• Title/Summary/Keyword: and western blot analysis

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Effects of Essential Oils Extracted from Cnidii Rhizoma on Differentiation and Adipogenesis in 3T3-L1 Adiopocytes (천궁(川芎)의 정유 추출물이 3T3-L1 세포의 분화 및 지방 생성에 미치는 영향)

  • Choi, Soo-Min;Kim, So-Young;Park, Na-Ri;Kim, Jung-Min;Yang, Doo-Hwa;Woo, Chang-Hoon;Kim, Mi-Ryeo;An, Hee-Duk
    • Journal of Korean Medicine Rehabilitation
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    • v.28 no.3
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    • pp.13-25
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    • 2018
  • Objectives We investigated anti-obesity effects of essential oils extracted from Cnidii Rhizoma (CR) in immature adipocytes to magnify it's clinical therapeutic usage. Methods Essential oil of CR was extracted with ethyl acetate or petroleum ether and through steam distillation, respectively. Oil red-O staining for monitoring its inhibition effect on adipogenesis and differentiation in murine 3T3-L1 adipocytes and 3-(4,5-methylthiazol-2-yl)-2,5-diphenyletetra zolium bromide (MTT) assay for cell safety were done. Also phospho-adenosine monophosphate (AMP)-activted protein kinase (P-AMPK), AMP-activated protein kinase, phospho-acetyl-CoA carboxylase (P-ACC), acetyl-CoA carboxylase, peroxisome proliferator-activated receptor-${\alpha}$ (PPAR-${\alpha}$), peroxisome proliferator-activated receptor-${\gamma}$ (PPAR-${\gamma}$) and CCAAT/enhancer binding protein ${\alpha}$ (C/EBP-${\alpha}$) expressions as obesity-related factors were measured by western blot analysis. Results Protein expressions of P-AMPK, P-ACC and PPAR-${\alpha}$ were increased in essential oils-treated adipocytes compared to those of control group, respectively. Furthermore, protein expressions of PPAR-${\gamma}$ and C/EBP-${\alpha}$ were decreased in essential oils-treated adipocytes compared to those of control group, respectively. Conclusions These results demonstrate that essential oils of CR inhibit adipogenesis and differentiation. Also they promote the oxidation of fatty acids in adipocytes. Thus, results suggest that essential oils of CR could be used as a valuable material for anti-obesity therapeutics via control of lipid metabolism.

Expression of Nerve Growth Factor in Estradiol Valerate-induced Polycystic Ovary Syndrome (Estradiol valerate에 의해 유발되는 다낭성난소증후군의 Nerve Growth Factor 변화)

  • Kim, Se-Eun;Lim, Sung-Chul;Jeong, Moon-Jin;Choi, Baik-Dong;Kim, Seung-Hyun;Ko, A-Ra;Seo, Seung-Yong;Kang, Seong-Soo;Moon, Chang-Jong;Kim, Jong-Choon;Kim, Sung-Ho;Bae, Chun-Sik
    • Journal of Veterinary Clinics
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    • v.28 no.4
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    • pp.403-407
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    • 2011
  • A form of polycystic ovary (PCO) resembling some aspects of the human PCO syndrome (PCOS) can be induced in rats by a single injection of estradiol valerate (EV). An increase in sympathetic outflow to the ovary precedes, by several weeks, the appearance of cysts, suggesting the involvement of a neurogenic component in the pathology of this ovarian dysfunction. To test the hypotheses that the change in sympathetic tone is related to an augmented production of hippocampal and/or ovarian nerve growth factor (NGF), and that this abnormally elevated production of NGF contributes to the induction of PCOS induced by EV. The animals were sacrificed after PCOS induction and the ovaries and hippocampus were sectioned and compared to the normal control. The expression of NGF was measured by immunohistochemical staining and Western blot analysis in the ovaries and hippocampus. EV-induced PCOS showed significant increase of ovarian NGF expression. Immunohistochemical expression of NGF was confined to the follicular cells and interstitial cells. Hippocampal NGF expression was not significantly changed. In conclusion EV-induced PCOS was related to the ovarian sympathetic activation which was mediated by NGF.

