• Natural Product Sciences
• /
• 제3권1호
• /
• pp.29-37
• /
• 1997

Protein Kinase C (PKC) is generally believed to play a central role in signal transduction, cellular growth control, gene expression, and tumor promotion. And it has been suggested that inhibitors of PKC might play important roles for the prevention and treatment of cancer. In order to investigate the possible inhibitors of PKC from natural products, PKC receptor binding assay was performed using bovine brain particulate as a source of PKC and the amount of $[^3H]Phorbol$ 12,13-dibutyrate (PDBu) bound to PKC was measured in the presence of test materials. Total methanol extracts from 100 kinds of natural products were partitioned into 3 fractions (n-hexane, ethyl acetate and aqueous layer) and their binding ability to the regulatory domain of PKC was evaluated. The ethyl acetate fractions of Morus alba $(roots,\;IC_{50}:\;156.6\;{\mu}g/ml)$, Rehmannia glutinosa $(roots,\;IC_{50}:\;134.3\;{\mu}g/ml)$, Lysimachia foenum-graecum $(roots,\;IC_{50}:\;167.8\;{\mu}g/ml)$, Polygonum cuspidata $(roots,\;IC_{50}:\;157.3\;{\mu}g/ml)$, Cnidium officinale $(aerial\;parts,\;IC_{50}:\;145.2\;{\mu}g/ml)$, and the hexane $(IC_{50}:\;179.3\;{\mu}g/ml)$ and the EtOAc fraction of Symplocarpus nipponicus $(roots,\;IC_{50}:\;155.9\;{\mu}g/ml)$ showed inhibitory activity of $[^3H]PDBu$ binding to PKC.

• Mycobiology
• /
• 제47권4호
• /
• pp.473-482
• /
• 2019

Rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most important diseases in rice production. PAS (period circadian protein, aryl hydrocarbon receptor nuclear translocator protein, single-minded protein) domains are known to be involved in signal transduction pathways, but their functional roles have not been well studied in fungi. In this study, targeted gene deletion was carried out to investigate the functional roles of the PAS-containing gene MoPAS1 (MGG_02665) in M. oryzae. The deletion mutant ΔMopas1 exhibited easily wettable mycelia, reduced conidiation, and defects in appressorium formation and disease development compared to the wild type and complemented transformant. Exogenous cAMP restored appressorium formation in ΔMopas1, but the shape of the restored appressorium was irregular, indicating that MoPAS1 is involved in sensing the hydrophobic surface. To examine the expression and localization of MoPAS1 in M. oryzae during appressorium development and plant infection, we constructed a MoPAS1:GFP fusion construct. MoPAS1:GFP was observed in conidia and germ tubes at 0 and 2 h post-infection (hpi) on hydrophobic cover slips. By 8 hpi, most of the GFP signal was observed in the appressoria. During invasive growth in host cells, MoPAS1:GFP was found to be fully expressed in not only the appressoria but also invasive hyphae, suggesting that MoPAS may contribute to disease development in host cells. These results expand our knowledge of the roles of PAS-containing regulatory genes in the plant-pathogenic fungus M. oryzae.

• 한국발생생물학회지:발생과생식
• /
• 제17권4호
• /
• pp.299-309
• /
• 2013

Transforming growth factor (TGF) family is well known to induce the chondrogenic differentiation of mesenchymal stem cells (MSC). However, the precise signal transduction pathways and underlying factors are not well known. Thus the present study aims to evaluate the possible role of C2 domain in the chondrogenic differentiation of human mesenchymal stem cells. To this end, 145 C2 domains in the adenovirus were individually transfected to hMSC, and morphological changes were examined. Among 145 C2 domains, C2 domain of protein kinase C eta ($PKC{\eta}$) was selected as a possible chondrogenic differentiation factor for hMSC. To confirm this possibility, we treated $TGF{\beta}3$, a well known chondrogenic differentiation factor of hMSC, and examined the increased-expression of glycosaminoglycan (GAG), collagen type II (COL II) as well as $PKC{\eta}$ using PT-PCR, immunocytochemistry and Western blot analysis. To further evaluation of C2 domain of $PKC{\eta}$, we examined morphological changes, expressions of GAG and COL II after transfection of $PKC{\eta}$-C2 domain in hMSC. Overexpression of $PKC{\eta}$-C2 domain induced morphological change and increased GAG and COL II expressions. The present results demonstrate that $PKC{\eta}$ involves in the TGF-${\beta}3$-induced chondrogenic differentiation of hMSC, and C2 domain of $PKC{\eta}$ has important role in this process.

