• IMMUNE NETWORK
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• 제4권2호
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• pp.81-87
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• 2004

Background: In the thymus, developing thymocytes continually interact with thymic epithelial cell components. Self MHC restriction of mature T cells are imposed in the thymus through interaction of immature double positive thymocytes and thymic cortical epithelial cells. The site of negative selection, however, is a matter of debate. Through systemic injection of anti-TCR antibody or antigenic peptides, investigators suggested that most of the negative selection occurs in the thymic cortex. But the requirements for negative selection, i.e cellular counterparts and costimulatory molecules are more available in the medulla or cortico-medullary junction rather than in the thymic cortex. Methods: The direct and indirect pathways of thymocyte death after systemic anti-TCR antibody injection were separated through several experimental systems. B6 mice were either adrenalectomized or sham-adrenalectomized to evaluate the role of endogenous glucocorticoids from adrenal gland. Role of TNF were evaluated through using TNF receptor double knockout mice. Results: We found that without indirectly acting mediators such as $TNF-\alpha$ or corticosteroid, double positive thymocyte death were minimal by systemic injection of anti-TCR antibody in TNF receptor double knockout neonatal mice. Also by analyzing neonatal wild-type mice with adoptively transferred mature T cells, only peripheral activation of mature T cells could induce extensive double positive thymocyte death. Conclusion: Thus, systemically injected anti-TCR antibody mediated thymocyte death are mostly induced through indirect pathway.

• Journal of Ginseng Research
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• 제21권3호
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• pp.160-168
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• 1997

The present study was carried out to investigate the effects of Panax ginseng saponin extracts on the dexamethasone-induced apoptosis of mouse thymus in vivo and mouse thymocytes in vitro. The saponin fractions of red ginseng (R-SAP) and white ginseng (Wl-SAP) were provided by the Korea Ginseng & Tobacco Research Institute, and the other saponin fraction of white ginseng (W2-SAP) was extracted in our laboratory. 1. The male ICR mice (3~4 wk old; weighing 15$\pm$2 g) were given by each saponin fraction of 5 mg/kg/ day for 4 days, and at one hour after the last treatment, they were injected by deuamethasone (5 mg/kg : DX). The mouse thymus was extracted at 6 hours after DX injection, and they were stained with hematoxylin-eosin reagents and an Apop-Tag kit, respectively, and the thymocytes prepared from it were labelled with anti-mouse FITC-anti-CD4 and anti-mouse PE-anti-CD8 and then analyzed by fluorescence activated cell sorter (FACS). DX-induced reduction of thymus weight was significantly attenuated by W2- SAP but was not affected by other saponin fractions. And DX-induced apoptotic death of thymocytes, appeared in the histologic findings of the thymus, was inhibited by the saponin fractions and the order of these inhibitory potencies was R-SAP》W2-SAP>Wl-SAP. However, in respect of T cell receptors, the differentiation of thymocytes seems not to be changed by treatments with DX or/and the saponin fractions. 2. In the primary thymocyte culture, the DX-induced reduction of thymocyte MTT values was rather greater in RPMI 1640 medium of IWc fetal bovine serum (FBS) or horse serum (HS). In addition, the DX-Induced MTT reduction was significantly inhibited by R-SAP or W2-SAP, in the culture using that medium of 5% FBS or HS. But these saponin fraction did not effected the DX-induced reduction of thymocyte MTT value in primary culture of 10% FBS or 10% HS. These results suggest that R-SAP and some W-SAP fractions may protect thymocyte from stress or glucocorticoisteroid-induced death of them.

• 약학회지
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• 제46권1호
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• pp.69-74
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• 2002

The effects of genistein on murine thymocytes for inducing apoptotic cell death and phagocytic activity of peritoneal macrophage were studied in vitro. Addition of genistein (10 and 50$\mu$M) to cultured thymocytes from BALB/c mice definitely promoted DNA fragmentation. Also, cytofluorometric analysis of these cells demonstrated a reduction in mitochondrial transmembrane potential ($\Delta$Ψm). But, repeated administration of genistein (1 mg/mouse/day) to mice for 7 days did not cause any detectable DNA fragmentation. Genistein decreased lucigenin chemiluminescence and engulfment of fluorescein-conjugated E. coli particles in peritoneal macrophage. These results suggest that genistein induce an apoptosis of thymocyte via reduction in $\Delta$Ψm and decrease phagocytic activity of peritoneal macrophage in vitro.

• 대한약리학회지
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• 제32권1호
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• pp.113-123
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• 1996