Construction of a Transgenic Plant to Develop a New Method for the Isolation of Calmodulin-Binding Proteins (새로운 방법을 이용한 칼모둘린 결합 단백질 분리를 위한 형질 전환 식물체의 구축)

  • Kim, Sun-Ho;Lee, Kyung-Hee;Kim, Kyung-Eun;Jung, Mi-Soon;Lim, Chae-Oh;Lee, Shin-Woo;Chung, Woo-Sik
    • Journal of Life Science
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    • v.17 no.9 s.89
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    • pp.1177-1181
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    • 2007
  • Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety CaM-binding proteins (CaMBPs). Because eukaryotes have multiple CaMBPs, it is important to isolate and characterize them in different tissues and conditions. So far a number of CaMBPs have been identified through classical screening methods. Many classes of proteins have been predicted to bind CaMs based on their structural homology with already known targets. In an effort to develop a method for large-scale analysis of CaMBPs in Arabidopsis, we have generated a transgenic plants overexpressing AtCaM2-GFP. We performed protein pull-down assay to test whether exogenously expressed AtCaM2-GFP proteins can interact with CaMBPs. The exogenously expressed AtCaM2-GFP could strongly interact with a CaMBP, AS1 protein. This result suggests that AtCaM2-GFP in transgenic plants may interact with many CaMBPs in plant cell. Therefore, we will be able to isolate kinds of CaMBPs by using these transgenic plants in many different tissue and environments.

The Effect of Polycan-Calcium Gluconate Complex on Inflammatory Mediators from Periodontitis Patients (Polycan-Calcium Gluconate 복합제에 의한 염증인자의 감소 효과)

  • Kim, Ji-Hye;Kim, Ki-Rim;Jin, Hye-Jung;Im, Sang-Uk;Song, Keun-Bae;Choi, Youn-Hee
    • Journal of dental hygiene science
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    • v.14 no.2
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    • pp.223-229
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    • 2014
  • The aim of this study was to examine the effect of polycan-calcium gluconate complex on the levels of immune and inflammatory mediators in gingival crevicular fluid and serum from patients with periodontitis. A total of 39 patients with periodontitis took placebo (placebo group) or polycan-calcium gluconate complex (treatment group) twice a day for 4 weeks. At baseline and 4 weeks, gingival crevicular fluid (GCF) and blood was collected from each subjects. The secretion level of interleukin $(IL)-1{\beta}$ in GCF was measured by enzyme-linked immunosorbent assay (ELISA). Western blot analysis was conducted to determine the expression of matrix metalloproteinase (MMP)-8 and tumor necrosis factor $(TNF)-{\alpha}$ in GCF. Serum samples were analysed for MMP-8 by ELISA and C-reactive protein (CRP) by turbidimetric immuno assay. MMP-8 and $TNF-{\alpha}$ were significantly decreased in the treatment group at 4 weeks. The level of $IL-1{\beta}$ in the treatment group was significantly lower than those of the placebo group. No differences were observed in CRP levels. Taken together, these data indicate that taking of polycan-calcium gluconate complex led to reduction of inflammatory biomarkers in serum and GCF of periodontitis patients.

Cell Migratory Induction by Expression of Angiogenin and Vascular Endothelial Growth Factor in Resveratrol Treated HeLa Cells (Resveratrol 처리한 HeLa세포에서 angiogenin과 vascular endothelial growth factor 발현유도에 따른 세포이동촉진)

  • Joe, I-Seul;Jeong, Sin-Gu;Cho, Goang-Won
    • Journal of Life Science
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    • v.24 no.4
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    • pp.337-342
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    • 2014
  • Resveratrol (RSV), a natural polyphenolic compound, is a modulator for cell division and cell migration, and has diverse beneficial properties. Angiogenin (ANG) and vascular endothelial growth factor (VEGF) are considered to be important mechanisms for cell proliferation, angiogenesis, the formation of tubular structures, and migration. In this study, we investigated whether RSV has a migratory effect in HeLa cells. When cells were treated with $0{\sim}50{\mu}M$ of RSV for 24 hr, the expression of ANG and VEGF was significantly increased in a dose dependent manner measured by real-time PCR. Similarly, we performed time dependent experiments for $50{\mu}M$ RSV treated cells and identified the optimized time at 24 hr. The increased expression in RSV treated cells was confirmed by Western blot analysis. To examine the toxic effects of RSV at the determined conditions, MTT assays were performed. The viabilities were unchanged for $0{\sim}50{\mu}M$ RSV treated cells, while they decreased at $100{\mu}M$ RSV. To examine the effect of migration in RSV treated cells, we performed a wound-healing assay. The migratory rates were significantly enhanced in the RSV treated group. In this study, we found that RSV induces an increase in the expression of migration factors ANG, VEGF, and enhances cell migration for the determined conditions.

