• 대한화장품학회지
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• 제27권1호
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• pp.17-27
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• 2001

A cell-based assay system for monitoring NF-$textsc{k}$B activity was developed to determine the influence of activated NF-$textsc{k}$B in human HaCaT cells. The pNF-$textsc{k}$B-SEAP-NPT plasmid that permits expression of the secreted alkaline phosphatase (SEAP) reported gene in response to the NF-$textsc{k}$B activity and contains neomycin phosphotransferase (NPT) gene for the geneticin resistance in host cells was constructed and transfected into human keratinocyte cell line HaCaT. Human HaCaT transfectant cells secreted the SEAP enzyme into the culture medium in a time-dependent manner until 72h. NF-$textsc{k}$B activities were measured in the SEAP reporter gene assay using a fluorescent detection method. The treatment of HaCaT cell transfectants with known antioxidants [e.g., N-acetyl-L-cysteine and vitamin C] showed inhibition of NF-$textsc{k}$B activity in a time-and concentration-dependent manner. The phorbol 12-myristate 13-acetate (PMA) known as a stimulator of NF-$textsc{k}$B expression demonstrated that it increased NF-$textsc{k}$B activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-$textsc{k}$B activity in the human skin and allow the screening of anti-inflammatory agents from various synthetic chemicals and natural products for dermatological purpose. Abbrevitions used: NF-$textsc{k}$B, nuclear factor kappa B; I-$textsc{k}$B, Inhibitory kappa B; SEAP, secreted alkaline phosphatase; NPT, neomycin phosphotransferease; PCR, polymerase chain reaction: dNTP, deoxynucleoside triphosphates; DMEM, dulbecco’s modified eagle medium; FBS, fetal bovine serum; PBs, phosphate-buffered saline; MUP, 4-methylumbellifery phosphate; NAC, N-acetyl-L-cysteine; DMSO, dimethyl sulfoxide; PMA, phorbol 12-myristate 13-acetate.

• 대한미생물학회지
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• 제35권1호
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• pp.9-18
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• 2000

Immunological mechanisms involving the release of inflammatory factors by HIV-1 infected microglia in the brain have been implicated in the pathogenesis of HIV dementia (HIVD). Since the regulation of matrix metalloproteinases (MMPs) activity can be influenced by variety of inflammatory mediators, this study was undertaken to look for a correlation between the MMP-9 release and the production of TNF-${\alpha}$ in response to HIV-1 p24 in the human monocyte cell line THP-1 as a model for microglia. First, it was shown that HIV-l core p24 antigen induced THP-1 to secrete MMP-9 in a dose response manner while it elicited a little effect on MMP-2 release in human astroglial cell line T98G. Next, it was found that p24 induced THP-1 to secrete TNF-${\alpha}$ without prior differentiation into macrophages by phorbol myristate acetate (PMA) treatment. Furthermore, anti-TNF-${\alpha}$ neutralizing antibodies significantly blocked p24-induced MMP-9 release in a dose dependent manner. Our data indicate that p24 antigen induces monocytic MMP-9 release by triggering up-regulation of TNF-${\alpha}$ secretion.

• The Korean Journal of Physiology and Pharmacology
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• 제5권1호
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• pp.79-86
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• 2001

Recent clinical studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (ATRA). However, most patients who receive continuous treatment with ATRA relapse and develop ATRA-resistant leukemia. Dendritic cells (DCs) are important antigen-presenting cells in the development of antileukemic T-cell responses. In this study, we investigated the strategies to overcome ATRA resistance of APL cells by inducing the differentiation of DCs from human leukemic cell lines for the developtment of adoptive immunotherapy. CD83 was used as a mature DC marker in this study and the expression of CD83 mRNA was determined by RT-PCR method. The promyelocytic leukemic cell line HL-60, B lymphoblast cell lines RPMI 7666 and NC-37 could be induced to dendritic cells in vitro. Treatment of HL-60 with phorbol 12-myristate 13-acetate (PMA) resulted in the expression of myeloid-related DC phenotypes, while treatment of RPMI 7666 with fms-like tyrosine kinase 3 ligand (Flt3-ligand, FL) and treatment of NC-37 with PMA and FL led to the expression of lymphoid-related DC phenotypes. In conclusion, myeloid-related DC phenotypes and lymphoid-related DC phenotypes could be generated from HL-60, NC-37 and RPMI 7666 cell lines, respectively. These DC phenotypes can potentially be used to generate antileukemic T cells in vitro for adoptive immunotherapy.