세포내 polyamine은 DNA 구조 뿐 아니라 전사과정, 세포의 성장, 분화, 및 증식 등에 간여하는 바, 배양 흉선세포의 apoptosis 을 억제한다고 한다. 따라서 dexamethasone에 의한 생쥐 흉선세포의 apoptosis 반응에 대한 polyamine의 억제작용을, polyamine 생성과 대사억제제들로 처치한 흉선세포의 일차배양실험에서 관찰하여, 그 결과를 A23187과 DHEA의 작용과 비교하였다. 1) 흉선세포 생존율이 dexamethasone, DHEA, A23187, DFMO, MGBG들에 의하여 직접 현저히 억제되며, aminoguanidine, putrescine, spermidine, 및 spermine들에 의해서는 영향을 받지 않았다. 2) 흉선세포 DNA의 분절화가 dexamethasone과 A2318T에 의하여 유의하게 증강되어 있으며 DHEA에 의하여도 다소 증가되었으나, DFMO, MGBG, aminoguanidine, putrescine, spermidine, 및 spermine들에 의하여는 크게 영향을 받지 않았다. 3) Dexamethasone에 의한 흉선세포의 apoptosis는 DHEA에 의하여 억제된 반면, DFMO, MGBG, 및 aminoguanidine에 의하여는 영향을 받지 않았다. Spermine은 dexamethasone과 A23187에 의한 세포생존율 감소를 유의하게 억제하였으며, A23187에 의한 세포생존율 감소는 putrescine과 spermidine에 의하여도 억제되는 경향을 보였다. 4) DFMO 및 MGBG에 의한 흉선세포 생존율 감소는 spermine에 의해 현저히 억제되었으나, putrescine과 spermidine에 의하여는 영향을 받지 않았다. 5) Dexamethasone을 DFMO 또는 MGBG와 병합처치하여 나타나는 흉선세포 생존율 감소는 각각 spermine과 putrescine에 의하여 유의하게 억제되었으나, aminoguanidine 또는 DHEA와 dexamethasone의 병합처치에 의한 생존율 감소는 polyamine 전처치에 의해 감소되지 않았다. 이상의 결과는 polyamine이 흉선세포의 apoptosis 반응을 억제할 수 있고, 이같은 억제효과의일부가 $[Ca^{2+}]_i$ 증가에 관련되는 신호전달과정과 연관될 뿐 아니라, 세포막의 polyamine transporter를 통한 polyamine 섭취가 이들의 생합성 또는 유리기능과 함께 세포내 polyamine 함량을 조정하므로, 흉선세포의 apoptosis에 억제적으로 작용할 수 있음을 시사하는 것으로 사료된다.

• Toxicological Research
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• 제13권1_2호
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• pp.39-48
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• 1997

Cyclophosphamide(25, 50 or 100 mg/kg), orally administered to male Sprague-Dawley rats, caused a time- and dose-dependent thymic atrophy. In the light microscopic examination of the atrophic thymus, thymocytes with condensed or fragmented nucleus were multifocally observed in the cortical region, started to increase 8 hr after CPA treatment and reached to the maximal level at 16 hr, although such cells were not seen after 48 hr when the severe depletion of thymocytes were marked. In agarose gel electrophoresis to analyze the DNA changes, DNA extracted from atrophic thymus showed a oligonucleosomal laddering at the corresponding time to morphological changes. In an additional supportive experiment, thymocytes showing morphological changes, nuclear condensation or apoptotic body, exhibited a positive reaction to immunoperoxidase staining using in situ apoptosis detection kit. Separately, agarose gel electrophoresis of DNA from bone .marrow cells was performed to investigate the involvement of bone marrow cells in the process of thymocyte apoptosis. Although DNA laddering was slightly increased 2 and 4 hr after treatment, no clear correlation was inferred. Taken togather, it is concluded that thymocytes showing morphological changes in thymic atrophy induced by cyclophosphamide administration represent an apoptosis having biochemical nature of programmed cell death.

• Animal cells and systems
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• 제5권4호
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• pp.267-274
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• 2001

Calpains are a family of cysteine proteases existing primarily in two forms designated by the $Ca^{2+}$ concentration needed for activation in vitro, $\mu$-calpain (calpain-I) and m-calpain (calpain-II). The physiologica1 roles of calpains remain unclear. Many groups have proposed a role for calpains In apoptosis, but their patterns of activation are not well characterized. Calpains have been implicated in neutrophil apoptosis, glucocorticoid-induced thymocyte apoptosis, as well as many other apoptotic pathways. Calpain activation in apoptosis is usually linked upstream or downstream to caspase activation, or in a parallel pathway alongside caspase activation. Calpains have been suggested to be involved in DNA fragmentation (via endonuclease activation), but also as effector proteases that cleave cellular proteins involved in DNA repair, membrane associated proteins and other homeostatic regulatory proteins. Recently, our laboratory demonstrated $\mu$-calpain activation in NAD(P)H: quinone oxidoreducatse 1 (NQO1)-expressing cells after exposure to $\beta$-lapachone, a novel quinone and potential chemo- and radio-therapeutic agent. Increased cytosolic $Ca^{2+}$ in NQO1-expressing cells after $\beta$-lapachone exposures were shown to lead to $\mu$-calpain activation. In turn, $\mu$-calpain activation was important for substrate proteolysis and DNA fragmentation associated with apoptosis. Upon activation, $\mu$-calpain translocated to the nucleus where it could proteolytically cleave PARP and p53. We provided evidence that $\beta$-lapachone-induced, $\mu$-calpain stimulated, apoptosis did not involve any of the known caspases; known apoptotic caspases were not activated after $\beta$-lapachone treatment of NQO1-expressing cells, nor did caspase inhibitors have any effect on $\beta$-1apachone-induced cell death. Elucidation of processes by which $\beta$-1apachone-stimulated $\mu$-calpain activation and calpains ability to activate endonucleases and induce apoptosis independent of caspase activity will be needed to further develop/modulate $\beta$-lapachone for treatment of human cancers that over-express NQO1.