• 제목/요약/키워드: E6 protein

검색결과 1,910건 처리시간 0.034초

Eucommia ulmoides Extract Stimulates Glucose Uptake through PI 3-kinase Mediated Pathway in L6 Rat Skeletal Muscle Cells

  • Hong, Eui-Jae;Hong, Seung-Jae;Jung, Kyung-Hee;Ban, Ju-Yeon;Baek, Yong-Hyeon;Woo, Hyun-Su;Park, Dong-Suk
    • Molecular & Cellular Toxicology
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    • 제4권3호
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    • pp.224-229
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    • 2008
  • Eucommia ulmoides (Duchung) is commonly used for treatment of diabetes in Korean traditional medicine. However, the exact mechanism of its anti-diabetic effect has not yet been fully elucidated. In this study, the effect of E. ulmoides extract on glucose uptake was investigated in L6 rat skeletal muscle cells. E. ulmoides extract stimulated the activity of phosphatidylinositol (PI) 3-kinase that is a major regulatory molecule in glucose uptake pathway. Protein kinase B (PKB) and protein kinase C-${\xi}$ (PKC-${\xi}$), downstream mediators of PI 3-kinase, were also activated by E. ulmoides extract. We assessed the activity of AMP-activated protein kinase (AMPK), another regulatory molecule in glucose uptake pathway. Phosphorylation level of AMPK did not change with treatment of E. ulmoides extract. Phosphorylations of p38 mitogen activated protein kinase (p38 MAPK) and acetyl-CoA carboxylase (ACC), downstream mediators of AMPK, were not significantly different. Taken together, our results suggest that E. ulmoides may stimulate glucose uptake through PI 3-kinase but not AMPK in L6 skeletal muscle cells.

Secretion of MCP-1, IL-8 and IL-6 Induced by House Dust Mite, Dermatophagoides pteronissinus in Human Eosinophilic EoL-1 Cells

  • Lee, Ji-Sook;Kim, In-Sik;Yun, Chi-Young
    • Animal cells and systems
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    • 제13권4호
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    • pp.391-397
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    • 2009
  • The house dust mite (Dermatophagoides pteronissinus) is an important factor in triggering allergic diseases. The function of eosinophils, particularly in the production of cytokine or chemokine, is critical in understanding the pathogenesis of inflammatory diseases. In this study, we examined whether D. pteronissinus extract (DpE) induces the expression of monocyte chemotactic protein 1 (MCP-1)/CCL2, IL-8/CXCL8, and IL-6 that mediate in the infiltration and activation of immune cells and in its signaling mechanism in the human eosinophilic cell line, EoL-1. DpE increased the mRNA and protein expression of MCP-1, IL-8, and IL-6 in a time- and dose-dependent course in EoL-1 cells. In our experiments using signal-specific inhibitors, we found that the increased expression of MCP-1, IL-8, and IL-6 due to DpE is associated with Src family tyrosine kinase and protein kinase C $\delta$ (PKC $\delta$). In addition, the activation of extracellular signal-regulated kinase (ERK) is required for MCP-1 and IL-8 expression while p38 mitogen-activated protein kinase (MAPK) is involved in IL-6 expression. DpE induced the phosphorylation of ERK and p38 MAPK. PP2, an inhibitor of Src family tyrosine kinase, and rottlerin, an inhibitor of PKC $\delta$, blocked the activation of ERK and p38 MAPK. DpE induces the activation of ERK and p38 MAPK via Src family tyrosine kinase and PKC $\delta$ for MCP-1, IL-8, or IL-6 production. Increased cytokine release due to the house dust mite and the characterization of its signal transduction may be valuable in understanding the eosinophil-related pathogenic mechanism of inflammatory diseases.

Metabolic Elasticity and Induction of Heat Shock Protein 70 in Labeo rohita Acclimated to Three Temperatures