• 생명과학회지
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• 제23권5호
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• pp.650-656
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• 2013

내피세포 단백질 C 수용체(EPCR)가 트롬빈-트롬보모듈린 복합체에 의한 단백질 C (PC) 활성 증가에 중요한 역할을 한다. EPCR의 활성은 ecodomain의 분열과 수용성 단백질(sEPCR)로 분비함으로써 현저하게 변화한다. EPCR의 탈락은 tumor necrosis factor-${\alpha}$ converting enzyme (TACE)에 의해 매개된다. 토마토에서 발견된 라이코펜은 항산화 효과, 항암 효과, 항염증 효과를 가지고 있다. 그러나 EPCR 탈락에서의 라이코펜의 효과는 알려지지 않았다. 우리는 라이코펜이 PMA, TNF-${\alpha}$, IL-$1{\beta}$와 CLP에 의해 유도된 EPCR 탈락에 미치는 영향을 연구했다. 그 결과, 라이코펜은 TACE의 발현을 억제시켜 PMA, TNF-${\alpha}$, IL-$1{\beta}$와 CLP에 의해 매개된 EPCR 탈락을 저해함을 보여준다. 또한 라이코펜은 PMA가 유발한 p38, ERK1/2, JNK의 인산화를 감소시켰다. 이러한 결과를 토대로, 라이코펜은 EPCR 탈락의 저해를 통해 다양한 중증 혈관 염증 질병 치료를 위한 후보 물질이 될 수 있을 것이다.

• 대한약리학회지
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• 제27권2호
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• pp.125-133
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• 1991

내피세포가 제거된 토끼의 적출 장간막 동맥에서 토끼의 대동맥 내피세포로 부터 A23187과 acetylcholine은 NO와 유사한 혈관 이완성 물질 (EDRF)을 유리한다. 이에 첨가하여 A23187은 acetylcholine과는 달리 superoxide anion에 의하여 파괴되지 않는 EDRF도 유리시킴을 확인하고 이의 특성에 대하여 연구하였다. 정상적인 생리영양액에서는 A23187과 acetylcholine의 용량-반응 곡선은 내피세포에 의존하지 않는 sodium nitroprusside의 곡선과 유사하였다. 이들은 methylene blue에 의하여 억제되었다. Hypoxanthine (HX)과 xanthine oxidase (XO)를 bath내로 투여시 phenylephrine에 의한 수축이 일과성으로 증가한 후 지속적으로 이완되었다. HX-XO 반응중에는 A23187은 장간막 동맥을 즉각적으로 이완시켰으나 acetylcholine의 이완작용은 소실되었다. A23187에 의하여 야기되는 장간막동맥의 이완은 50 mM $K^+-PSS$로 수축을 야기시켰을 때에는 나타나지 아니하였다. Superoxide dismutase를 전처치하였을 때는 HX-XO 반응중에도 acetylcholine 뿐만아니라 A23187에 의한 장간막 동맥의 수축은 이완되었다. 한편, acetylcholine에 의하여 야기되는 장간막 동맥의 이완은 A23187에 의하여 야기되는 이완에 비하여 phorbol 12-myristate 13-acetate (PMA)에 훨씬 더 민감하게 억제되었다. 내피세포의 기능과는 무관한 sodium nitroprusside에 의하여 야기되는 이완은 PMA에 의하여 영향을 받지 아니하였다. 이상의 결과로 미루어 볼때, A23187과 acetylcholine은 methylene blue에 의하여 억제되는 내피세포 의존성 이완을 야기시키고, 첨가하여 A23187은 어떤 병적 환경 아래서는 superoxide anion과 PMA에 저항성을 지닌 혈관이완성 물질을 유리하는 것으로 사료된다. 앞으로 A23187에 의하여 유리되는 혈관 이완성 물질이 superoxide anion에 의존하여 생성된 것인지에 대하여는 추후의 연구과제이다.

• The Korean Journal of Physiology and Pharmacology
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• 제1권2호
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• pp.185-193
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• 1997

The characteristics of $Na^+$-dependent cycloleucine uptake was investigated in OK cells with regard to substrate specificity and regulation by protein kinase C (PKC). Inhibition studies with different synthetic and natural amino acids showed a broad spectrum affinity to neutral amino acids regardless of their different side chains including branched or aromatic, indicating that the $Na^+$-dependent cycloleucine uptake in OK cells is mediated by System $B^o$ or System $B^o$-like transporter rather than the classical System A or ASC. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate, but not $4{\alpha}-PMA$ elicited a time-dependent biphasic stimulation of $Na^+$-dependent cycloleucine uptake, which produced early transient peak at 30 min and late sustained peak at 180min. Both the early and late stimulations by PMA were due to an increase in Vmax and not due to a change in Km. PKC inhibitors blocked both the early and late stimulation by PMA, while protein synthesis inhibitors blocked the late stimulation only. These results suggest the existence and regulation by PKC of System $B^o$ or System $B^o$-like broad spectrum transport system for neutral amino acids in OK cells.

