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Silencing
Kinds of Silencing Terms modified by Silencing Selected AbstractsWhen is high-Ca2+ microdomain required for mitochondrial Ca2+ uptake?,ACTA PHYSIOLOGICA, Issue 1 2009A. Spät Abstract Ca2+ release from IP3 -sensitive stores in the endoplasmic reticulum (ER) induced by Ca2+ -mobilizing agonists generates high-Ca2+ microdomains between ER vesicles and neighbouring mitochondria. Here we present a model that describes when such microdomains are required and when submicromolar [Ca2+] is sufficient for mitochondrial Ca2+ uptake. Mitochondrial Ca2+ uptake rate in angiotensin II-stimulated H295R adrenocortical cells correlates with the proximity between ER vesicles and the mitochondrion, reflecting the uptake promoting effect of high-Ca2+ peri-mitochondrial microdomains. Silencing or inhibition of p38 mitogen-activated protein kinase (MAPK) or inhibition of the novel isoforms of protein kinase C enhances mitochondrial Ca2+ uptake and abolishes the positive correlation between Ca2+ uptake and ER-mitochondrion proximity. Inhibition of protein phosphatases attenuates mitochondrial Ca2+ uptake and also abolishes its positive correlation with ER-mitochondrion proximity. We postulate that during IP3 -induced Ca2+ release, Ca2+ uptake is confined to ER-close mitochondria, because of the simultaneous activation of the protein kinases. Attenuation of Ca2+ uptake prevents Ca2+ overload of mitochondria and thus protects the cell against apoptosis. On the other hand, all the mitochondria accumulate Ca2+ at a non-inhibited rate during physiological Ca2+ influx through the plasma membrane. Membrane potential is higher in ER-distant mitochondria, providing a bigger driving force for Ca2+ uptake. Our model explains why comparable mitochondrial Ca2+ signals are formed in response to K+ and angiotensin II (equipotent in respect to global cytosolic Ca2+ signals), although only the latter generates high-Ca2+ microdomains. [source] Signaling events leading to the curative effect of cystatin on experimental visceral leishmaniasis: Involvement of ERK1/2, NF-,B and JAK/STAT pathwaysEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2009Susanta Kar Abstract Curative effect of cystatin, a natural cystein protease inhibitor, on experimental visceral leishmaniasis was associated with strong upregulation of iNOS. The transductional mechanisms underlying this cellular response was investigated in the murine macrophage cell line RAW 264.7 and in the BALB/c mouse model of visceral leishmaniasis. Cystatin synergizes with IFN-, in inducing ERK1/2 phosphorylation and NF-,B DNA-binding activity. Pretreatment of cells with specific inhibitors of NF-,B or ERK1/2 pathway blocked the cystatin plus IFN-,-inducible NF-,B activity and markedly reduced the expression of iNOS at both mRNA and protein levels. Silencing of mitogen- and stress-activated protein kinase 1 significantly reduced cystatin-mediated NF-,B-dependent iNOS gene transcription suggesting the involvement of mitogen- and stress-activated protein kinase 1 activation in ERK1/2 signaling. DNA binding as well as silencing experiments revealed the requirement of IFN-,-mediated JAK-STAT activation even though cystatin did not modulate this signaling cascade by itself. In the in vivo situation, key steps in the activation cascade of NF-,B, including nuclear translocation of NF-,B subunits, I,B phosphorylation and I,B kinase, are all remarkably enhanced in Leishmania -infected mice by cystatin. Understanding the molecular mechanisms through which cystatin modulates macrophage effector responses will contribute to better define its potential for macrophage-associated diseases, in general. [source] RNA interference reveals a role for TLR2 and TLR3 in the recognition of Leishmania donovani promastigotes by interferon,,-primed macrophagesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2006Jean-Frédéric Flandin Abstract Leishmania donovani promastigotes evade the induction of a proinflammatory response during their invasion of naive macrophages. However, their entry into IFN-,-primed macrophages is accompanied by the secretion of nitric oxide (NO) and proinflammatory cytokines. In the present study, we addressed the hypothesis that priming with IFN-, induces the expression of a receptor that enables mouse macrophages to recognize L. donovani promastigotes. We observed that in IFN-,-primed macrophages, L. donovani promastigotes stimulated Interleukin-1 receptor-associated kinase-1 (IRAK-1) activity. We next showed that Toll-like receptor (TLR)3 is barely detectable in naive macrophages but is expressed in IFN-,-treated macrophages. Silencing of TLR3, TLR2, IRAK-1 and myeloid differentiation factor 88 (MyD88) expression by RNA interference revealed that both TLR are involved in the secretion of NO and TNF-, induced by L. donovani promastigotes. Using L. donovani mutants, we showed that TLR2-mediated responses are dependent on Gal,1,4Man,-PO4 -containing phosphoglycans, whereas TLR3-mediated responses are independent of these glycoconjugates. Furthermore, our data indicate a participation of TLR2 and TLR3 in the phagocytosis of L. donovani promastigotes and a role for TLR3 in the leishmanicidal activity of the IFN-,-primed macrophages. Collectively, our data are consistent with a model