  • Das, T.;Pal, A.K.;Chakraborty, S.K.;Manush, S.M.;Chatterjee, N.;Apte, S.K.
    • Asian-Australasian Journal of Animal Sciences
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    • 제19권7호
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    • pp.1033-1039
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    • 2006
  • The metabolic response of Labeo rohita to thermal acclimation was assessed. Advanced fingerlings of L. rohita (average weight $31{\pm}1.4g$) were acclimated to 31, 33 and $36^{\circ}C$ compared with ambient temperatures ($26^{\circ}C$) for 30 days and different enzymes associated with stress response were estimated. Glycolytic enzyme-Lactate dehydrogenase, (LDH, E.C.1.1.1.27), TCA cycle enzyme-Malate dehydrogenase (MDH, E.C.1.1.1.37), Protein metabolizing enzymes-Aspartate amino transferase (AST, E.C.2.6.1.1) and Alanine amino transferase (ALT, E.C.2.6.1.2) of liver, gill and muscle, Gluconeogenic enzymes-Fructose 1,6 Bi phosphatase (FBPase, E.C. 3.1.3.11) and Glucose 6 phosphatase (G6Pase, E.C. 3.1.3.9) of liver and kidney were significantly (p<0.05) different with increasing acclimation temperatures. Heat Shock Protein-70 (HSP-70) was expressed in increasing intensity at 31, 33 and $36^{\circ}C$ but was not expressed at $26^{\circ}C$. Results suggest that higher acclimation temperatures enhance metabolism and L. rohita maintains homeostasis between $26-36^{\circ}C$ via an acclimation episode. Such adaptation appears to be facilitated by resorting to gluconeogenic and glycogenolytic pathways for energy mobilization and induction of HSPs.

A Novel Mutant of Human Papillomavirus Type 18 E6E7 Fusion Gene and its Transforming Activity

  • Zhou, Zhi-Xiang;Zhao, Chen;Li, Qian-Qian;Zeng, Yi
    • Asian Pacific Journal of Cancer Prevention
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    • 제15권17호
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    • pp.7395-7399
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    • 2014
  • Background: Persistent human papillomavirus (HPV) infection, especially with high-risk types such as HPV16 and HPV18, has been identified as the primary cause of cervical cancer. E6 and E7 are the major onco-proteins of high-risk HPVs, which are consistently expressed in HPV infected tissues but absent in normal tissues and represent ideal therapeutic targets for immunotherapy of cervical cancer. Materials and Methods: In this study, the optimized fusion gene HPV18 E6E7 (HPV18 ofE6E7) was constructed according to genetic codon usage for human genes. At the same time, for safety future clinical application, a mutant of HPV18 ofE6E7 fusion gene was generated by site-directed mutagenesis at L52G for the E6 protein and C98G for the E7 protein. Results: HPV18-E6E7 mutant (HPV18 ofmE6E7) constructed in this work not only lost the transformation capability for NIH 3T3 cells and tumorigenicity in BALB/c nude mice, but also maintained very good stability and antigenicity. Conclusion: These results suggest that the mutant should undergo further study for application as a safe antigenspecific therapeutic vaccine for HPV18-associated tumors.

Protein Evaluation of Dry Roasted Whole Faba Bean (Vicia faba) and Lupin Seeds (Lupinus albus) by the New Dutch Protein Evaluation System: the DVE/OEB System

  • Yu, P.;Egan, A.R.;Leury, B.J.
    • Asian-Australasian Journal of Animal Sciences
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    • 제12권6호
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    • pp.871-880
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    • 1999
  • The effects of dry roasting (110, 130, $150^{\circ}C$ for 15, 30, 45 min) on potential ruminant protein nutritional values in terms of: a), rumen bypass protein (BCP); b), rumen bypass starch (BST); c), fermented organic matter (FOM); d), true absorbed bypass protein (ABCP); e) microbial protein synthesized in the rumen based on available energy (E_MP); f), microbial protein synthesized in the rumen based on available nitrogen (N_MP); g), true protein supplied to the small intestine (TPSI); h), true absorbed rumen synthesized microbial protein (AMP); i), endogenous protein losses (ENDP); j), true digested protein in the small intestine (DVE); k), degraded protein balance (OEB) of whole lupin seeds (WLS) and faba beans (WFB) were evaluated by the new Dutch DV/OEB protein evaluation system. Dry roasting significantly increased BCP, BST, TPSI, ABCP, DVE (p<0.001) and decreased FOM, E_MP, AMP, N_MP and OEB (p<0.001) with increasing temperatures and times except that when temperature was at $110^{\circ}C$. The values of BCP, BST, TPSI, ABCP and DVE at $150^{\circ}C/45min$ for WLS and WFB were increased 2.2, 3.7; -, 2.0; 1.7, 1.7; 2.3, 3.7 and 1.7, 1.7 times and the values of FOM, E_MP, AMP, N_MP and OEB at $150^{\circ}C/45min$ for WLS and WFB were decreased by 15.3, 25.8; 18.1, 25.8; 18.7, 25.8; 54.6, 41.6 and 82.3% 54.7%, respectively, over the raw WLS and WFB. The results indicated that though dry roasting reduced microbial protein synthesis due to reducing FOM, TPSI didn't decrease but highly increased due to increasing BCP more than enough for compensation of the microbial protein decreasing. Therefore the net absorbable DVE in the small intestine was highly increased. The OEB values were significantly reduced for both WLS and WFB but not to the level of negative. It indicated that microbial protein synthesis might not be impaired due to the sufficient N supplied in the rumen, but the high positive OEB values in the most treatments except of $150^{\circ}C$ for 30 and 45 min of WLS (The OEB values: 54.8 and 26.0 g/kg DM) indicated that there were the large amounts of N loss in the rumen. It was concluded that dry roasting at high temperature was effective in shifting protein degradation from rumen to intestines and it increased the DVE values without reaching the negative OEB values. No optimal treatment was found in WLS due to the too high OEB values in all treatments. But dry roasting at $150^{\circ}C$ for 30 and 45 min might be optimal treatments for WLS due to the very lower OEB values.

