• Laboraroty Animal Research
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• 제34권4호
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• pp.166-175
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• 2018

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. Rag-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of Rag-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of Rag-2, we recently established a new Rag-2 knockout FVB mouse line ($Rag-2^{-/-}$) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. $Rag-2^{-/-}$ mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in $Rag-2^{-/-}$ mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in $Rag-2^{-/-}$ mice. These findings indicate that our $Rag-2^{-/-}$ mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

• IMMUNE NETWORK
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• 제1권1호
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• pp.61-69
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• 2001

Background: To determine the molecular structure of type II collagen-specific T-cell receptors associated with rheumatoid arthritis (RA). Methods: We generated CII-specific T-cell lines of 8 RA patients by prolonged in vitro culture with bovine CII (bCII) and the immunogenic peptide (256-270) of human CII. The proliferation response towards CII stimulation was measured from the uptake of 3H-thymidine. Changes in the secretion of Th 1 and Th2 cytokines in the culture supernatent were measured by ELISA. The TCR clonotypes of these T-cells were examined by RT-PCR/SSCP analyses of all 22 $V_{\beta}$ chains. Results: T-cells from patients' tissue exhibited strong proliferation index upon CII stimulation, which was maintained up to 6 months in the culture. The secretion of INF-$\gamma$from these T-cells increased along with the duration of culture time, while the amount of IL-4 production did not show significant changes. The SSCP band patterns of patients' T-cells appear as discrete bands unlike the smeary streak produced from normal samples. Some SSCP bands, each representing selected expansion of a TCR containing certain subtype of $V_{\beta}$ peptides, appeared to be identical in more than one patients. Among these, the expansion of SSCP band representing the $V_{\beta}$ 14 CDR3 region persisted after switching the antigen to the immunogenic human peptide (256-270). Conclusion: CII-reactive T-cells expressing distinct CDR3 motifs are selectively expanded in the peripheral blood and synovial fluid of RA patients, and their persistent proliferation upon CII stimulation, as well as the production Th 1-type cytokines, may play pivotal roles in RA pathogenesis.

• Journal of Korean Neurosurgical Society
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• 제30권8호
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• pp.1037-1041
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• 2001

결합조직형성 섬유종은 드문 골종양으로 조직학적으로는 양성이나 국소적으로 빨리 자라는 질환이다. 두개골에 발생한 결합조직형성 섬유종은 지금까지 9례가 보고되고 있으며, 이 중 8명은 여자에서 발생하다. 이 논문은 남자에서 발생한 두개골 결합조직형성 섬유종을 보고하며, 문헌을 검토하여, 이 질환의 임상양상 및 조직학적소견, 그리고 치료방법등을 보고하고자 한다. 환자는 21세 남자로 두통을 호소하으며, 두부 전산화 단층 촬상 뇌을 침습한 소견이 없는 단일 골용해성 부위가 관찰되었으며, 조직학적 검사상 결합조직형성 섬유종으로 밝혀졌다. 면역조직화학적 염색상, 종양의 에스트로겐과 프로게스테론 수용기는 음성이었다. 정상골을 포함한 종양을 전적출하으며, 35개월간 추적관찰결과 재발소견은 없었다. 다른 부위의 결합조직형성 섬유종과 비교했을 때, 종양의 정상골을 포함한 전적출이 두개골에서 가능하므로, 재발이 없는 것으로 생각되며, 남자에서 발생한 결합조직형성 섬유종을 치험하기에 문헌검토와 함께 보고하는 바이다.

• The Korean Journal of Physiology
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• 제29권2호
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• pp.269-277
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• 1995