where recognition of L. donovani promastigotes depends on the macrophage activation status and requires the expression of TLR3. [source] Silencing of an abdominal Hox gene during early development is correlated with limb development in a crustacean trunkEVOLUTION AND DEVELOPMENT, Issue 2 2010Cheryl C. Hsia SUMMARY We tested whether Artemia abd-A could repress limbs in Drosophila embryos, and found that although abd-A transcripts were produced, ABD-A protein was not. Similarly, developing Artemia epidermal cells showed expression of abd-A transcripts without accumulation of ABD-A protein. This finding in Artemia reveals a new variation in Hox gene function that is associated with morphological evolution. In this case, a HOX protein expression pattern is completely absent during early development, although the HOX protein is expressed at later stages in the central nervous system in a "homeotic-like" pattern. The combination of an absence of ABD-A protein expression in the Artemia limb primordia and the weak repressive function of Artemia UBX protein on the limb-promoting gene Dll are likely to be two reasons why homonomous limbs develop throughout the entire Artemia trunk. [source] Increased bacterial load in shrimp hemolymph in the absence of prophenoloxidaseFEBS JOURNAL, Issue 18 2009Fernand F. Fagutao Invertebrates rely on their innate immune responses to protect themselves from pathogens, one of which is melanization of bacteria mediated by the activation of phenoloxidase (PO). Furthermore, invertebrate hemolymph, even that of healthy individuals, has been shown to contain bacterial species. The mechanisms that prevent these bacteria from proliferating and becoming deleterious to the host are, however, poorly understood. Here, we show that knocking down the activity of the inactive precursor of PO [prophenoloxidase (proPO)] by RNA interference resulted in a significant increase in the bacterial load of kuruma shrimp, Marsupenaeus japonicus, even in the absence of a bacterial or viral challenge. Silencing of proPO also led to a sharp increase in shrimp mortality. In addition, the hemolymph of proPO-depleted shrimp had significantly lower hemocyte counts and PO activity than control samples. Microarray analysis after proPO silencing also showed a decrease in the expression of a few antimicrobial peptides, but no effect on the expression of the genes involved in the clotting system. Treatment with antibiotics prior to and after proPO dsRNA injection, to counteract the loss of proPO, resulted in a significant increase in shrimp survival. Our results therefore show that the absence of proPO renders the shrimp incapable of controlling bacteria present in the hemolymph, and that proPO is therefore essential for its survival. [source] TRB3, upregulated by ox-LDL, mediates human monocyte-derived macrophage apoptosisFEBS JOURNAL, Issue 10 2009Yuan-yuan Shang Tribble3 (TRB3), a mammalian homolog of Drosophila tribbles, slows cell-cycle progression, and its expression is increased in response to various stresses. The aim of this study was to investigate the role of the TRB3 gene in macrophage apoptosis induced by oxidized low-density lipoprotein (ox-LDL). We found that, in human monocyte-derived macrophages, TRB3 is upregulated by ox-LDL in a dose- and time-dependent manner. The cell viability of TRB3-overexpressing macrophages was decreased, but apoptosis was increased and the level of activated caspase-3 increased. Factorial analyses revealed no significant interaction between TRB3 overexpression and ox-LDL stimulation with respect to macrophage apoptosis. Furthermore, TRB3-silenced macrophages showed decreased apoptosis, and TRB3-silenced cells treated with ox-LDL showed significantly increased apoptosis. Silencing of TRB3 and ox-LDL stimulation showed significant interaction for macrophage apoptosis, suggesting that TRB3 knockdown resisted the macrophage apoptosis induced by ox-LDL. Therefore, TRB3 in part mediates the macrophage apoptosis induced by ox-LDL, which suggests that TRB3 might be involved in vulnerable atherosclerotic plaque progression. [source] Lipid-Like Nanoparticles for Small Interfering RNA Delivery to Endothelial CellsADVANCED FUNCTIONAL MATERIALS, Issue 19 2009Seung-Woo Cho Abstract Here, nanoparticles composed of lipid-like materials (lipidoids) to facilitate non-viral delivery of small interfering RNA (siRNA) to endothelial cells (ECs) are developed. Nanoparticles composed of siRNA and lipidoids with small size (,200,nm) and positive charge (,34,mV) are formed by self-assembly of lipidoids and siRNA. Ten lipidoids are synthesized and screened for their ability to facilitate the delivery of siRNA into ECs. Particles composed of leading lipidoids show significantly better delivery to ECs than a leading commercially available transfection reagent, Lipofectamine 2000. As a model of potential therapeutic application, nanoparticles composed of the top performing lipidoid, NA114, are studied for their ability to deliver siRNA targeting anti-angiogenic factor (SHP-1) to human ECs. Silencing of SHP-1 expression significantly enhances EC proliferation and decreases EC apoptosis under a simulated ischemic condition. [source] MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells,HEPATOLOGY, Issue 1 2009Teng Xu Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Down-regulation