The Effects of High Temperature on Infection by Potato virus Y, Potato virus A, and Potato leafroll virus

  • Chung, Bong Nam;Canto, Tomas;Tenllado, Francisco;Choi, Kyung San;Joa, Jae Ho;Ahn, Jeong Joon;Kim, Chun Hwan;Do, Ki Seck
    • The Plant Pathology Journal
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    • v.32 no.4
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    • pp.321-328
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    • 2016
  • We examined the effects of temperature on acquisition of Potato virus Y-O (PVY-O), Potato virus A (PVA), and Potato leafroll virus (PLRV) by Myzus persicae by performing transmission tests with aphids that acquired each virus at different temperatures. Infection by PVY-O/PVA and PLRV increased with increasing plant temperature in Nicotiana benthamiana and Physalis floridana, respectively, after being transmitted by aphids that acquired them within a temperature range of $10-20^{\circ}C$. However, infection rates subsequently decreased. Direct qRT-PCR of RNA extracted from a single aphid showed that PLRV infection increased in the $10-20^{\circ}C$ range, but this trend also declined shortly thereafter. We examined the effect of temperature on establishment of virus infection. The greatest number of plants became infected when N. benthamiana was held at $20^{\circ}C$ after inoculation with PVY-O or PVA. The largest number of P. floridana plants became infected with PLRV when the plants were maintained at $25^{\circ}C$. PLRV levels were highest in P. floridana kept at $20-25^{\circ}C$. These results indicate that the optimum temperatures for proliferation of PVY-O/PVA and PLRV differed. Western blot analysis showed that accumulations of PVY-O and PVA coat proteins (CPs) were lower at $10^{\circ}C$ or $15^{\circ}C$ than at $20^{\circ}C$ during early infection. However, accumulation increased over time. At $25^{\circ}C$ or $30^{\circ}C$, the CPs of both viruses accumulated during early infection but disappeared as time passed. Our results suggest that symptom attenuation and reduction of PVY-O and PVA CP accumulation at higher temperatures appear to be attributable to increased RNA silencing.

Berberine Induces p53-Dependent Apoptosis through Inhibition of DNA Methyltransferase3b in Hep3B Cells (Hep3B 세포에서 베르베린은 DNA methyltransferase3b 억제를 통해 p53을 발현시켜 세포사멸을 유도)

  • Kim, Dae-Yeon;Kim, Seon-Hyoung;Cheong, Hee-Tae;Ra, Chang-Six;Rhee, Ki-Jong;Jung, Bae Dong
    • Korean Journal of Clinical Laboratory Science
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    • v.52 no.1
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    • pp.69-77
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    • 2020
  • The tumor suppressor gene, p53, is inactivated in the human hepatocellular carcinoma cells line, Hep3B. Berberine has been reported to inhibit the proliferation of cancer cells. This study examined whether apoptosis was induced in berberine-treated Hep3B cells and observed the association between apoptosis and the expression of p53 and DNA methyltransferase (DNMT). The cell viability was measured using an MTT assay. Apoptosis of Hep3B was measured using annexin V flow cytometry. Berberine-treated cells were examined for their DNMT enzymatic activity, mRNA expression, and protein synthesis. The p53 levels were examined by Western blot analysis. The berberine treatment resulted in increased Hep3B cell death and apoptosis in a time- and dose-dependent manner. The DNMT3b activity, mRNA expression, and protein levels all decreased after the berberine treatment. In contrast, the p53 protein levels increased with a concomitant decrease in DNMT3b. No change in the expression of ERK was observed, but the P-ERK levels decreased in a dose dependent manner. These results indicate that a treatment of Hep3B cells with berberine can reduce the expression of DNMT3b, leading to an increase in the tumor suppressant gene p53 and an increase in cell apoptosis. This shows that berberine can effectively suppress the proliferation of liver cancer cells.

The alterations of $Ca^{2+}$-activated $K^+$ channels in coronary artery during cardiac hypertrophy

  • Kim, Nari;Lee, Sang-Kyeong;Chung, Joon-Yong;Seog, Dae-Hyun;Kim, Euiyong;Jin Han
    • Proceedings of the Korean Biophysical Society Conference
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    • 2003.06a
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    • pp.23-23
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    • 2003
  • It has been suggested that the impairment of smooth muscle cell (SMC) function by alterations in the $Ca^{2+}$-activated $K^{+}$ ( $K_{Ca}$ ) channels accounts for the reduction in coronary reserve during left ventricular hypertrophy (LVH). However, this hypothesis has not been fully investigated. The main goal of this study was to assess whether the properties of $K_{Ca}$ channels in coronary SMCs were altered during LVH. New Zealand white rabbits (0.8-1.0 kg) and Sprague-Dawley rats (300-400 g) were randomly selected to receive either an injection of isoproterenol (300 $\mu\textrm{g}$/kg body weight) or an equal volume of 0.9% saline (1 mL/kg body weight). The animals developed LVH 10 days after injection. In patch-clamp experiments, the unitary current amplitude and open probability for the $K_{Ca}$ channels were significantly reduced in LVH patches compared with control patches. The concentration-response curve of the $K_{Ca}$ channel to [C $a^{2+}$]$_{i}$ was shifted to the right. Inhibition of the $K_{Ca}$ channels with TEA was more pronounced in LVH cells than in the control cells. The whole-cell currents of $K_{Ca}$ channels were reduced during LVH. Western blot analysis indicated no differences in $K_{Ca}$ channel expression between the control and LVH coronary SM membranes. In contraction experiments, the effect of a high $K^{+}$concentration on the resting tension of the LVH coronary artery was greater than on that of the control. The effect of TEA on the resting tension of the LVH coronary artery was reduced as compared with the effect on the control. Our findings imply a novel mechanism for reduced coronary reserve during LVH.ing LVH.