Membrane vesicles were prepared by differential centrifugation from epithelial cells of porcine trachea. Total activity of microsomal ATPases was measured spectrophotometrically by a coupled enzyme assay. The steady-state activity of the enzyme was $329{\pm}10$ nmol/min mg protein. Thapsigargin, a specific antagonist of intracellular $Ca^{2+}-ATPase$, inhibited about 50% of the activity, leaving $178{\pm}18\;nmol/min .mg$ protein (n=6), indicating that the $Ca^{2+}-ATPase$ is one of the major microsomal ATPases. The microsomes used in this study appeared to be tight-sealed vesicles since they showed saturation in $^{45}Ca^{2+}$ uptake experiments. Inositol 1,4,5-trisphosphate $InsP_{3}, 4\;{\mu}M$, an agonist of $InsP_{3}$-sensitive $Ca^{2+}$ release channel ($InsP_{3}$, receptor), and Ca-ionophore A23187 $(10\;{\mu}M)$ induced $^{45}Ca^{2+}$ releases of 20% and 50% of stored $^{45}Ca^{2+}$, respectively. The addition of $(10\;{\mu}M\;InsP_{3}$ also increased the microsomal ATPase activity from $282{\pm}8$ nmol/min mg protein to $334{\pm}21$ nmol/min . mg protein in the intact vesicles. Similar increase in the activity was observed by making microsomes leaky (uncoupling) using the Ca-ionophore A23187. ;$InsP_{3}-induced$ effects were blocked by either thapsigargin or heparin suggesting that: 1) the $InsP_{3}-induced$ increase in ATPase activity is mediated by microsomal $Ca^{2+}-ATPase$, and 2) dissipation of $Ca^{2+}$ gradient across the microsomal membrane is responsible for the $InsP_{3}-induced$ effect. In order to test the dependence of the $Ca^{2+}-ATPase$ activity on the activity of $InsP_{3}-induced$ the activity of ATPases was monitored in various concentrations of free $Ca^{2+}$ using $EGTA-Ca^{2+}$ buffers. The $Ca^{2+}$-dependent biphasic change is the well-known character of $InsP_{3} receptor but not of microsomal $Ca^{2+}-ATPase$ in non-excitable cells; however, the activity of microsomal ATPase appeared biphasic and a maxim진 activity of $397{\pm}36nmol/min\;.mg$ protein was obtained in the solution containing 100 nM free $Ca^{2+}$. Below or above this concentration, the activity of ATPases was lower. These results strongly support a positive correlation of microsomal $Ca^{2+}-ATPase$ to the $InsP_{3}$ receptors in epithelial microsomes.

• The Korean Journal of Pain
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• 제14권1호
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• pp.7-11
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• 2001

Background: Although several mechanisms of causalgia, which results from a partial injury to the peripheral nerve trunk, have been proposed, whether or not antidromic impulses from the injured neurons contribute to the development of the mechanical hyperalgesia has not been studied. The purpose of this experiment is was to investigate the role of antidromic impulses to the peripheral sensory receptor site on the development of mechanical hyperalgesia in a rat model of peripheral neuropathy. Methods: Rats were prepared with tight ligation of by tightly ligating the left fifth and sixth lumbar spinal nerves. The effect of bupivacaine pretreatment on the development of mechanical hyperalgesia was evaluated by injecting 0.5% bupivacaine (0.3 ml) into the plantar surface of the left hind paw before the skin incision was made. For the control group, normal saline (0.3 ml) was injected instead of bupivacaine. To measure the mechanical hyperalgesia, paw withdrawal thresholds were measured using a series of von Frey hairs. Mechanical hyperalgesia was measured a the day before, and 1, 2, 3, 4, 7, and 14 days after the surgery. Results: The control group showed decreased withdrawal thresholds from the day after the surgery (the values were $14.0{\pm}0.5$, $8.9{\pm}1.3$, $8.4{\pm}1.6$, $6.9{\pm}1.2$, $8.8{\pm}1.5$, $10.5{\pm}1.3$, and $8.6{\pm}1.3$ g; at -1, 1, 2, 3, 4, 7, and 14 days after the surgery, respectively). However, withdrawal thresholds of the bupivacaine-pretreated group showed increased withdrawal thresholds for three days after the surgery ($14.5{\pm}0.3$, $12.6{\pm}1.4$, $12.7{\pm}1.1$, $10.5{\pm}1.3$ g; at 1, 1, 2, 3 days after the surgery). Conclusions: Our result suggests that antidromic impulses to the peripheral sensory receptors are at least partly responsible for the initial development of mechanical hyperalgesia in a rat model of peripheral neuropathy.

