• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.21-29
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• 2016

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of $Na^+$ and $H^+$ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of $Ca^{2+}$. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.

• Molecules and Cells
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• 제39권5호
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• pp.426-438
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• 2016

Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant immune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVR-Pii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

• IMMUNE NETWORK
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• 제2권3호
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• pp.142-149
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• 2002

Background: Our previous study showed that purified protein derivative (PPD)-stimulated pleural mononuclear cells (PMC) from tuberculous pleurisy (Tbp) produced significantly more $IFN-{\gamma}$ (10- to 70-fold) after in vitro PPD stimulation than freshly isolated pleural cells from malignant pleurisy. The present study was designed to determine whether blocking the CD40-CD40 ligand (CD40L) interaction decreases $IFN-{\gamma}$ production by altering IL-12 levels. Methods: IL-12 and $IFN-{\gamma}$ production after neutralizing anti-CD40L antibody treatment was compared to the efficacy of anti-CD80, anti-CD86, and a combination of anti-CD80 and CD86 (CD80+86) monoclonal antibodies (mAb). These activities were measured by enzyme-linked immunosorbent assays (ELISAs) and reverse transcription-polymerase chain reaction (RT-PCR), after in vitro stimulation with PPO antigen (Ag). Results: Neutralization of CD80, CD86 and CD80+86 did not decrease $IFN-{\gamma}$ and IL-12 production in Tbp-PMC, whereas neutralization of CD40L significantly depressed IL-12 p40 and $IFN-{\gamma}$. In addition, neutralization of CD40L completely inhibited IL-12 p40 and $IFN-{\gamma}$ mRNA expression. Conclusion: The CD40-CD40L interaction might play a major role in IL-12 and $IFN-{\gamma}$ production in Tbp-PMC, thus contributing to protective immunity in human tuberculosis.

• IMMUNE NETWORK
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• 제2권2호
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• pp.102-108
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• 2002

Background: This study was aimed to differentiate two forms of CTLA-4 (CD152) in activated peripheral blood lymphocyte and clarify the mechanism how cytoplasmic form of this molecule is targeted to cell surface. Methods: For this purpose we generated 2 different anti-human CD152 peptide antibodies and 5 different N'-terminal deletion mutant CTLA4Ig fusion proteins and carried out a series of Western blot and ELISA analyses. Antipeptide antibodies made in this study were anti-CTLA4pB and anti-CTLA4pN. The former recognized a region on extracellular single V-like domain and the latter recognized N'-terminal sequence of leader domain of human CD152. Results: In Western blot, the former antibody recognized recombinant human CTLA4Ig fusion protein as an antigen. And this recognition was completely blocked by preincubating antipeptide antibody with the peptide used for the antibody generation at the peptide concentration of 200 ug/ml. These antibodies were recognized human CD152 as a cytoplasmic sequestered- and a membrane bound- forms in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte (PBL). These two forms of CD152 were further differentiated by using anti-CTLA4pN and anti-CTLA4pB antibodies such that former recognized cytosolic form only while latter recognized both cytoplasmic- and membraneforms of this molecule. Furthermore, in a transfection expression study of 5 different N'-terminal deletion mutant CTLA4Ig, mutated proteins were secreted out from transfected cell surface only when more than 6 amino acids from N'-terminal were deleted. Conclusion: Our results implies that cytosolic form of CTLA-4 has leader sequence while membrane form of this molecule does not. And also suggested is that at least N'-terminal 6 amino acid residues of human CTLA-4 are required for regulation of targeting this molecule from cytosolic- to membrane- area of activated human peripheral blood T lymphocyte.

• IMMUNE NETWORK
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• 제15권2호
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• pp.66-72
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• 2015

Currently, detecting biochemical differences before and after allogeneic stem cell transplantation (SCT) for improved prediction of acute graft-versus-host disease (aGVHD) is a major clinical challenge. In this pilot study, we analyzed the kinetics of circulating adipokine levels in patients with or without aGVHD before and after allogeneic SCT. Serum samples were obtained and stored at $-80^{\circ}C$ within 3 hours after collection, prior to conditioning and at engraftment after transplantation. A protein array system was used to measure the levels of 7 adipokines of patients with aGVHD (n=20) and without aGVHD (n=20). The resistin level at engraftment was significantly increased (p<0.001) after transplantation, regardless of aGVHD occurrence. In the non-aGVHD group, the concentrations of the hepatocyte growth factor (HGF) (mean values${\pm}$SD; $206.6{\pm}34.3$ vs. $432.3{\pm}108.9pg/ml$, p=0.040) and angiopoietin-2 (ANG-2) (mean values${\pm}$SD; $3,197.2{\pm}328.3$ vs. $4,471.8{\pm}568.4pg/ml$, p=0.037) at engraftment were significantly higher than those of the pre-transplant period, whereas in the aGVHD group, the levels of adipokines did not change after transplantation. Our study suggests that changes in serum HGF and ANG-2 levels could be considered helpful markers for the subsequent occurrence of aGVHD.