of miR-195 has been observed in various types of cancers. However, the biological function of miR-195 is still largely unknown. In this study we aimed to elucidate the pathophysiologic role of miR-195. Our results showed that miR-195 expression was significantly reduced in as high as 85.7% of hepatocellular carcinoma (HCC) tissues and in all of the five HCC cell lines examined. Moreover, introduction of miR-195 dramatically suppressed the ability of HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in nude mice. Furthermore, ectopic expression of miR-195 blocked G1/S transition, whereas inhibition of miR-195 promoted cell cycle progression. Subsequent investigation characterized multiple G1/S transition-related molecules, including cyclin D1, CDK6, and E2F3, as direct targets of miR-195. Silencing of cyclin D1, CDK6, or E2F3 phenocopied the effect of miR-195, whereas overexpression of these proteins attenuated miR-195-induced G1 arrest. In addition, miR-195 significantly repressed the phosphorylation of Rb as well as the transactivation of downstream target genes of E2F. These results imply that miR-195 may block the G1/S transition by repressing Rb-E2F signaling through targeting multiple molecules, including cyclin D1, CDK6, and E2F3. Conclusion: Our data highlight an important role of miR-195 in cell cycle control and in the molecular etiology of HCC, and implicate the potential application of miR-195 in cancer therapy. (HEPATOLOGY 2009.) [source] Despotism without Bounds: The French Secret Police and the Silencing of Dissent in London, 1760,1790HISTORY, Issue 296 2004SIMON BURROWS Through an examination of the policing of dissident French refugees in London between 1760 and 1790, this article contends that recent historians have tended to over-emphasize the reforming nature of the Bourbon government in the decades prior to the French Revolution, especially under Louis XVI, and overlooked the more repressive and ,despotic' aspects of the regime. It reveals that the Paris police or French secret agents adopted a variety of clandestine methods in their attempts to silence dissident exiles, including attempts at kidnap, and allegedly murder. As much of this police activity was reported in the British press and French printed texts, both before and during the French Revolution, and several of the exiles were celebrated writers or future revolutionary leaders, it was widely known among informed contemporaries. The article therefore contends that the French revolutionaries' allegations of despotism and suspicions of monarchic conspiracies were more deeply rooted in experience than recent historiography has tended to suggest. At the same time, reports of the attempts of the ,despotic' French government to suppress the activities of Frenchmen on British soil helped to reinforce a British national identity based on the celebration of the liberties France lacked. [source] Gastrin suppresses the interdependent expression of p16 and anion exchanger 1 favoring growth inhibition of gastric cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 6 2010Hua Tian Abstract Our previous studies demonstrated that expression and interaction of p16 with anion exchanger 1 (AE1) in gastric cancer cells is correlated with progression and shorter survival of the cancer. In this article, the effects of gastrin on p16 and AE1 and its implication in prevention and treatment of gastric cancer were studied by molecular biology techniques, animal experiment and clinical analysis. The results showed that expression of p16 in human gastric body carcinoma was downregulated along with the progression of the cancer, suggesting the reverse correlations between gastrin and p16 in vivo. Further experiments indicated that gastrin suppressed the expression of p16 via the p16 promoter and thereafter resulted in the degradation of AE1 in gastric cancer cells. Silencing of AE1 or p16 significantly inhibited the proliferation of the cancer cells. Using a xenograft tumor model in nude mice, we showed that experimental systemic hypergastrinemia induced by the administration of omeprazole led to decreased expression of AE1 and p16 as well as to a marked growth inhibition of SGC7901 tumors. It is concluded that a moderate plasma gastrin level is beneficial to the growth inhibition of gastric cancer by suppressing the expression of AE1 and p16. This finding may have an important implication for the prevention and treatment of cancers arise in the gastric antrum. [source] Biological indicators of prognosis in Ewing's sarcoma: An emerging role for lectin galactoside-binding soluble 3 binding protein (LGALS3BP)INTERNATIONAL JOURNAL OF CANCER, Issue 1 2010Diana Zambelli Abstract Starting from an experimental model that accounts for the 2 most important adverse processes to successful therapy of Ewing's sarcoma (EWS), chemoresistance and the presence of metastasis at the time of diagnosis, we defined a molecular signature of potential prognostic value. Functional annotation of differentially regulated genes revealed 3 major networks related to cell cycle, cell-to-cell interactions and cellular development. The prognostic impact of 8 genes, representative of these 3 networks, was validated in 56 EWS patients. High mRNA expression levels of HINT1, IFITM2, LGALS3BP, STOML2 