• The Korean Journal of Physiology and Pharmacology
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• 제15권1호
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• pp.53-59
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• 2011

The secretion of insulin from pancreatic ${\beta}$-cells is triggered by the influx of $Ca^{2+}$ through voltage-dependent $Ca^{2+}$ channels. The resulting elevation of intracellular calcium ($[Ca^{2+}]_i$) triggers additional $Ca^{2+}$ release from internal stores. Less well understood are the mechanisms involved in $Ca^{2+}$ mobilization from internal stores after activation of $Ca^{2+}$ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic ${\beta}$-cell line, INS-1 cells. To measure cytosolic and stored $Ca^{2+}$, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. $[Ca^{2+}]_i$ was repetitively increased by caffeine stimulation in normal $Ca^{2+}$ buffer. However, peak $[Ca^{2+}]_i$ was only observed after the first caffeine stimulation in $Ca^{2+}$ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in $[Ca^{2+}]_i$ were reduced by pretreatment with ruthenium red, as well as by depletion of internal $Ca^{2+}$ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced $Ca^{2+}$ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells,$Ca^{2+}$ release from internal stores was activated by caffeine, $Ca^{2+}$, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in perrneabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic ${\beta}$-cells.

• 한국미생물·생명공학회지
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• 제39권2호
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• pp.140-145
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• 2011

돼지유행성설사 바이러스(porcine epidemic diarrhea virus: PEDV)는 자돈에게 감염 시 수양성설사를 동반한 급성 장염을 유발하며 매우 높은 폐사율을 보이는 그룹 1 코로나바이러스이다. PEDV는 다른 그룹 1 코로나바이러스와 마찬가지로 숙주 세포에 감염 시 aminopeptidase N (APN)을 세포 수용체로 이용한다고 알려져 있다. 코로나바이러스의 spike(S) 단백질은 숙주세포의 표면에 부착과 관련하여 감염 개시에 있어 중요한 역할을 하는 것으로 알려져 있으며 특히 S 단백질의 S1 도메인은 세포 수용체에 특이적인 결합을 매개하는 수용체 결합 도메인(receptor binding domain: RBD)을 포함하고 있는 것으로 알려져 있다. 이미 많은 코로나바이러스의 RBD의 위치가 확인되어져 있지만 PEDV의 RBD에 대해서는 아직까지 알려진 바가 없다. 본 연구에서는 돼지 APN 수용체와 결합을 매개하는 PEDV의 RBD를 규명하기 위해 S1 도메인을 주형으로 하는 일련의 재조합 truncated variant들을 제작하였고 각각의 truncated들이 실제로 pAPN과의 결합을 이루는지에 대하여 실험을 통해 확인하였다. 그 결과 S1 도메인의 N 말단 부분이 pAPN과의 결합에서 중요한 부위임을 확인할 수 있었다. 본 연구에서 도출된 결과는 향후 PEDV의 S 단백질과 pAPN간의 분자적 상호작용을 이해하는 데에 도움을 줄 것으로 판단된다.

• Applied Microscopy
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• 제38권1호
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• pp.29-34
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• 2008

본 연구자는 Zinc Selenium autometallography ($ZnSe^{AMG}$) (Danscher et al., 1997) 염색법을 중심으로 중추신경계통 내 zinc ($Zn^{2+}$)의 분포와 이들을 함유하고 있는 신경종말, 소위 ZEN(zinc-enriched) terminals의 미세구조에 관하여 보고한 바 있다. 이번 연구에서는 다른 몇 가지, 즉 Neo-Timm staining (Danscher, 1982), TSQ fluorescence staining (Frederickson et al.,1987), Zinc transporter-3 Immunohistochemistry ($ZnT3^{IHC}$) (Palmiter et at., 1997) 염색법으로 흰쥐 해마복합체에 분포하는 $Zn^{2+}$를 염색한 후 이들의 염색패턴에서 차이점을 밝히고자 하였다. $ZnSe^{AMG}$ 염색법은 $Zn^{2+}$에 대한 특이성은 다소 떨어지나 광학 및 전자현미경하에서 관찰이 가능하며, 반영구적인 표본으로 보관이 가능하다는 장점이 있었고, TSQ는 $Zn^{2+}$에 대한 특이성이 매우 높을 뿐 아니라 그 염색법이 매우 간단하다는 장점이 있는 반면 형광물질의 안정성과 표본보관이 용이하지 않다는 단점이 있다. 그 외 Neo-Timm 염색법은 TSQ형광염색법과 유사한 염색 패턴을 보였으며, $ZnT3^{IHC}$염색법은 오히려 $ZnSe^{AMG}$에 가까운 염색의 결과를 보였다. 본 연구의 결과는 중추신경계통 내 $Zn^{2+}$에 관한 형태학적 연구에서 기초자료로 활용될 수 있을 것이다.

