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北京早园竹叶不同提取组分对cho细胞akt信号通路的影响  [PDF]
梅晶,祖桂芳,赵晓红,何颖,孙健
食品科学 , 2012,
Abstract: ?通过比较北京早园竹叶提取物中的主要活性组分黄酮苷类、酚酸类及总提取物3个组分对中国仓鼠卵巢细胞(cho)的增殖、凋亡及akt信号通路作用差别,考察竹叶提取物对正常细胞可能产生有害作用的活性组分。采用mtt法检测细胞增殖作用、annexinv-fitc/pi双染流式法测细胞凋亡、incellanalyzer1000活细胞图像分析系统测定akt信号通路的活化。结果显示:1)50~400μg/ml范围内3种组分对细胞存活率的影响无明显变化,当质量浓度≥800μg/ml时细胞增殖率明显下降(p<0.05),细胞毒性作用明显,且以酚酸类组分作用最强;2)不同组分的竹叶提取物在200~800μg/ml范围内与空白组比较均可降低细胞凋亡率(p<0.05),但总提取物和黄酮苷类在质量浓度为1600μg/ml时本身可诱导cho细胞的凋亡增加,差异显著(p<0.05),且以黄酮苷类诱导的细胞凋亡率最高,达14.21%;3)与对照组比较,黄酮苷类在100~800μg/ml剂量条件下,对akt的活性无明显影响,总提取物在400、800μg/ml时akt的激活率明显增加(p<0.01),而酚酸类在100~800μg/ml范围内,随剂量增加,激活率增高,呈明显的剂量-反应关系(r=0.996,p<0.01)。由此可见3种不同提取物组分对cho细胞的作用是有差别的,其中酚酸类可能是产生有害作用的主要组分。
Vectors Encoding Seven Oikosin Signal Peptides Transfected into CHO Cells Differ Greatly in Mediating Gaussia luciferase and Human Endostatin Production although mRNA Levels are Largely Unaffected
Christiane Tr??e,Hanne Ravneberg,Beate Stern,Ian F. Pryme
Gene Regulation and Systems Biology , 2007,
Abstract: The signal peptide of the luciferase secreted by the marine copepod Gaussia princeps has been shown to promote high-level protein synthesis/secretion of recombinant proteins, being far superior to mammalian counterparts. The main aim of the present study was to investigate the effects of seven selected signal peptides derived from oikosins, house proteins of the marine organism Oikopleura dioica, on synthesis/secretion of recombinant proteins. Vector constructs were made in which the coding regions of two naturally secreted proteins, Gaussia luciferase and human endostatin (hEndostatin), were “seamlessly” fused to the signal peptide coding sequences of interest. CHO cells were transfected with the plasmids and populations of stably transfected cells established. The amounts of reporter proteins in cell extract and medium samples were determined and the results compared to those obtained from cells stably transfected with a reference vector construct. In addition, the amounts of luciferase or hEndostatin encoding mRNAs in the cells were determined and related to the protein levels obtained. The levels of reporter protein produced varied greatly among the seven oikosin signal peptides tested. Whereas the oikosin 1 signal peptide resulted in about 40% production of Gaussia luciferase compared to the reference construct, oikosins 2–7 were extremely ineffective (<1%). mRNA levels were not dramatically affected such that inadequate availability of transcript for translation was not the underlying reason for the observations. The oikosin 1 signal peptide was also the most effective regarding synthesis/secretion of hEndostatin. No secreted product was observed using the oikosin 3 signal peptide. Interestingly, the molecular weight of hEndostatin in cell extracts prepared from cells transfected with oikosin 2 and 3 constructs was higher than that using the oikosin 1 signal peptide. The overall fi ndings indicate that the signal peptide affects the efficiency of protein synthesis and secretion through a mechanism operating at the posttranscriptional level. The results described here provide substantial support to our previous observations which suggested that the choice of the signal peptide is imperative when aiming to achieve optimal synthesis and secretion of a recombinant protein using transfected mammalian cells.
