), 석화(石火), 고토등(苦土等)은 밭의 경우 화산회토양(火山灰土壌)에서 높은 편이나 삼림지(森林地) 및 유휴지토양(遊休地土壌)은 낮은 경향(傾向)을 보였다. 3) 화산회토양(火山灰土壌)은 규반비(珪礬比) 및 규산(珪酸)/유기물비(有機物比)가 낮은 반면(反面) K/Ca+Mg, Al/Fe(활성(活性)) 및 C/P비(比)가 현저(顕著)히 높았다. 나. 유기물(有機物) 및 질소분별정량(窒素分別定量) 1) 화산회토양(火山灰土壌)은 유기물(有機物)의 총탄소중(総炭素中) Humin-C의 비율(比率), 유기물중(有機物中) Humin산(酸) 그리고 Humin중(中) C/N율(率)이 비화산회토양(非火山灰土壌)보다 현저(顕著)히 높았다. 2) 화산회토양(火山灰土壌)은 비화산회토양(非火山灰土壌)에 비(比)하여 총질소(総窒素), 산(酸) 및 알카리가용성(可溶性) 질소함량(窒素含量)이 현저(顕著)히 높은 반면(反面) 총질소중(総窒素中) 무기태질소(無機態窒素)로 방출(放出)될 수 있는 무기화율(無機化率)은 높지 않았다. 다. 토양부식(土壌腐植)의 형태(形態) 1) 화산회토양(火山灰土壌)은 비화산회토양(非火山灰土壌)에 비(比)하여 광흡(光吸) 수능(收能)이 높고(K600, RF치(値), ${\delta}logK$) 부식화도(腐植化度)가 진전(進展)될수록 색농도(色濃度)가 짙은 것으로 나타났다. 2) 화산회토양(火山灰土壌)은 산화제(酸化劑)에 대(対)한 저항성(抵抗性)이 높고 산(酸) 및 알카리 가수분해성(加水分解性)이 강(强)하여 부식화도(腐植化度)가 높아 부식(腐植)의 자연분해(自然分解)가 극(極)히 어려운 것으로 나타났다. 라. Humin의 관능기조사(官能基調査) 화산회토양(火山灰土壌)의 유출부식산(油出腐植酸)의 관능기(官能基)는 phenolic-OH기(基), Alcoholic-OH기(基) 및 Carboxyl기(基)가 많고 비화산회토양(非火山灰土壌)은 Methoxyl기(基) 및 Carbonyl기(基)가 많았다. 마. 부식산(腐植酸)의 흡광도(吸光度) 1) 공시토양(供試土壌)의 가시광역(可視光域)은 200~500nm부근의 단파장영역(短波長領域)이었으며 주로 350, 420, 450 및 480nm 에서 4개(個)의 흡광곡선(吸光曲線)을 나타내었다. 2) 화산회토양(火山灰土壌)인 흑악통(黑岳統)은 362nm부근에서 단일(單一)의 높은 흡광도(吸光度)를 보였으며 비화산회토양(非火山灰土壌)인 영악통(永楽統)은 360nm와 390nm에서 2개(個)의 단순(單純)한 높은 흡광대(吸光帶)를 나타내었다. 마. 분해촉진제(分解促進剤) 처리효과(處理効果) 화산회토양(火山灰土壌)에 대(対)한 분해촉진효과(分解促進効果)는 이도통(統)은 역분해성(易分解性) 유기물(有機物) 첨가(添加)에 따른 "Priming Effect"가 증가(增加)되었으며 남원(南元)과 흑악통(黑岳統)은 Na-Pyrophosphate의 첨가효과(添加効果)가 있었다.