• Biomolecules & Therapeutics
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• 제21권1호
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• pp.29-34
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• 2013

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family of cytokines. TRAIL selectively induces apoptotic cell death in various tumors and cancer cells, but it has little or no toxicity in normal cells. Agonism of TRAIL receptors has been considered to be a valuable cancer-therapeutic strategy. However, more than 85% of primary tumors are resistant to TRAIL, emphasizing the importance of investigating how to overcome TRAIL resistance. In this report, we have found that nemadipine-A, a cell-permeable L-type calcium channel inhibitor, sensitizes TRAIL-resistant cancer cells to this ligand. Combination treatments using TRAIL with nemadipine-A synergistically induced both the caspase cascade and apoptotic cell death, which were blocked by a pan caspase inhibitor (zVAD) but not by autophagy or a necrosis inhibitor. We further found that nemadipine-A, either alone or in combination with TRAIL, notably reduced the expression of survivin, an inhibitor of the apoptosis protein (IAP) family of proteins. Depletion of survivin by small RNA interference (siRNA) resulted in increased cell death and caspase activation by TRAIL treatment. These results suggest that nemadipine-A potentiates TRAIL-induced apoptosis by down-regulation of survivin expression in TRAIL resistant cells. Thus, combination of TRAIL with nemadipine-A may serve a new therapeutic scheme for the treatment of TRAIL resistant cancer cells, suggesting that a detailed study of this combination would be useful.

• Journal of Ginseng Research
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• 제12권2호
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• pp.104-113
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• 1988

여러가지 농도의 cholesterol을 함유한 배지에서 배양한 CHO 세포내로 흡입된 cholesterol 양을 조사한 결과 흡입량이 배지의 cholesterol 농도에 비례하였으므로 cholesterol 흡입은 확산에 의한 것으로 생각된다. $^{125}I$으로 표지된 저밀도 지방단백질($^{125}I$-LDL)을 이용하여 여러가지 농도의 cholesterol을 함유한 배지에서 배양한 CHO 세포와 cholesterol이 없는 정상배지에서 배양한 CHO 세포에서의 LDL 수용체의 합성양상을 조사한 결과 배지에 가해준 cholesterol이 LDL 수용체 합성을 억제함을 확인하였다. Cholesterol의 LDL 수용체 생합성 억제작용에 미치는 ginsenoside의 영향을 조사하기 위해 ginsenoside와 cholesterol을 함께 함유한 배지에서 배양한 CHO 세포(시험군)과 cholesterol만을 포함한 배지에서 배양한 CHO 세포(대조군)에서의 LDL 수용체의 활성양상을 분석한 결과 대조군에 비해 시험군에서의 LDL 수용체 활성이 크게 증가하였다. RNA 및 단백질 합성도 시험군이 대조군보다 증가하였음을 관찰하였다. 그러나 이와 같은 실험조건하에서의 대조군과 시험군의 cholesterol농도를 측정한 결과 시험군의 cholesterol 농도가 대조군보다 훨씬 저하되고 있었다. Ginsenoside의 cholesterol농도 저하작용을 관찰하기 위해 CHO세포에서의 cholesterol의 steroid hormone(estradiol, progesterone)으로의 전환에 미치는 ginsenoside의 영향을 조사한 결과 ginsenoside는 cholesterol의 hormone으로의 전환을 촉진하였음이 확인되었다. 위와 같은 실험결과로 볼 때 ginsenoside는 CHO 세포내부에서의 cholesterol의 LDL 수용체 합성억제를 완화시켜 주는 것이라고 생각된다.