and c-MYC were associated with reduced risk to death and lower risk to develop metastasis. At multivariate analysis, LGALS3BP, a matricellular protein with a role in tumor progression and metastasis, was the most important predictor of event-free survival and overall survival. The association between LGALS3BP and prognosis was confirmed at protein level, when expression of the molecule was determined in tumor tissues but not in serum, indicating a role for the protein at local tumor microenvironment. Engineered enhancement of LGALS3BP expression in EWS cells resulted in inhibition of anchorage independent cell growth and reduction of cell migration and metastasis. Silencing of LGALS3BP expression reverted cell behavior with respect to in vitro parameters, thus providing further functional validation of genetic data obtained in clinical samples. Thus, we propose LGALS3BP as a novel reliable indicator of prognosis, and we offer genetic signatures to the scientific communities for cross-validation and meta-analysis, which are indispensable tools for a rare tumor such as EWS. [source] Silencing of hSlo potassium channels in human osteosarcoma cells promotes tumorigenesisINTERNATIONAL JOURNAL OF CANCER, Issue 2 2008Béatrice Cambien Abstract Potassium channels, the most diverse superfamily of ion channels, have recently emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large conductance Ca2+ -activated K+ channels, often referred to as BK channels, are at the crossroads of several tumor-associated processes such as cell proliferation, survival, secretion and migration. Despite the high BK channel expression in osteosarcoma (OS), their function has not yet been investigated in this malignant bone pathology. Here, using stable RNA interference to reduce the expression of hSlo, the human pore-forming ,-subunit of the BK channel, in human Cal72 OS cells, we show that BK channels play a functional role in carcinogenesis. Our results reveal for the first time that BK channels exhibit antitumoral properties in OS in vivo and affect the tumor microenvironment through the modulation of both chemokine expression and leukocyte infiltration. © 2008 Wiley-Liss, Inc. [source] Silencing of APAF-1 in B-CLL results in poor prognosis in the case of concomitant p53 mutationINTERNATIONAL JOURNAL OF CANCER, Issue 9 2006Isrid Sturm Abstract Apoptosis protease-activating factor 1 (APAF-1), a transcriptional target of p53, is a cytosolic adaptor protein that links the mitochondrial apoptosis pathway to the caspase cascade. Here, we aimed to study the impact of APAF-1 expression levels on cell death induced by anticancer drugs or ionizing irradiation (IR) and disease prognosis in B-type chronic lymphocytic leukemia (B-CLL) patients. Samples from 138 patients with B-CLL were investigated for APAF-1 expression and p53 mutations. The results were related to survival data, in vitro cytotoxicity of various cytotoxic drugs and IR and clinico-pathological data. Variable APAF-1 expression was observed in all investigated B-CLL samples. Reduction in APAF-1 expression was observed at both mRNA and protein level indicating transcriptional silencing whereas mutation of p53 or the immunoglobulin heavy chain variable genes (IgHV) had no impact on APAF-1 expression. Surprisingly, APAF-1 loss did not result in resistance to cytotoxic therapies. Likewise, APAF-1 downregulation on its own showed no impact on disease prognosis. Nevertheless, a poor prognosis was observed in patients with loss of APAF-1 expression and additional p53 mutation. Thus, loss of APAF-1 may become relevant when additional core apoptosis signaling components are disrupted. © 2005 Wiley-Liss, Inc. [source] Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma,INTERNATIONAL JOURNAL OF CANCER, Issue 3 2005Joseph Kwong Abstract Tazarotene-induced gene 1 (TIG1) and Tazarotene-induced gene 3 (TIG3) are retinoid acid (RA) target genes as well as candidate tumor suppressor genes in human cancers. In our study, we have investigated the expression of TIG1 and TIG3 in nasopharyngeal carcinoma (NPC). Loss of TIG1 expression was found in 80% of NPC cell lines and 33% of xenografts, whereas TIG3 was expressed in all NPC samples and immortalized nasopharyngeal epithelial cells. In order to elucidate the epigenetic silencing of TIG1 in NPC, the methylation status of TIG1 promoter was examined by genomic bisulfite sequencing and methylation-specific PCR (MSP). We have detected dense methylation of TIG1 5,CpG island in the 5 TIG1 -negative NPC cell lines and xenograft (C666-1, CNE1, CNE2, HONE1 and X666). Partial methylation was observed in 1 NPC cell line HK1 showing dramatic decreased in TIG1 expression. Promoter methylation was absent in 2 TIG1 -expressed NPC xenografts and the normal epithelial cells. Restoration of TIG1 expression and unmethylated alleles were observed in NPC cell lines after 5-aza-2,-deoxycytidine treatment. Moreover, the methylated TIG1 sequence was detected in 39 of 43 (90.7%) primary NPC tumors by MSP. In conclusion, our results showed that TIG1 expression is lost in the majority of NPC cell lines and xenografts, while promoter hypermethylation is the major mechanism for TIG1 silencing. Furthermore, the frequent epigenetic inactivation of TIG1 in primary NPC tumors implied that it may play an important role in NPC