Periostin Facilitates Skin Sclerosis via PI3K/Akt Dependent Mechanism in a Mouse Model of Scleroderma  [PDF]
Lingli Yang, Satoshi Serada, Minoru Fujimoto, Mika Terao, Yorihisa Kotobuki, Shun Kitaba, Saki Matsui, Akira Kudo, Tetsuji Naka, Hiroyuki Murota, Ichiro Katayama
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0041994
Abstract: Objective Periostin, a novel matricellular protein, is recently reported to play a crucial role in tissue remodeling and is highly expressed under fibrotic conditions. This study was undertaken to assess the role of periostin in scleroderma. Methods Using skin from patients and healthy donors, the expression of periostin was assessed by immunohistochemistry and immunoblotting analyses. Furthermore, we investigated periostin?/? (PN?/?) and wild-type (WT) mice to elucidate the role of periostin in scleroderma. To induce murine cutaneous sclerosis, mice were subcutaneously injected with bleomycin, while untreated control groups were injected with phosphate-buffered saline. Bleomycin-induced fibrotic changes were compared in PN?/? and WT mice by histological analysis as well as by measurements of profibrotic cytokine and extracellular matrix protein expression levels in vivo and in vitro. To determine the downstream pathway involved in periostin signaling, receptor neutralizing antibody and signal transduction inhibitors were used in vitro. Results Elevated expression of periostin was observed in the lesional skin of patients with scleroderma compared with healthy donors. Although WT mice showed marked cutaneous sclerosis with increased expression of periostin and increased numbers of myofibroblasts after bleomycin treatment, PN?/? mice showed resistance to these changes. In vitro, dermal fibroblasts from PN?/? mice showed reduced transcript expression of alpha smooth actin and procollagen type-I alpha 1 (Col1α1) induced by transforming growth factor beta 1 (TGFβ1). Furthermore, recombinant mouse periostin directly induced Col1α1 expression in vitro, and this effect was inhibited by blocking the αv integrin-mediated PI3K/Akt signaling either with anti-αv functional blocking antibody or with the PI3K/Akt kinase inhibitor LY294002. Conclusion Periostin plays an essential role in the pathogenesis of Bleomycin-induced scleroderma in mice. Periostin may represent a potential therapeutic target for human scleroderma.
Norcantharidin Facilitates LPS-Mediated Immune Responses by Up-Regulation of AKT/NF-κB Signaling in Macrophages  [PDF]
Qufei Zhao,Yu Qian,Ruimei Li,Binghe Tan,Honghui Han,Mingyao Liu,Min Qian,Bing Du
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0044956
Abstract: Norcantharidin (NCTD), a demethylated analog of cantharidin, is a common used clinical drug to inhibit proliferation and metastasis of cancer cells. But the role of NCTD in modulating immune responses remains unknown. Here, we investigated the function and mechanism of NCTD in regulation of TLR4 associated immune response in macrophages. We evaluated the influence of NCTD on host defense against invaded pathogens by acute peritonitis mouse model, ELISA, Q-PCR, nitrite quantification, phagocytosis assay and gelatin zymography assay. Our data showed that the survival and the serum concentrations of IL-6 and TNF-α were all enhanced by NCTD significantly in peritonitis mouse model. Accordingly, LPS-induced cytokine, nitric oxide and MMP-9 production as well as the phagocytosis of bacteria were all up-regulated by NCTD in a dose dependent manner in both RAW264.7 cells and bone marrow-derived macrophages (BMMs). Then we further analyzed TLR4 associated signaling pathway by Western blot, Immunofluorescence and EMSA in the presence or absence of LPS. The phosphorylation of AKT and p65 at serine 536 but not serine 468 was enhanced obviously by NCTD in a dose dependent manner, whereas the degradation of IκBα was little effected. Consequently, the nuclear translocation and DNA binding ability of NF-κB was also increased by NCTD obviously in RAW264.7 cells. Our results demonstrated that NCTD could facilitate LPS-mediated immune response through promoting the phosphorylation of AKT/p65 and transcriptional activity of NF-κB, thus reprofiling the traditional anti-tumor drug NCTD as a novel immune regulator in promoting host defense against bacterial infection.