• IMMUNE NETWORK
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• 제8권1호
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• pp.29-37
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• 2008

Interleukin-23 (IL-23) is a novel pro-inflammatory cytokine which has been implicated to play a pathogenic role in rheumatoid arthritis (RA). This study was undertaken to investigate the IL-23 inductive activity of the proinflammatory cytokine IL-17, $IL-1{\beta}$ and tumor necrosis factor (TNF-${\alpha}$) in RA synovial fluid mononuclear cells (SFMC). Expression of IL-23p19, IL-17, $IL-1{\beta}$ and TNF-${\alpha}$ in joint was examined by immunohistochemistry (IHC) of patients with RA and osteoarthritis (OA). The effects of IL-17 and $IL-1{\beta}$ on expression of IL-23p19 in human SFMC from RA patients were determined by reverse transcriptase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). IL-23p19 was expressed in the RA fibroblast like synoviocyte (FLS), but not from OA FLS. Similar to the protein expression, IL-23p19 mRNA could be detected by RT-PCR in RA SFMC. IL-17 and $IL-1{\beta}$ could induce RA SFMC to produce the IL-23p19. The effects of IL-17 were much stronger than $IL-1{\beta}$ or TNF-${\alpha}$. These responses were observed in a doseresponsive manner. In addition, IL-17 or $IL-1{\beta}$ neutralizing antibody down-regulated the expression of IL-23p19 induced by LPS in RA-SFMC. Our results demonstrate that IL-23p19 is overexpressed in RA synovium and IL-17 and $IL-1{\beta}$ appears to upregulate the expression of IL-23p19 in RA-SFMC.

• 생명과학회지
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• 제30권3호
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• pp.267-277
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• 2020

이 연구는 설치류에서 양파추출물의 효능을 평가하기 위하여 실시하였다. 실험동물은 대조군(control)과 양파추출물 투여군(ACE)으로 나누어서 진행하였다. ACE그룹은 대조군에 비해 적혈구(RBC), 헤모글로빈(HGB), 헤마토크릿(Hct) 수준이 약간 증가하는 것을 보였다(p<0.05). 반면, 헤모글로빈, 단핵구, 림프구 및 호중구는 대조군과 비교하였을 경우 유의미한 변화(p<0.05)가 없었다. GOT (glutamic oxaloacetic transaminase) 및 GPT (glutamic pyruvic transaminase) 수준을 분석한 결과 대조군에 비해 ACE그룹이 유의적으로 감소하였다(p<0.05). 혈당, 총단백질, HDL-콜레스테롤은 ACE 그룹에서 약간 높았고, 트리글리세라이드, 총콜레스테롤 수치는 대조군에 비해 ACE그룹에서 더 낮았다(p<0.05). 간 조직과 혈액의 면역과 염증에 관련된 사이토카인(인터루킨 1알파(IL-1α), 인터루킨 1베타(IL-1β), 종양괴사인자알파(TNF-α), 인터루킨 6(IL-6), 인터루킨 10(IL-10), 인터페론감마(INF-γ), 인터루킨 18(IL-18), 인터루킨 2(IL-2), 과립구 대식구 집락자극인자(GM-CSF)) 수준은 모두 정상 범위 내에 있는 것으로 확인되었다. 본 연구결과는 고농축 식이성 양파추출물은 혈액학적 지표와 면역 기능에 독성이 없다는 것을 보여주는 기초데이터로 활용할 수 있을 것이다.

• 디지털융복합연구
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• 제14권10호
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• pp.515-520
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• 2016

본 연구는 명월초 추출물의 Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB)의 농도 및 Nitric Oxide(NO) 측정을 통하여 현대인들의 피부염증 및 감염질환 예방에 도움이 되는 천연원료 개발을 위하여 수행하였다. 명월초 추출물의 cytosol과 nucleus의 분리과정을 거쳐 Human Dermal Fibroblasts(HDF cell)을 이용하여 다양한 염증조절 인자 중 대표적인 NF-kB 농도를 측정하였고 RAW264.7 세포에 대하여 신경전달 매개체로 피부면역 반응에 중요한 역할을 하는 NO의 양을 분석하였다. 명월초 추출물을 투여한 결과, NF-kB의 단백질과 mRNA 밴드가 감소하여 명월초의 항염 효과가 피부염증 및 질환예방에 효과가 있을 것으로 기대할 수 있었다. 또한 명월초 추출물의 농도가 증가함에 따라 NO는 유의적으로 감소하여 농도 의존적 결과를 보였다. 따라서, 향후 임상적 측면에서의 더 세밀한 항염 효과에 대한 연구가 지속된다면 명월초는 식품으로 뿐 아니라 화장품 등 현대인들이 넓게 적용할 수 있는 융합소재로 이용될 수 있으리라 사료된다.