tumorigenesis. [source] Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell linesINTERNATIONAL JOURNAL OF CANCER, Issue 1 2004Aniruddha Choudhury Abstract Silencing of a specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. We designed 3 such siRNA against the Her2/neu (HER2) gene. The HER2 gene is known to play an important role in the oncogenesis of several types of cancers, such as breast, ovarian, colon and gastric cancers. Introduction of the siRNA into HER2 positive tumor lines in vitro greatly reduced the cell surface expression of the HER2 protein. Concurrently, a range of effects on cell physiology, such as growth inhibition or apoptosis, was observed. The expression of HLA class I was observed to be upregulated when HER2 was silenced with siRNA. Treatment of SKBr3 and MCF7/HER2 tumor cell lines with the HER2 siRNA resulted in growth arrest of cells in the late G1/S-phase. Our results suggest that siRNA may be an effective method of abrogating the effect of HER2 in tumorigenesis. © 2003 Wiley-Liss, Inc. [source] Reciprocal regulation of transcription factors and PLC isozyme gene expression in adult cardiomyocytesJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6b 2010Tushi Singal Abstract By employing a pharmacological approach, we have shown that phospholipase C (PLC) activity is involved in the regulation of gene expression of transcription factors such as c-Fos and c-Jun in cardiomyocytes in response to norepinephrine (NE). However, there is no information available regarding the identity of specific PLC isozymes involved in the regulation of c-Fos and c-Jun or on the involvement of these transcription factors in PLC isozyme gene expression in adult cardiomyocytes. In this study, transfection of cardiomyocytes with PLC isozyme specific siRNA was found to prevent the NE-mediated increases in the corresponding PLC isozyme gene expression, protein content and activity. Unlike PLC ,1 gene, silencing of PLC ,1, ,3 and ,1 genes with si RNA prevented the increases in c-Fos and c-Jun gene expression in response to NE. On the other hand, transfection with c-Jun si RNA suppressed the NE-induced increase in c-Jun as well as PLC ,1, ,3 and ,1 gene expression, but had no effect on PLC ,1 gene expression. Although transfection of cardiomyocytes with c-Fos si RNA prevented NE-induced expression of c-Fos, PLC ,1 and PLC ,3 genes, it did not affect the increases in PLC ,1 and PLC ,1 gene expression. Silencing of either c-Fos or c-Jun also depressed the NE-mediated increases in PLC ,1, ,3 and ,1 protein content and activity in an isozyme specific manner. Furthermore, silencing of all PLC isozymes as well as of c-Fos and c-Jun resulted in prevention of the NE-mediated increase in atrial natriuretic factor gene expression. These findings, by employing gene silencing techniques, demonstrate that there occurs a reciprocal regulation of transcription factors and specific PLC isozyme gene expression in cardiomyocytes. [source] ADAM8 expression is associated with increased invasiveness and reduced patient survival in pancreatic cancerJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 5 2007N. Valkovskaya Abstract ADAM8 belongs to a family of transmembrane proteins implicated in cell,cell interactions, proteolysis of membrane proteins, and various aspects of carcinogenesis. In the present study, we aimed to evaluate the expression and function of ADAM8 in pancreatic cancer. ADAM8 mRNA levels were analysed by quantitative RT-PCR and correlated to patient survival. Immunohistochemistry was performed to localize ADAM8 in pancreatic tis-sues. Silencing of ADAM8 expression was carried out by transfection with specific siRNA oligonucleotides. Cell growth and invasion assays were used to assess the functional consequences of ADAM8 silencing. SELDI-TOF-MS was performed to detect the proteolytic activity of ADAM8 in pancreatic cancer cells. ADAM8 mRNA was significantly overexpressed in pancreatic ductal adenocarcinoma (PDAC) compared with normal pancreatic tissues (5.3-fold increase; P= 0.0008), and high ADAM8 mRNA and protein expression levels correlated with reduced survival time of PDAC patients (P= 0.048 and P= 0.065, respectively). Silencing of ADAM8 expression did not significantly influence pancreatic cancer cell growth but suppressed invasiveness. In addition, decreased proteolytic activity was measured in cell culture supernatants following silencing of ADAM8. In conclusion, ADAM8 is overexpressed in PDAC, influences cancer cell invasiveness and correlates with reduced survival, suggesting that ADAM8 might be a potential target in pancreatic cancer therapy. [source] Expression of RIZ1 protein (Retinoblastoma-interacting zinc-finger protein 1) in prostate cancer epithelial cells changes with cancer grade progression and is modulated in vitro by DHT and E2JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009Valentina Rossi The nuclear protein methyl-transferase Retinoblastoma-interacting zinc-finger protein 1 (RIZ1) is considered to be a downstream effector of estrogen action in target tissues. Silencing of RIZ1 expression is common in many tumors. We analyzed RIZ1 expression in normal and malignant prostate tissue and evaluated whether estradiol (E2) or dihydrotestosterone (DHT) treatment modulated RIZ1 in cultured prostate epithelial cells (PEC). Moreover, we studied the possible involvement of RIZ1 in estrogen action on the EPN prostate cell line, constitutively expressing both estrogen receptor (ER)-, and ,. RIZ1 protein, found in the nucleus of normal PECs by immunohistochemistry, was progressively lost in cancer tissues as the Gleason score increased and was only detected in the cytoplasmic compartment. RIZ1 transcript levels, as assayed by semi-quantitative RT-PCR in primary PEC cultures, were significantly reduced in cancer cells (P,<,0.05). In EPN DHT treatment significantly increased RIZ1 transcript and protein levels (P,<,0.05); E2 induced a reduction of S phase without significant changes of RIZ1 expression. In E2-treated EPN cell extracts RIZ co-immunoprecipitated with ER, and ER,. Our data demonstrate that RIZ1 is expressed in normal PECs and down-regulated in cancer cells, with a switch of its sub-cellular localization from the nucleus to the cytoplasm upon cancer grade progression. RIZ1 expression levels in the PECs were modulated by DHT or E2 treatment in vitro. Furthermore, the E2 effects on ER-expressing prostate cells involve RIZ1, which confirms a possible role for ER-mediated pathways in a non-classic E2 -target tissue. J. Cell. Physiol. 221: 771,777, 2009. © 2009 Wiley-Liss, Inc. [source] Tomato Fruit Development and Ripening Are Altered by the Silencing of LeEIN2 GeneJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 12 2006Hong-Liang Zhu Abstract Loss-of-function ethylene insensitive 2 (EIN2) mutations showed ethylene insensitivity in Arabidopsis, which indicated an essential role of EIN2 in ethylene signaling. However, the function of EIN2 in fruit ripening has not been investigated. To gain a better understanding of EIN2, the temporal regulation of LeEIN2 expression during tomato fruit development was analyzed. The expression of LeEIN2 was constant at different stages of fruit development, and was not regulated by ethylene. Moreover, LeEIN2-silenced tomato fruits were developed using a virus-induced gene silencing fruit system to study the role of LeEIN2 in tomato fruit ripening. Silenced fruits had a delay in fruit development and ripening, related to greatly descended expression of ethylene-related and ripening-related genes in comparison with those of control fruits. These results suggested LeEIN2 positively mediated ethylene signals during tomato development. In addition, there were fewer seeds and locules in the silenced fruit than those in the control fruit, like the phenotype of parthenocarpic tomato fruit. The content of auxin and the expression of auxin-regulated gene were declined in silenced fruit, which indicated that EIN2 might be important for crosstalk between ethylene and auxin hormones. (Managing editor: Li-Hui Zhao) [source] Sp proteins play a critical role in histone deacetylase inhibitor-mediated derepression of CYP46A1 gene transcriptionJOURNAL OF NEUROCHEMISTRY, Issue 2 2010Maria João Nunes J. Neurochem. (2010) 113, 418,431. Abstract We investigated whether the CYP46A1 gene, a neuronal-specific cytochrome P450, responsible for the majority of brain cholesterol turnover, is subject to transcriptional modulation through modifications in histone acetylation. We demonstrated that inhibition of histone deacetylase activity by trichostatin A (TSA), valproic acid and sodium butyrate caused a potent induction of both CYP46A1 promoter activity and endogenous expression. Silencing of Sp transcription factors through specific small interfering RNAs, or impairing Sp binding to the proximal promoter, by site-directed mutagenesis, led to a significant decrease in TSA-mediated induction of CYP46A1 expression/promoter activity. Electrophoretic mobility shift assay, DNA affinity precipitation assays and chromatin immunoprecipitation assays were used to determine the multiprotein complex recruited to the CYP46A1 promoter, upon TSA treatment. Our data showed that a decrease in Sp3 binding at particular responsive elements, can shift the Sp1/Sp3/Sp4 ratio, and favor the detachment of histone deacetylase (HDAC) 1 and HDAC2 and the recruitment of p300/CBP. Moreover, we observed a dynamic change in the chromatin structure upon TSA treatment, characterized by an increase in the local recruitment of euchromatic markers and RNA polymerase II. Our results show the critical participation of an epigenetic program in the control of CYP46A1 gene transcription, and suggest that brain cholesterol catabolism may be affected upon treatment with HDAC inhibitors. [source] Silencing of choline acetyltransferase expression by lentivirus-mediated RNA interference in cultured cells and in the adult rodent brainJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2009Julie Santamaria Abstract RNA interference (RNAi) is a potent mechanism for local silencing of gene expression and can be used to study loss-of-function phenotypes in mammalian cells. We used RNAi to knockdown specifically the expression of choline acetyltransferase (ChAT), the enzyme of acetylcholine biosynthesis, both in cultured cells and in the adult brain. We first identified a 19-nucleotide sequence in the coding region of rat and mouse ChAT transcripts that constitutes a target for potent silencing of ChAT expression by RNAi. We generated a lentiviral vector that produces both a small hairpin RNA (shRNA) targeting ChAT