Inhibition of Oxidative Stress-Elicited AKT Activation Facilitates PPARγ Agonist-Mediated Inhibition of Stem Cell Character and Tumor Growth of Liver Cancer Cells  [PDF]
Lanlan Liu, Zhaojuan Yang, Yingqian Xu, Jingyi Li, Dongxu Xu, Li Zhang, Jiabin Sun, Suhua Xia, Feiyan Zou, Yongzhong Liu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0073038
Abstract: Emerging evidence suggests that tumor-initiating cells (TICs) are the most malignant cell subpopulation in tumors because of their resistance to chemotherapy or radiation treatment. Targeting TICs may be a key innovation for cancer treatment. In this study, we found that PPARγ agonists inhibited the cancer stem cell-like phenotype and attenuated tumor growth of human hepatocellular carcinoma (HCC) cells. Reactive oxygen species (ROS) initiated by NOX2 upregulation were partially responsible for the inhibitory effects mediated by PPARγ agonists. However, PPARγ agonist-mediated ROS production significantly activated AKT, which in turn promoted TIC survival by limiting ROS generation. Inhibition of AKT, by either pharmacological inhibitors or AKT siRNA, significantly enhanced PPARγ agonist-mediated inhibition of cell proliferation and stem cell-like properties in HCC cells. Importantly, in nude mice inoculated with HCC Huh7 cells, we demonstrated a synergistic inhibitory effect of the PPARγ agonist rosiglitazone and the AKT inhibitor triciribine on tumor growth. In conclusion, we observed a negative feedback loop between oxidative stress and AKT hyperactivation in PPARγ agonist-mediated suppressive effects on HCCs. Combinatory application of an AKT inhibitor and a PPARγ agonist may provide a new strategy for inhibition of stem cell-like properties in HCCs and treatment of liver cancer.
Impact of an Altered PROX1 Expression on Clinicopathology, Prognosis and Progression in Renal Cell Carcinoma  [PDF]
Tao Lv, Yanfeng Liu, Jianping Zhang, Le Xu, Yu Zhu, Hankun Yin, Huimin An, Zongming Lin, Youhua Xie, Lian Chen
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0095996
Abstract: The transcription factor PROX1 (prospero homeobox 1) has a critical role in the development of various organs, and has been implicated in both oncogenic and tumor-suppressive functions in human cancers. However, the role of PROX1 in the development of renal cell carcinomas (RCCs) has not yet been studied. Here, we reported that PROX1 expression was decreased in human RCC tissues compared with adjacent normal tissues. In RCC tissues, however, poorly differentiated RCC expressed higher PROX1 levels compared with well-differentiated RCC. In addition, the PROX1 immunostaining levels were positively correlated with tumor nuclear grade and lymph node metastasis. Further, high PROX1 expression indicated poor survival for patients. These findings imply that in the different developmental stages of RCC, PROX1 may exert distinct functions according to the specific microenvironment of tumor. Moreover, in vitro experiments revealed that PROX1 overexpression enhanced the proliferation and migration of RCC cells; conversely, PROX1 depletion by siRNA attenuated the proliferation and migration of RCC cells. Collectively, these observations suggest that PROX1 plays an important role in RCC development and progression, and PROX1 may be a novel target for prevention and treatment of RCC.