Direct characterization of E2-dependent target specificity and processivity using an artificial p27-linker-E2 ubiquitination system

  • Ryu, Kyoung-Seok;Choi, Yun-Seok;Ko, Jun-Sang;Kim, Seong-Ock;Kim, Hyun-Jung;Cheong, Hae-Kap;Jeon, Young-Ho;Choi, Byong-Seok;Cheong, Chae-Joon
    • BMB Reports
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    • 제41권12호
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    • pp.852-857
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    • 2008
  • Little attention has been paid to the specificity between E2 and the target protein during ubiquitination, although RING-E3 induces a potential intra-molecular reaction by mediating the direct transfer of ubiquitin from E2 to the target protein. We have constructed artificial E2 fusion proteins in which a target protein (p27) is tethered to one of six E2s via a flexible linker. Interestingly, only three E2s (UbcH5b, hHR6b, and Cdc34) are able to ubiquitinate p27 via an intra-molecular reaction in this system. Although the first ubiquitination of p27 (p27-Ub) by Cdc34 is less efficient than that of UbcH5b and hHR6b, the additional ubiquitin attachment to p27-Ub by Cdc34 is highly efficient. The E2 core of Cdc34 provides specificity to p27, and the residues 184-196 are required for possessive ubiquitination by Cdc34. We demonstrate direct E2 specificity for p27 and also show that differential ubiquitin linkages can be dependent on E2 alone.

히스티딘으로 표지된 람다 박테리오파아지 꼬리 단백질 J와 대장균 K-12와의 결합 (Binding of the His-tagged Tail Protein J of Bacteriophage Lambda with Escherichia coli K-12)

  • 신혜자
    • 생명과학회지
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    • 제28권1호
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    • pp.78-82
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    • 2018
  • 본 연구에서는 미생물 검출용 바이오센서 제작을 위해 재조합 람다 파아지 꼬리 단백질 J을 센싱 요소로 활용가능한지를 조사하였다. 융합 단백질의 입체 장애를 최소화하기 위해 J 단백질 절편의 N-말단을 6X His-tag로 융합하고 HisTALONTM 컬럼으로 정제하였다. 정제 단백질은 약 38 kDa 크기를 SDS-PAGE에서 나타내며 anti-His 단클론 항체와 반응하였다. Anti-His 단클론 항체는 6HN-J를 처리한 E. coli K-12와 특이적으로 결합하나 BSA 처리하거나 6HN-J 처리한 다른 미생물들(Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa)과는 결합되지 않음을 보여주었다. 또한 정제 단백질과 숙주세포의 결합은 온전한 람다 파아지의 in vivo 숙주 표면 흡착을 약 $1{\mu}g/ml$ 단백질 농도에서 50%, $25{\mu}g/ml$ 단백질 농도에서 거의 완전히 방해하였다. 결론적으로 재조합 6HN-J 단백질은 탁월한 선택성과 선별성으로 인해 바이오센서의 제작에서 센싱 요소로 활용 가능함을 시사한다.

Cytotoxic T Lymphocytes Elicited by Dendritic Cell-Targeted Delivery of Human Papillomavirus Type-16 E6/E7 Fusion Gene Exert Lethal Effects on CaSki Cells

  • Wu, Xiang-Mei;Liu, Xing;Jiao, Qing-Fang;Fu, Shao-Yue;Bu, You-Quan;Song, Fang-Zhou;Yi, Fa-Ping
    • Asian Pacific Journal of Cancer Prevention
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    • 제15권6호
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    • pp.2447-2451
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    • 2014
  • Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Consideration of safety and non human leukocyte antigen restriction, protein vaccine has become the most likely form of HPV therapeutic vaccine, although none have so far been reported as effective. Since tumor cells consistently express the two proteins E6 and E7, most therapeutic vaccines target one or both of them. In this study, we fabricated DC vaccines by transducing replication-defective recombinant adenoviruses expressing E6/E7 fusion gene of HPV-16, to investigate the lethal effects of specific cytotoxic T lymphocytes (CTL) against CaSki cells in vitro. Mouse immature dendritic cells (DC) were generated from bone marrow, and transfected with pAd-E6/E7 to prepare a DC vaccine and to induce specific CTL. The surface expression of CD40, CD68, MHC II and CD11c was assessed by flow cytometry (FCM), and the lethal effects of CTL against CaSki cells were determined by DAPI, FCM and CCK-8 methods. Immature mouse DC was successfully transfected by pAd-E6/E7 in vitro, and the transfecting efficiency was 40%-50%. A DC vaccine was successfully prepared and was used to induce specific CTL. Experimental results showed that the percentage of apoptosis and killing rate of CaSki cells were significantly increased by coculturing with the specific CTL (p <0.05). These results illustrated that a DC vaccine modified by HPV-16 E6/E7 gene can induce apoptosis of CaSki cells by inducing CTL, which may be used as a new strategy for biological treatment of cervical cancer.