mRNAs and the enhanced green fluorescent protein (EGFP) reporter protein to facilitate identification of transduced cells. In the cholinergic cell line NG108-15, there was at least 90% less of the ChAT protein, as measured by assaying its enzymatic activity, 3 days postinfection with this vector than in cells infected with a control vector. The vector was used to transduce cholinergic neurons in vivo and reduced ChAT expression strongly and specifically in the cholinergic neurons of the medial septum in adult rats, without affecting the expression of the vesicular acetylcholine transporter. This lentiviral vector is thus a powerful tool for specific inactivation of cholinergic neurotransmission and can therefore be used to study the role of cholinergic nuclei in the brain. This lentiviral-mediated RNAi approach will also allow the development of new animal models of diseases in which cholinergic neurotransmission is specifically altered. © 2008 Wiley-Liss, Inc. [source] SGT1 positively regulates the process of plant cell death during both compatible and incompatible plant,pathogen interactionsMOLECULAR PLANT PATHOLOGY, Issue 5 2010KERI WANG SUMMARY SGT1 (suppressor of G2 allele of Skp1), an interactor of SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes that mediate protein degradation, plays an important role at both G1,S and G2,M cell cycle transitions in yeast, and is highly conserved throughout eukaryotes. Plant SGT1 is required for both resistance (R) gene-mediated disease resistance and nonhost resistance to certain pathogens. Using virus-induced gene silencing (VIGS) in Nicotiana benthamiana, we demonstrate that SGT1 positively regulates the process of cell death during both host and nonhost interactions with various pathovars of Pseudomonas syringae. Silencing of NbSGT1 in N. benthamiana plants delays the induction of hypersensitive response (HR)-mediated cell death against nonhost pathogens and the development of disease-associated cell death caused by the host pathogen P. syringae pv. tabaci. Our results further demonstrate that NbSGT1 is required for Erwinia carotovora - and Sclerotinia sclerotiorum -induced disease-associated cell death. Overexpression of NbSGT1 in N. benthamiana accelerates the development of HR during R gene-mediated disease resistance and nonhost resistance. Our data also indicate that SGT1 is required for pathogen-induced cell death, but is not always necessary for the restriction of bacterial multiplication in planta. Therefore, we conclude that SGT1 is an essential component affecting the process of cell death during both compatible and incompatible plant,pathogen interactions. [source] DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell deathMOLECULAR PLANT PATHOLOGY, Issue 3 2007CHANG-SIK OH SUMMARY DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato, which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1, the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1, or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora. E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana, and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1 -silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth. [source] Involvement of the p110, isoform of PI3K in early development of mouse embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2009Xiao-yan Xu Class I of phosphoinositide 3-kinases (PI3Ks) is characterized as a group of intracellular signal proteins possessing both protein and lipid kinase activities. Recent studies implicate class I of PI3Ks acts as indispensable mediators in early development of mouse embryos, but the molecular mechanisms are poorly defined. In this paper, mouse one-cell embryos were used to investigate a possible contribution of the catalytic subunit of PI3K, p110,, to cell cycle progression. The expression level of p110, was determined in four phases of one-cell embryos. Silencing of p110, by microinjection of p110, shRNA into one-cell embryos resulted in a G2/M arrest and prevented the activation of Akt and M-phase promoting factor (MPF). Further, microinjection of the synthesized mRNA coding for a constitutively active p110, into one-cell embryos induced cell cleavage more effectively than microinjection of wild-type p110, mRNA, whereas microinjection of mRNA of kinase-deficient p110, delayed the first mitotic cleavage. Taken together, this study demonstrates that p110, is significant for G2/M transition of mouse one-cell embryos and further emphasizes the importance of Akt in PI3K pathway. Mol. Reprod. Dev. 76: 389,398, 2009. © 2008 Wiley-Liss, Inc. [source] DNA Methylation, Genomic Silencing, and Links to Nutrition and CancerNUTRITION REVIEWS, Issue 6 2005Dale C. McCabe DNA methylation is a heritable epigenetic feature that is associated with transcriptional silencing, X-chromosome inactivation, genetic imprinting, and genomic stability. The addition of the methyl group is catalyzed by a family of DNA methyltransferases whose cosubstrates are DNA and S-adenosylmethionine, the latter being derived from the methionine cycle. Aberrant DNA methylation is linked to numerous pathologies, including cancer. The purpose of this review is to describe DNA methylation and its functions, to examine the relationship between dietary methyl insufficiency and DNA methylation, and to