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Antimelanogenic Effect of Isomaltol Glycoside from Red Ginseng Extract (홍삼추출물에 함유된 이소말톨 글리토시드의 멜라닌 생성저해 효과)

  • Lee, Sang Myung
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.45 no.3
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    • pp.255-263
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    • 2019
  • Isomaltol glycoside is a hydrophilic furanic glycoside in which the amino acids and sugars of ginseng are thermally denatured during red ginseng production. Various skin whitening tests were conducted on isomaltol glycoside containing a lot of red ginseng extract in order to investigate the skin whitening effect as a cosmetic raw material. We have tested melanin content assay in B16-F10 cells, zebrafish embryo pigmentation assay, mushroom tyrosinase inhibitory activity, western blot analysis to determine skin whitening activity of isomaltol glycosides. In the zebrafish melanin content assay, isomaltol glycoside decreased total melanin content by about 20% and zebrafish tyrosinase activity by about 10% after treatment with 50 and $100{\mu}g/mL$ compared to the untreated control group. Isomaltol glycoside also showed a concentration-dependent decrease in melanin content in B16-F10 melanoma. Furthermore, it increased the expression of MITF phosphorylation factors p-AKT and p-ERK in B16-F10 melanoma and decreased the concentration of MITF. It also inhibited tyrosinase, TRP-1 and TRP-2 expression. The content of isomaltol glycoside was about 3% in the ginseng extract and about 1% in the ginseng root. Thus, isomaltol glycoside is considered as one of the main components that exhibit the whitening activity of ginseng when considered quantitatively as whitening activity.

Anti-inflammatory Effect of Yukil-san Water Extract on LPS-induced RAW 264.7 Cells (LPS로 활성화된 RAW 264.7 cell에서 NF-𝜅B억제를 통한 육일산(六一散) 물추출물의 염증억제효과)

  • Lee, Chang Wook;Park, Sang Mi;Kim, Eun Ok;Byun, Sung Hui;Kim, Sang Chan
    • Herbal Formula Science
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    • v.30 no.2
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    • pp.45-57
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    • 2022
  • Objectives : Yukil-san (YIS, 六一散; Liu yi san) is composed of Talcum and Glycyrrhizae Radix, the name is said to be derived from the proportion of the two herbal components of the formula. The YIS originated from 'Formulas from the discussion illuminating the Yellow Emperor's Basic Question'(黃帝素問宣明論方; Huang di su wen xuan ming lun fang) written by Liu Wan-Su (劉完素). YIS could clear summerheat, resolve dampness, and augment the qi. This formula may be used to treat the common cold, influenza, acute gastroenteritis, cystitis, urethritis and bacillary dysentery. But, there is insufficient of study about the effects of YIS on the anti-inflammatory activities. The present study evaluated the anti-inflammatory effects of YIS on lipopolysaccharide (LPS)-activated RAW 264.7 cells. Methods : Cell viability was assessed by MTT assay and nitric oxide (NO) was evaluated by measuring the nitrite content in culture medium. Pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β and IL-6 were quantified by ELISA kit. The expression of proteins related with nuclear factor-κB (NF-κB) pathway and inducible NO synthase (iNOS) were assessed by western blot analysis. Results : YIS significantly inhibited the expression of iNOS increased by LPS, and thus significantly inhibited the production of NO. In addition, YIS significantly inhibited pro-inflammatory cytokines. In the regulation of inflammation, NF-κB pathway plays a crucial role. YIS inhibited the expression of p-IκBα and thus inhibited the translocation of NF-κB to the nucleus. Conclusions : These results suggest that YIS ameliorates inflammatory response in LPS-activated RAW 264.7 cells through the inhibition of inflammatory mediators, via suppression of the NF-κB pathway. Therefore, this study provides objective evidence for the anti-inflammatory effect of YIS including the underlying mechanisms.