• Title/Summary/Keyword: 4Pi microscopy

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Src Family Kinase Inhibitor PP2 Induces LC3 Conversion in a Manner That is Uncoupled from Autophagy and Increases Apoptosis in Multidrug-Resistant Cells

  • Kim, Yun-Ki;Ahn, Jun-Ho;Lee, Mi-Chael
    • Biomolecules & Therapeutics
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    • v.20 no.4
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    • pp.393-398
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    • 2012
  • Recently, we reported that defective autophagy may contribute to the inhibition of the growth in response to PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a selective SFK inhibitor, in multidrug-resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr). In this study, we demonstrated that PP2 induces LC3 conversion via a mechanism that is uncoupled from autophagy and increases apoptosis in Ras-NIH 3T3/Mdr cells. PP2 preferentially induced autophagy in Ras-NIH 3T3 cells rather than in Ras-NIH 3T3/Mdr cells as determined by LC3-I to LC3-II conversion and GFP-LC3 fluorescence microscopy. Beclin 1 knockdown experiments showed that, regardless of drug resistance, PP2 induces autophagy via a Beclin 1-dependent mechanism. PP2 induced a conformational change in Beclin 1, resulting in the enhancement of the pro-autophagic activity of Beclin 1, in Ras-NIH 3T3 cells. Further, PI3K inhibition induced by wortmannin caused a significant increase in apoptosis in Ras-NIH 3T3 cells, as demonstrated by flow cytometric analysis of Annexin V staining, implying that autophagy inhibition through PI3K increases apoptosis in response to PP2 in Ras-NIH 3T3 cells. However, despite the fact that wortmannin abrogates PP2-induced GFP-LC3 punctae formation, some LC3 conversion remains in Ras-NIH 3T3/Mdr cells, suggesting that LC3 conversion may occur in an autophagy-independent manner. Taken together, these results suggest that PP2 induces LC3 conversion independent of PI3K, concomitant with the uncoupling of LC3 conversion from autophagy, in multidrug-resistant cells.

Colorless Copolyimide Films: Thermo-mechanical Properties, Morphology, and Optical Transparency (무색 투명한 폴리이미드 공중합체 필름 : 열적-기계적 성질, 모폴로지, 및 광학 투명성)

  • Jin, Hyo-Seong;Chang, Jin-Hae
    • Polymer(Korea)
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    • v.32 no.3
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    • pp.256-262
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    • 2008
  • Copolyimides containing pendant trifluoromethyl ($CF_3$) groups were synthesized from 4,4'-(hexafluoroisopropylidene)diphthalic anhydride (6FDA) and bis[4-(3-aminophenoxy)phenyl]sulfone (BAPS) with various concentrations of 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane(BAPP) to poly(amic acid)(PAA), followed by thermal imidization. These copolyimides were readily soluble in N,N'-dimethylacetamide (DMAc) and could be solution-cast into a flexible and tough film. The thermomechanical properties, morphology and an optical transparency of the copolyimide films were determined using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), wide angle X-ray diffraction (XRD), scanning electron microscopy (SEM), universal tensile machine (UTM), and a UV-Vis spectrometer. The cast copolyimide films exhibited high optical transparency with a cut-off wavelength (${\lambda}_0$) of $275{\sim}319\;nm$ in UV-vis absorption and a low yellow index(YI) value of $3.65{\sim}10.37$. The thermo-mechanical properties of copolyimide films were enhanced linearly with increasing a BAPP content. In contrast, the optical transparency of the copolyimide films was found to get worse with increasing a BAPP content.

Vanillic Acid Stimulates Anagen Signaling via the PI3K/Akt/β-Catenin Pathway in Dermal Papilla Cells

  • Kang, Jung-Il;Choi, Youn Kyung;Koh, Young-Sang;Hyun, Jin-Won;Kang, Ji-Hoon;Lee, Kwang Sik;Lee, Chun Mong;Yoo, Eun-Sook;Kang, Hee-Kyoung
    • Biomolecules & Therapeutics
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    • v.28 no.4
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    • pp.354-360
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    • 2020
  • The hair cycle (anagen, catagen, and telogen) is regulated by the interaction between mesenchymal cells and epithelial cells in the hair follicles. The proliferation of dermal papilla cells (DPCs), mesenchymal-derived fibroblasts, has emerged as a target for the regulation of the hair cycle. Here, we show that vanillic acid, a phenolic acid from wheat bran, promotes the proliferation of DPCs via a PI3K/Akt/Wnt/β-catenin dependent mechanism. Vanillic acid promoted the proliferation of DPCs, accompanied by increased levels of cell-cycle proteins cyclin D1, CDK6, and Cdc2 p34. Vanillic acid also increased the levels of phospho(ser473)-Akt, phospho(ser780)-pRB, and phospho(thr37/46)-4EBP1 in a time-dependent manner. Wortmannin, an inhibitor of the PI3K/Akt pathway, attenuated the vanillic acid-mediated proliferation of DPCs. Vanillic acid-induced progression of the cell-cycle was also suppressed by wortmannin. Moreover, vanillic acid increased the levels of Wnt/β-catenin proteins, such as phospho(ser9)-glycogen synthase kinase-3β, phospho(ser552)-β-catenin, and phospho(ser675)-β-catenin. We found that vanillic acid increased the levels of cyclin D1 and Cox-2, which are target genes of β-catenin, and these changes were inhibited by wortmannin. To investigate whether vanillic acid affects the downregulation of β-catenin by dihydrotestosterone (DHT), implicated in the development of androgenetic alopecia, DPCs were stimulated with DHT in the presence and absence of vanillic acid for 24 h. Western blotting and confocal microscopy analyses showed that the decreased level of β-catenin after the incubation with DHT was reversed by vanillic acid. These results suggest that vanillic acid could stimulate anagen and alleviate hair loss by activating the PI3K/Akt and Wnt/β-catenin pathways in DPCs.