Generation and characterization of a monoclonal antibody against MERS-CoV targeting the spike protein using a synthetic peptide epitope-CpG-DNA-liposome complex

  • Park, Byoung Kwon;Maharjan, Sony;Lee, Su In;Kim, Jinsoo;Bae, Joon-Yong;Park, Man-Seong;Kwon, Hyung-Joo
    • BMB Reports
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    • 제52권6호
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    • pp.397-402
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    • 2019
  • Middle East respiratory syndrome coronavirus (MERS-CoV) uses the spike (S) glycoprotein to recognize and enter target cells. In this study, we selected two epitope peptide sequences within the receptor binding domain (RBD) of the MERS-CoV S protein. We used a complex consisting of the epitope peptide of the MERS-CoV S protein and CpG-DNA encapsulated in liposome complex to immunize mice, and produced the monoclonal antibodies 506-2G10G5 and 492-1G10E4E2. The western blotting data showed that both monoclonal antibodies detected the S protein and immunoprecipitated the native form of the S protein. Indirect immunofluorescence and confocal analysis suggested strong reactivity of the antibodies towards the S protein of MERS-CoV virus infected Vero cells. Furthermore, the 506-2G10G5 monoclonal antibody significantly reduced plaque formation in MERS-CoV infected Vero cells compared to normal mouse IgG and 492-1G10E4E2. Thus, we successfully produced a monoclonal antibody directed against the RBD domain of the S protein which could be used in the development of diagnostics and therapeutic applications in the future.

Effects of Down-regulation of HDAC6 Expression on Proliferation, Cell Cycling and Migration of Esophageal Squamous Cell Carcinoma Cells and Related Molecular Mechanisms

  • Li, Ning;Tie, Xiao-Jing;Liu, Pei-Jie;Zhang, Yan;Ren, Hong-Zheng;Gao, Xin;Xu, Zhi-Qiao
    • Asian Pacific Journal of Cancer Prevention
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    • 제14권2호
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    • pp.685-689
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    • 2013
  • Objective: To study the effects of down-regulation of HDAC6 expression on proliferation, cell cycling and migration of esophageal squamous cell carcinoma (ESCC) cells and related molecular mechanisms. Methods: ESCC cell line EC9706 cells were randomly divided into untreated (with no transfection), control siRNA (transfected with control siRNA) and HDAC6 siRNA (transfected with HDAC6 small interfering RNA) groups. Effects of HDAC6 siRNA interference on expression of HDAC6 mRNA and protein in EC9706 cells were investigated by semi-quantitative RT-PCR, Western blotting and immunocytochemistry methods. Effects of down-regulation of HDAC6 expression on cell proliferation, cell cycle, and cell migration were studied using a CCK-8 kit, flow cytometry and Boyden chambers, respectively. Changes of mRNA and protein expression levels of cell cycle related factor (p21) and cell migration related factor (E-cadherin) were investigated by semi-quantitative RT-PCR and Western blotting methods. Results: After transfection of HDAC6 siRNA, the expression of HDAC6 mRNA and protein in EC9706 cells was significantly downregulated. In the HDAC6 siRNA group, cell proliferation was markedly inhibited, the percentage of cells in G0/G1 phase evidently increased and the percentage of cells in S phase decreased, and the number of migrating cells significantly and obviously decreased. The mRNA and protein expression levels of p21 and E-cadherin in the HDAC6 siRNA group were significantly higher than those in the untreated group and the control siRNA group, respectively. Conclusions: HDAC6 siRNA can effectively downregulate the expression of HDAC6 mRNA and protein in EC9706 cells. Down-regulation of HDAC6 expression can obviously inhibit cell proliferation, arrest cell cycling in the G0/G1 phase and reduce cell migration. The latter two functions may be closely related with the elevation of mRNA and protein expression of p21 and E-cadherin.