Crucial role of zebrafish prox1 in hypothalamic catecholaminergic neurons development
Anna Pistocchi, Germano Gaudenzi, Silvia Carra, Erica Bresciani, Luca Del Giacco, Franco Cotelli
BMC Developmental Biology , 2008, DOI: 10.1186/1471-213x-8-27
Abstract: Here we report that, in zebrafish, prox1 is widely expressed in several districts of the Central Nervous System (CNS). Specifically, we evidenced prox1 expression in a group of neurons, already positive for otp1, located in the hypothalamus at the level of the posterior tuberculum (PT). Prox1 knock-down determines the severe loss of hypothalamic catecholaminergic (CA) neurons, identified by tyrosine hydroxylase (TH) expression, and the synergistic prox1/otp1 overexpression induces the appearance of hypothalamic supernumerary TH-positive neurons and ectopic TH-positive cells on the yolk epitelium.Our findings indicate that prox1 activity is crucial for the proper development of the otp1-positive hypothalamic neuronal precursors to their terminal CA phenotype.The catecholaminergic neurons of the CNS of vertebrates participate in a wide variety of tasks, including motor coordination, mood regulation, and cognitive function, among others. Neurotransmitters catecholamines (CA), namely Dopamine (DA), Adrenaline (AD), and Noradrenaline (NA), are neuroactive molecules that exert strong influence on vertebrates behavior [1] and serve a variety of central and peripheral functions [2].Embryological studies indicate that several extracellular signals, as Hedgehog and FGF, are vital to define the development of the prosencephalic CA neurons [3-7]. The homeodomain transcription factor Orthopedia (Otp), regulated by such signaling pathways [8], is crucial in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates [9,10]. Specifically, Otp is required for the correct differentiation of the CA neurons positioned in the zebrafish Posterior Tuberculum (PT) and hypothalamus [8,11]. Despite all these evidences, the role of specific transcription factors leading to the proper differentiation of the hypothalamic CA neurons remains largely unclear [7].Prox1 homeobox gene is the vertebrate homologous of prospero in Drosophila melanogas
Knockdown of Akt Sensitizes Osteosarcoma Cells to Apoptosis Induced by Cisplatin Treatment  [PDF]
Guoyou Zhang,Ming Li,Xiaodong Zhu,Yushu Bai,Changwei Yang
International Journal of Molecular Sciences , 2011, DOI: 10.3390/ijms12052994
Abstract: Akt plays an important role in the inhibition of apoptosis induced by chemotherapy and other stimuli. We therefore investigated if knockdown of Akt2 promoted drug-induced apoptosis in cultured osteosarcoma cells in vitro. SAOS-2 cells were transfected with Akt2 siRNA. The sensitivity of the transformed cell line to the chemotherapeutic drug cisplatin was assessed. Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased their sensitivity to cisplatin. Knockdown of Akt2, together with cisplatin treatment, promoted the expression of p53 up-regulated modulator of apoptosis (PUMA). It is possible that the augmentation of cisplatin cytotoxicity may be mediated by PUMA activation. The results of this study suggest that knockdown of Akt2 expression may have therapeutic applications in enhancing the efficacy of chemotherapy in patients with osteosarcoma.
Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity  [PDF]
Bernd Fritzsch,Miriam Dillard,Alfonso Lavado,Natasha L. Harvey,Israt Jahan
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0009377
Abstract: The homeobox gene Prox1 is required for lens, retina, pancreas, liver, and lymphatic vasculature development and is expressed in inner ear supporting cells and neurons.
金鱼Prox1基因cDNA的克隆及其在眼睛发育中的表达分析
马冬梅,朱华平,桂建芳
动物学研究 , 2007,
Abstract: 脊椎动物的Prox1基因,与果蝇的转录因子prospero同源。为了探讨Prox1基因在金鱼眼睛发生过程中的表达图式,我们从金鱼眼睛SMART库中克隆了Prox1 cDNA。它全长共2 851bp,编码739个氨基酸。组织分布研究表明,Prox1主要分布于眼、脑、心、肝、脾和肾中。整体原位杂交显示,Prox1 mRNA首先是在晶体期的晶体原基中有转录,心跳期则在未成熟晶体的细胞中和视网膜的幼芽区可以检测到。晶体纤维形成后,它主要定位于视纤维层和内网织细胞层。免疫组化显示,心跳期Prox1蛋白的定位与mRNA相同,晶体纤维形成以后,Prox1蛋白主要定位在晶体上皮细胞内侧的晶体纤维上一个环状区域,与Prox1 mRNA的定位不同。这说明,Prox1 基因在晶体发生过程中有重要作用,且在晶体的不同发育时期起的作用可能有所不同。另外,Prox1在晶体发育过程中有一个从内向外的变化过程。
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