• 약학회지
• /
• 제44권1호
• /
• pp.52-59
• /
• 2000

An exceptionally conserved sequence that is shared among most G protein-coupled neurotransmitter receptors is an aspartate-arginine-tyrosine triplet that is located at the second cytoplasmic domain. Using the ml subtype of muscarinic acetylcholine receptors as an example, a point mutation of the arginine residue at position 123 into asparagine was induced. This mutation resulted in a complete blockade of the carbachol-induced increases of PI hydrolysis and intracellular $Ca^2$$^{+}$ level, in spite of the expression of the wild-type and mutant receptors at similar concentrations in Chinese hamster ovary cells. In marked contrast, the muscarinic agonist carbachol induced concentration-dependent enhancement of the activity of NO synthase at mutant ml receptors although the enhancement was significantly smaller than at wild-type ml receptors. These data suggest that this highly conserved arginine residue plays an important role in coupling of muscarinic receptors to the second messenger systems and the presence of alternate mechanisms of activation of neuronal NO synthase which might be operative in the absence of large changes in the concentration of cellular $Ca^{2+}$.2+/.

• Molecules and Cells
• /
• 제28권4호
• /
• pp.365-368
• /
• 2009

Toll-like receptors (TLRs) play an important role in host defense by sensing invading microbial pathogens and initiating innate immune responses. The stimulation of TLRs by microbial components triggers the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-${\beta}$ (TRIF)-dependent downstream signaling pathways. Isoliquiritigenin (ILG), an active ingredient of Licorice, has been used for centuries to treat many chronic diseases. ILG inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-${\kappa}B$ kinase. However, it is not known whether ILG inhibits the TRIF-dependent pathway. To evaluate the therapeutic potential of ILG, we examined its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by several agonists. ILG inhibited nuclear factor-${\kappa}B$ and interferon regulatory factor 3 activation induced by lipopolysaccharide or polyinosinic-polycytidylic acid. ILG inhibited the lipopolysaccharide-induced phosphorylation of interferon regulatory factor 3 as well as interferon-inducible genes such as interferon inducible protein-10, and regulated activation of normal T-cell expressed and secreted (RANTES). These results suggest that ILG can modulate TRIF-dependent signaling pathways of TLRs, leading to decreased inflammatory gene expression.