β-Elemene Induces Apoptosis in Human Renal-cell Carcinoma 786-0 Cells through Inhibition of MAPK/ERK and PI3K/Akt/mTOR Signalling Pathways

  • Zhan, Yun-Hong;Liu, Jing;Qu, Xiu-Juan;Hou, Ke-Zuo;Wang, Ke-Feng;Liu, Yun-Peng;Wu, Bin
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.6
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    • pp.2739-2744
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    • 2012
  • Background: Renal-cell carcinoma (RCC) is resistant to almost all chemotherapeutics and radiation therapy. ${\beta}$-Elemene, a promising anticancer drug extracted from a traditional Chinese medicine, has been shown to be effective against various tumors. In the present study, anti-tumor effects on RCC cells and the involved mechanisms were investigated. Methods: Human RCC 786-0 cells were treated with different concentrations of ${\beta}$-elemene, and cell viability and apoptosis were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Protein expression was assayed by western blotting. Autophagy was evaluated by transmission electron microscopy. Results: ${\beta}$-Elemene inhibited the viability of 786-0 cells in a dose- and time-dependent manner. The anti-tumor effect was associated with induction of apoptosis. Further study showed that ${\beta}$-elemene inhibited the MAPK/ERK as well as PI3K/Akt/mTOR signalling pathways. Moreover, robust autophagy was observed in cells treated with ${\beta}$-elemene. Combined treatment of ${\beta}$-elemene with autophagy inhibitors 3-methyladenine or chlorochine significantly enhanced the anti-tumor effects. Conclusions: Our data provide first evidence that ${\beta}$-elemene can inhibit the proliferation of RCC 786-0 cells by inducing apoptosis as well as protective autophagy. The anti-tumor effect was associated with the inhibition of MAPK/ERK and PI3K/Akt/mTOR signalling pathway. Inhibition of autophagy might be a useful way to enhance the anti-tumor effect of ${\beta}$-elemene on 786-0 cells.

Development of Cryptosporidium parvum in cell culture (세포배양에서 Cryptosporidium parvum의 발육)

  • Kim, Bo-sook;Joo, Hoo-don;Wee, Sung-hwan;Kim, Tae-jong
    • Korean Journal of Veterinary Research
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    • v.35 no.2
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    • pp.317-326
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    • 1995
  • The purpose of this study was to establish a method for in vitro culture of C parvum isolated in Korea by determination of suitable cell model to complete development of this parasite. The result obtained were summerized as follows: 1. To determine the most suitable cell line, six types of cell line were examined by microscopy. All cell lines were infected with C parvum and showed the highest infection score in HmLu cells. 2. The staining methods including DMSO-modified acid-fast(A-F) stain, hematoxylin-eosin(H & E) stain and immunofluorescence antibody(IFA) stain were applied to examine the infection of C parvum in cell culture. These staining methods were possible to examine the infection of C parvum in cell culture. The most sensitive one was IFA staining technique. 3. Developmental stages of C parvum in HmLu cell were observed. After the initial 8 hour incubation period, some trophozoites were observed. The meronts and gametes were appeared at 24-48 hour post inoculation(PI), and oocysts were observed firstly at 48-72 hour PI. 4. In H & E stain, the parasite appeared as basophilic within parasitophorous vacuole membrane(PVM) and lying in cytoplasm at near the nucleus of the host cells. It was able to distinguish the type I, type II meronts and gametes. 5. In DMSO-modified acid-fast stain, specific stained parasites were appeared firstly after 48 hour PI. The parasites were showed with different degrees of staining bright red color within PVM. 6. The endogenous stages of parasites in HmLu cell recovered at 48, 96, 120 and 144 hour after inoculation were reacted with rabbit immunized serum in immunofluorescence antibody and avidin-biotin complex peroxidase staining technique.