• 한국발생생물학회지:발생과생식
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• 제7권1호
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• pp.15-22
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• 2003

Fas는 세포자연사를 유도하는 세포 표면 수용체 단백질로서 면역계에서 중요한 역할을 한다. 이러한 Fas mRNA는 림프조직뿐만 아니 라 비 림프조직에서도 발현한다. 한편 대다수의 난포들은 세포자연사와 연관된 기 작을 통해 난포폐쇄로 진행되는 것으로 알려지고 있으나, 난포폐쇄에 관한 기작은 아직 규명되지 않았다. 따라서 본 연구의 목적은 먼저 생쥐의 난소의 과립세포와 난자에서 Fas와 Fas ligand의 발현 여부를 알아보고, Fas와 Fas ligand발현과 난포폐쇄와의 상관 관계를 알아보고자 하였다. RT-PCR을 수행한 결과, 난소내 과립세포와 난자에서 Fas와 Fas ligand mRNA가 모두 발현하는 것을 확인할 수 있었다. 면역조직화학적 염색 결과 또한, 원시 난포를 제외한 모든 발달 중인 난포의 과립세포와 난자들에서 Fas와 Fas ligand가 검출되는 것을 확인하였고, 특히 폐쇄를 보이는 난포에서 강한 발현을 보였다. 한편, 난소에서 세포자연사를 확인하기 위해 TUNEL 염색을 수행한 결과, TUNEL 양성을 보이는 과립세포와 난자의 수는 건강한 정상 난포에 비해 폐쇄난포에서 증가하는 것을 관찰할 수 있었다. 이상의 결과들은 난소내 과립세포와 난자에서 Fas와 Fas ligand 발현과 난포폐쇄와 상관 관계가 있음을 보여주고 있으며, 이러한 Fas와 Fas ligand가 생쥐 난소내 난포폐쇄 유발에 관여하고 있을 것으로 사료된다

• IMMUNE NETWORK
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• 제3권1호
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• pp.38-46
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• 2003

Background: Platelet-activating factor (PAF) induces nuclear factor $(NF)-{\kappa}B$ activation and angiogenesis and increases tumor growth and pulmonary tumor metastasis in vivo. The role of $NF-{\kappa}B$ activation in PAF-induced angiogenesis in a mouse model of Matrigel implantation, and in PAF-mediated pulmonary tumor metastasis were investigated. Methods: Angiogenesis using Matrigel and experimental pulmonary tumor metastasis were tested in a mouse model. Electrophoretic mobility shift assay was done for the assessment of $NF-{\kappa}B$ translocation to the nucleus. Expression of angiogenic factors, such as tumor necrosis factor $(TNF)-{\alpha}$, interleukin $(IL)-1{\alpha}$, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were tested by RT-PCR and ELISA. Results: PAF induced a dose- and time-dependent angiogenic response. PAF-induced angiogenesis was significantly blocked by PAF antagonist, CV6209, and inhibitors of $NF-{\kappa}B$ expression or action, including antisense oligonucleotides to p65 subunit of $NF-{\kappa}B$ (p65 AS) and antioxidants such as ${\alpha}$-tocopherol and N-acetyl-L-cysteine. In vitro, PAF activated the transcription factor, $NF-{\kappa}B$ and induced mRNA expression of $TNF-{\alpha}$, $IL-1{\alpha}$, bFGF, VEGF, and its receptor, KDR. The PAF-induced expression of the above mentioned factors was inhibited by p65 AS or antioxidants. Also, protein synthesis of VEGF was increased by PAF and inhibited by p65 AS or antioxidants. The angiogenic effect of PAF was blocked when anti-VEGF antibodies was treated or antibodies against $TNF-{\alpha}$, $IL-1{\alpha}$, and bFGF was co-administrated, but not by antibodies against $TNF-{\alpha}$, $IL-1{\alpha}$, and bFGF each alone. PAF-augmented pulmonary tumor metastasis was inhibited by p65 AS or antioxidants. Conclusion: These data indicate that PAF increases angiogenesis and pulmonary tumor metastasis through $NF-{\kappa}B$ activation and expression of $NF-{\kappa}B$-dependent angiogenic factors.