• Radiation Oncology Journal
• /
• 제17권4호
• /
• pp.299-306
• /
• 1999

목 적 : Ataxia-Telangiectasia (AT) 증은 여러 가지 유전적 결함을 갖는 질병으로 방사선 민감도가 비정상적으로 상승되어 있는 것이 특징이다 AT 환자에서 공통적으로 존재하는 ATM 유전자는 현재까지 방사선 신호전달에 관여하는 것으로 알려진 Pl-3 kinase와 유사한 구조임이 알려져 ATM이 방사선 신호전달경로에 중요한 작용을 할 것으로 추정하게 되었다. 본 연구에서는 AT 세포와 정상세포에 PKCI를 과발현 시킴으로써 방사선 신호전달에 관여하는 PKC를 억제하여 이것이 방사선 민감도에 미치는 영향을 관찰하고, 방사선에 의해 유도되는 early response gene인 c-fos transcription의 차이를 측정하여 ATM과 PKCI에 의한 신호전달이 c-fos 유전자 전사에 미치는 영향을 분석하고자 하였다. 대상 및 방법 : PKCI expression vector를 작제한 후 정상세포인 LM217과 AT세포인 AT5BIVA에 transfection 시킨 후 plasmid의 genomic DNA에 결합된 것은 polymerase chain reaction (PCR) 방법으로 확인하였고 PKCI의 mRNA 발현 여부는 northern blotting으로 확인하였다. 방사선 민감도는 아포토시스로 측정하였으며 PKCI가 과발현된 각 세포주에 5 Gy의 방사선을 조사한 후 48시간에 세포를 모아 TUNEL방법으로 아포토시스 세포의 수를 측정하였다. c-fos 유전자의 전사는 reporter 유전자로 c-fos CAT plsmid를 $\beta$-gal expression vector와 같이 각 세포주에 transfection 시키고 36시간이 지난 후 CAT assay를 하여 activity를 측정하고 동시에 $\beta$-gal assay를 시행하여 transfection 효율을 보정해 주었다. PKCI, Ras의 영향을 보기 위하여는 PKCI, Ras expression vector와 c-fos CAT plasmid를 cotransfection하고 CAT activity로 측정 하였다. 결 과 : 이 실험의 결과 LM과 AT 세포에서 PKCI가 방사선 민감도에 미치는 영향과 c-fos 전사에 미치는 영향을 처음으로 보여주었다. PKCI의 과발현이 LM 세포에서는 방사선 민감도를 증가시켰지만 AT세포에서는 오히려 약간 감소시키는 작용을 나타내었다. c-fos 전사는 AT 세포에서 LM 세포에 비하여 70배 낮게 나타났는데 PKCI가 과발현 됨으로써 LM 에서는 c-fos의 전사가 감소되었지만 AT 세포에서는 영향이 없었다. Ras 단백으로 c-fos를 유도시키고 여기에 PKCI 발현 백터를 contransfection 하면 LM세포에서는 induction 이 감소되었지만 AT 세포에서는 영향이 없었다. 즉 LM과 AT 세포에서의 PKCI에 의한 반응의 차이는 Ras와 관련된 signal transduction pathway라는 것을 알 수 있었다. 결 론 : PKCI는 정상세포에서는 방사선에 의한 세포 손상을 증가시키지만 AT 세포에서는 별 영향을 보이지 않는 것을 알 수 있었으며, 두 세포간의 이러한 차이는 c-fos proto-oncogene의 전사차이로 설명할 수 있겠다. 이러한 차이가 AT 세포의 방사선 민감도의 한 원인일 것으로 생각된다.

• 미생물학회지
• /
• 제47권1호
• /
• pp.1-6
• /
• 2011

암의 유전자치료에서 암세포로의 선택적 유전자전달 매체의 부족은 치료효과의 감소를 야기하는 문제이다. 본 연구에서는 난소암 유전자치료의 효율을 높이기 위한 목적으로 난소암세포로 선택적인 유전자전달을 하도록 개량된 아데노바이러스 벡터를 제조하고, 그 효율성을 난소암세포주를 이용하여 조사하였다. 난소암세포에 과다발현하는 분자인 ErbB receptor를 표적하도록 아데노바이러스 외피단백질 fiber에 ErbB receptor에 대한 ligand인 heregulin으로부터 유래한 펩티드를 부착하였다. 53개의 아미노산으로 구성된 외부 펩티드를 fiber에 부착하였을 때 바이러스 감염에 중요한 기능을 하는 fiber 삼량체 구조 형성에 영향을 미치지 않았다. Fiber를 조작한 개량 아데노바이러스는 야생형 fiber를 가진 1세대 아데노바이러스 벡터에 비해 선택적으로 난소암으로 유전자를 전달하는 비율이 증가하였다. 특히 항암제에 저항성을 가진 난소암세포주 OVCAR3에서 유전자전달 효율이 약 5배 증가되었다. 따라서 난소암의 유전자치료에서 개량된 아데노바이러스로 치료 유전자를 전달하면 치료의 효율성을 향상시킬 수 있을 것이다.