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Fabrication of Protein A-Viologen Hetero Langmuir- Blodgett Film for Fluorescence Immunoassay

  • Lee, Woochang;Chun, Bum-Suk;Oh, Byung-Keun;Lee, Won-Hong;Park, Jeong-Woo
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.9 no.4
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    • pp.241-244
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    • 2004
  • Protein A molecular thin film was fabricated as a platform of antibody-based biosensor. For the immobilization of the protein A thin film, a viologen multilayer was built up using the Langmuir-Blodgett (LB) technique, and then, protein A was adsorbed on the viologen LB film by an electrostatic interaction force, which was formed as a hetero-film structure. For the deposition of viologen, surface pressure area ($\pi$-A) isotherm was investigated. The fabricated protein A-viologen hetero LB film was investigated using atomic force microscopy (AFM). Using the developed molecular film, antibody immobilization and fluorescence measurement was carried out.

Study on morphology of silsesquioxane based polymer LB films by using AFM (AFM을 이용한 Silsesquioxane based polymer의 LB막 모폴로지 관찰)

  • Lee, Ji-Yoon;Yang, Chang-Heon;Kim, Chung-Kyun;Park, Jae-Chul;Kwon, Young-Soo
    • Proceedings of the KIEE Conference
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    • 2008.07a
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    • pp.1293-1294
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    • 2008
  • The Langmuir-Blodgett (LB) method has been one of the most suitable techniques for fabricating organic thin films with well-controlled structures, compositions and thickness at the molecular level. We investigated the surface activity of dendrimer films at air-water interface by ${\pi}$-A isotherm. Also, we attempted to fabricate a 1G(4,3)-chloride dendrimer LB films. And their surface morphologies were observed by atomic force microscopy (AFM).

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A study on Electrical Properties of the Polyamic Acid Alkylamine Salts(PAAS) LB films (Polyamic Acid Alkylamine Salts(PAAS) LB 박막의 전기적 특성에 관한 연구)

  • 이호식;이원재;김태완;강도열
    • Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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    • 1997.11a
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    • pp.298-301
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    • 1997
  • This paper describes the thermally stimulated current(TSC) measurements arachidic acid(AA) and polyamic acid alkylamine salts(PAAS) LB film, which is a precursor of polyimide(PI). The measurements were performed from room temperature to about 250$^{\circ}C$ and the temperature was increased at a rate of 0.02 $^{\circ}C$/s linearly[4]. It shows that peaks of TSC are observed at about 80$^{\circ}C$ in the arachidic acid and about 80$^{\circ}C$, 160$^{\circ}C$ in the PAAS LB films. The DSC and TGA of PAAS, arachidic acid are measure. Monolayer phases on the water subphase such as Langmuir(L) films and the phase transitions from gas phase to solid phase via liquid phase are observed using Brewster angle microscopy(BAM). BAM is also used to observe the Langemuir-Blodgett(LB) films.

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Preparation of Aminosiloxane-grafted Poly(imidesiloxane) Copolymer and its Morphology and Adhesive Properties in Film (아미노실록산이 그래프팅된 폴리(이미드실록산) 공중합체 제조와 필름 모폴로지 및 점착 특성 연구)

  • Lee, Ji Mok;Kwon, Eunjin;Lee, Sunyoung;Jung, Hyun Min
    • Polymer(Korea)
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    • v.37 no.4
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    • pp.547-552
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    • 2013
  • Polyimide (PI) containing carboxyl functional group was prepared and reacted with diaminosiloxane during high temperature film casting. The morphology of resulting film was observed by using transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX), which revealed that globular 100 nm-sized PI domains and continuous polysiloxane phase were formed. X-ray photoelectron spectroscopy (XPS) study indicated that air-film interface mainly consisted of polysiloxane blocks. Poly(imidesiloxane) thin layer was thermostable until $400^{\circ}C$ and its pressure- sensitive adhesive property was retained up to $300^{\circ}C$. The comparative experiments revealed that grafting between carboxyl groups of polyimide and aminosiloxane was crucial for formation of microdomain structure and pressure-sensitive adhesive property.

Surface Topography and Photoluminescence of Chemically Etched Porous Si (화학식각법에 의해 형성된 다공질실리콘의 표면형상 및 발광특성)

  • Kim, Hyeon-Su;Min, Seok-Gi
    • Korean Journal of Materials Research
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    • v.4 no.4
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    • pp.379-384
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    • 1994
  • Room-temperature photoluminescent porous Si has been formed by etching Si wafer u-ith the solution of $HF:HNO_{3}: H_{2}O$=l : 5 : 10. We have observed photoluminescence(PL) spectra similar to those reported recently for porous-Si films formed by anodic etching with HF solutions. We have also investigated the dependence of PI, spectra on the etching time which was varied from 1 to 10 minutes. We found that 5-minute etching gave us the strongest PL intensity. We also found by atomic force microscopy( AFM) measurements that the surface fearure size became smaller for longer etching time and the average feature size of the etched Si wafer for 5-minute was about 1, 500~2, 000$\AA$. This indicates that the surface feature of the etched porous Si affects the PL intensity of the sample.

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