• 약학회지
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• 제43권1호
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• pp.42-47
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• 1999

Phorbol ester, growth factors activities are mediated by unclear transcription factors, the c-Fos and c-Jun, which can regulate transcriptional activation through specific DNA sites and by forming the transcription factor AP-l, which usually mediates cell proliferation and differentiation signals. We explored effects of Pini Folium extract (API-l) on AP-l activity. Western blot analysis confirmed that API-l decreased levels of c-Fos or c-Jun protein induced by the tumor promoter Phorbol 12-myristate 13-acetate (PMA; 200 nM). Transient transfection assays with a c-fos promoter reporter construct showed that API-l decreased transcription activity by ore than 50~60%. However, treatment of API-l activity studied further. The main substances were fractionated into dichloromethane layer. Futhermore, API-l extract repressed the [$^3H$]-thymidine uptake in C6 glioma cells, indicating that this extract could be included in a new type of modulator in the mitogenesis.

• Molecules and Cells
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• 제23권2호
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• pp.138-144
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• 2007

Previously we showed that the human GM3 synthase gene was expressed during the induction of megakaryocytic differentiation in human leukemia K562 cells by phorbol 12-myristate 13-acetate (PMA). In this study we found that treatment of PMA-induced K562 cells with $G{\ddot{o}}6976$, a specific inhibitor of PKC, and U0126, an inhibitor of the extracellular signal-regulated kinase (ERK) reduced expression of GM3 synthase, whereas wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) did not. Moreover, activation of ERK and cAMP response element binding protein (CREB) was prevented by pretreatment with $G{\ddot{o}}6976$ and U0126. PMA stimulated the promoter activity of the 5'-flanking region from -177 to -83 region of the GM3 synthase gene, and mutation or deletion of a CREB site located around -143 of the promoter reduced PMA-stimulated promoter activity, as did the inhibitors $G{\ddot{o}}6976$ and U0126. Our results demonstrate that induction of GM3 synthase during megakaryocytic differentiation in PMA-stimulated human leukemia K562 cells depends upon the PKC/ERK/CREB pathway.

• Molecular & Cellular Toxicology
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• 제2권4호
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• pp.223-228
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• 2006

Several plant-derived cannabinoids and endogenous ligands for cannabinoid receptors such as 2-arachidonyl-glycerol have been known to inhibit interleukin-2 (IL-2) expression. In the present study, we utilized arachidonylethanolamide (AEA), a putative endogenous ligand for cannabinoid receptors, to determine whether AEA modulated the expression of IL-2. AEA inhibited phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io)-induced IL-2 protein secretion and mRNA expression in EL-4 mouse T-cells as determined by ELISA and RT-PCR, respectively. To further characterize the inhibitory mechanism of AEA at the transcriptional level, we performed promoter study for IL-2 gene in PMA/Io-stimulated EL-4 cells. AEA decreased the transcriptional activity of the nuclear factor of activated T-cells (NF-AT) as well as the IL-2 promoter activity. These results suggest that AEA suppresses IL-2 expression and that the inhibition is mediated, at least in part, through the down-regulation of NF-AT.

• Asian-Australasian Journal of Animal Sciences
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• 제19권7호
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• pp.958-963
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• 2006

The effect of ginsenosides (GS) on germ cell proliferation was evaluated with a chicken ovarian germ-somatic cell coculture model and the mechanism involving protein kinase C (PKC) pathway was investigated. Ovarian cells were cultured in serum-free McCoy's 5A medium and challenged with GS alone or in combinations with PKC activator (phorbol 12-myristate 13-acetate, PMA) or inhibitor ($H_7$) for 48 h. The number of germ cells was counted and the proliferating cells were identified by immunocytochemistry of proliferating cell nuclear antigen (PCNA). Results showed that GS significantly increased germ cell proliferation and this stimulating effect was further increased by PMA, but inhibited by H7, in a dose-dependent manner. Moreover, GS-elevated PCNA expression and the PCNA -labeling index of germ cells displayed similar changes with the increased numbers of germ cells. These results indicated that GS stimulated proliferation of ovarian germ cells with involvement of the PKC-mediated system.