• Toxicological Research
• /
• 제20권3호
• /
• pp.179-185
• /
• 2004

Helicobacter pylori (H. pylori) infects more than half of the people in the world as a major microbe to cause most of gastric diseases. Recently, cytotoxin associated-antigen A (CagA) is believed as one of the most important virulence factors of H. pylori. Molecular toxicological pathway of CagA is necessary to investigate for understanding the pathological and toxicological aspects of H. pylori, since this virulence protein harasses intercellular processes of host cells to get profit for the survival of H. pylori. CagA is coded from cag pathogenicity island (cag PAI) and translocated into host cells by Type 4 secretion system (TFSS). Tyrosine phosphorylation of CagA targets Src homology 2-containing phosphotyrosine phosphatase (SHP-2) to form a CagA-SHP-2 complex. This complex depends on the similarity of sequence between EPIYA motif and Src homology 2 domain (SH2 domain) of CagA. The generation of growth factors is an essential role of CagA in protecting and healing gastric mucosa for the survival of H. pylori. On the other hand, the activation of IL-8 by CagA induces neutrophils generating inflammation and free radicals. Indeed, free radicals are well known carcinogen to induce DNA damage. In addition, the transduction of mitogen-activation signal by CagA is one of the interesting features to understand how to cause cancer. The relationship between cancer and inflammation with CagA was mainly discussed in this review.

• BMB Reports
• /
• 제53권6호
• /
• pp.335-340
• /
• 2020

Since cancer is the leading cause of death worldwide, there is an urgent need to understand the mechanisms underlying cancer progression and the development of cancer inhibitors. Signal transducer and activator of transcription 3 (STAT3) is a major transcription factor that regulates the proliferation and survival of various cancer cells. Here, dual-specificity phosphatase 3 (DUSP3) was identified as a regulator of STAT3 based on an interaction screening performed using the protein tyrosine phosphatase library. DUSP3 interacted with the C-terminal domain of STAT3 and dephosphorylated p-Y705 of STAT3. In vitro dephosphorylation assay revealed that DUSP3 directly dephosphorylated p-STAT3. The suppressive effects of DUSP3 on STAT3 were evaluated by a decreased STAT3-specific promoter activity, which in turn reduced the expression of the downstream target genes of STAT3. In summary, DUSP3 downregulated the transcriptional activity of STAT3 via dephosphorylation at Y705 and also suppressed the migratory activity of cancer cells. This study demonstrated that DUSP3 inhibits interleukin 6 (IL-6)/STAT3 signaling and is expected to regulate cancer development. Novel functions of DUSP3 discovered in IL-6/STAT3 signaling regulation would help expand the understanding of cancer development mechanisms.

• 생명과학회지
• /
• 제22권4호
• /
• pp.456-465
• /
• 2012

유전자 재조합 이형이합체 인간 뼈 형성촉진인자(rhBMP)들은 뼈 재생을 위한 조직공학연구에 중요한 요소들이다. 그러나 실제 뼈 재생에 관한 연구를 수행함에 이들을 이용하는 것은 뼈 형성 촉진인자들이 짧은 반감기를 가지며 비교적 고농도의 단백질을 사용해야 하기 때문에 그만큼 많은 비용이 소요된다는 문제점을 가지고 있다. 이러한 한계를 뛰어넘기 위하여, 본 연구에서는 단백질 전달 서열(PTD)이 융합된 인간 뼈 형성촉진인자 2와 7이 이형이합체 유전자 재조합 단백질(rhBMP2/7-PTD)을 발현하는 세포주를 확립하였다. 이형이합체의 형성은 BMP 2와 BMP 7 단백질 및 PTD 영역의 사이에 각각 4개의 glycine 아미노산 염기서열이 첨가될 수 있도록 하여 각 단백질의 folding이 자유롭도록 디자인하였다. 이렇게 개발된 세포주는 고농도의 rhBMP2/7-PTD을 지속적으로 발현하여 배양액 내로 분비함으로 조직공학용 연구 및 개발에 효율적으로 이용 할 수 있다. 이상의 세포주에서 발현된 rhBMP2/7-PTD 단백질은 뼈 세포분화 유도를 확인 할 수 있는 ALP 활성을 나타냄으로써 뼈 성장촉진 단백질로서 생물학적인 활성을 가지고 있음을 보였다. 본 연구의 결과로 개발된 rhBMP2/7-PTD 형질전환 세포주는 향후 뼈 조직 재생과 같은 연구에 중요하고 효과적인 도구로 이용될 수 있을 것으로 사료된다.