evaluate the associations between DNA methylation and cancer. [source] Quantitative assessment of RUNX3 methylation in neoplastic and non-neoplastic gastric epithelia using a DNA microarrayPATHOLOGY INTERNATIONAL, Issue 10 2006Kanji So Silencing of the RUNX3 gene by hypermethylation of its promoter CpG island plays a major role in gastric carcinogenesis. To quantitatively evaluate RUNX3 methylation, a fiber-type DNA microarray was used on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the RUNX3 promoter CpG island, at which methylation is critical for gene silencing, was amplified by polymerase chain reaction using a Cy5 end-labeled primer. Methylation rates (MR) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Five gastric cancer cell lines were analyzed, as well as 26 primary gastric cancers and their corresponding non-neoplastic gastric epithelia. MR in four of the cancer cell lines that lost RUNX3 mRNA ranged from 99.0% to 99.7% (mean, 99.4%), whereas MR in the remaining cell line that expressed RUNX3 mRNA was 0.6%. In primary gastric cancers and their corresponding non-neoplastic gastric epithelia, MR ranged from 0.2% to 76.5% (mean, 22.7%) and from 0.7% to 25.1% (mean, 5.5%). Ten (38.5%) of the 26 gastric cancers and none of their corresponding non-neoplastic gastric epithelia had MR >30%. Most of the samples with MR >10% tested methylation-positive by conventional methylation-specific polymerase chain reaction (MSP). This microarray-based methylation assay is a promising method for the quantitative assessment of gene methylation. [source] Methylation-Associated Silencing of Death-Associated Protein Kinase Gene in Laryngeal Squamous Cell Cancer,THE LARYNGOSCOPE, Issue 8 2005Wei-Jia Kong MD Abstract Objectives/Hypothesis: Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated Ser/Thr kinase that functions as a positive mediator of programmed cell death. It has been found that DAPK gene is frequently inactivated by its promoter hypermethylation in some cancers and tumor cell lines. However, it is not clear whether promoter hypermethylation of DAPK gene exists in laryngeal squamous cell cancer (LSCC). The aim of this study was to investigate the promoter methylation status of the DAPK gene in LSCC and the effect of 5-Aza-2'-deoxycytidine (5-Aza-CdR), a demethylating agent, on Hep-2 cells, a human laryngeal cancer cell line, and on xenografts of Hep-2. Methods: Methylation-specific polymerase chain reaction (PCR) and reverse-transcription PCR techniques were used to determine the promoter methylation status and mRNA expression of DAPK gene in LSCC. Furthermore, Hep-2 cells in vitro and in vivo were treated by 5-Aza-CdR to explore the effect of demethylating agents on DAPK mRNA expression and tumor growth. Results: Hypermethylation of DAPK gene promoter was found in 39 (67.2%) of 58 LSCC samples. There was no significant difference in the promoter hypermethylation rate among the samples of different histologic grades or samples from patients with different T stages. However, there was significant difference in methylation status of DAPK gene between the samples from patients in N0 stages and those from patients in N1 stages. No promoter hypermethylation of DAPK gene was found in any of the five normal laryngeal tissue samples. DAPK mRNA expression was not detected in tumor specimens with promoter hypermethylation. On the contrary, DAPK mRNA expression was observed in the unmethylated tumor specimens, specimens from tissues adjacent to the tumor, and normal laryngeal tissues samples. Promoter hypermethylation of DAPK gene was found, and no DAPK mRNA expression was detected in Hep-2 cells. DAPK mRNA expression in Hep-2 cells and xenografts could be restored by treating cells and xenografts with 5-Aza-CdR. The tumors' xenografts, induced by way of Hep-2 cell injection in nude mice treated with 5-Aza-CdR, were obviously smaller than those in nude mice treated with phosphate-buffered saline. Conclusions: Abnormal loss of DAPK expression could be associated with aberrant promoter region methylation in the LSCC. 5-Aza-CdR may slow the growth of Hep-2 cells in vitro and in vivo by reactivating tumor suppressor gene DAPK silenced by de novo methylation. [source] The chloroplast protein RPH1 plays a role in the immune response of Arabidopsis to Phytophthora brassicaeTHE PLANT JOURNAL, Issue 2 2009Khaoula Belhaj Summary Plant immune responses to pathogens are often associated with enhanced production of reactive oxygen species (ROS), known as the oxidative burst, and with rapid hypersensitive host cell death (the hypersensitive response, HR) at sites of attempted infection. It is generally accepted that the oxidative burst acts as a promotive signal for HR, and that HR is highly correlated with efficient disease resistance. We have identified the Arabidopsis mutant rph1 (resistance to Phytophthora 1), which is susceptible to the oomycete pathogen Phytophthora brassicae despite rapid induction of HR. The susceptibility of rph1 was specific for P. brassicae and coincided with a reduced oxidative burst, a runaway cell-death response, and failure to properly activate the expression of defence-related genes. From these results, we conclude that, in the immune response to P. brassicae, (i) HR is not sufficient to stop the pathogen, (ii) HR initiation can occur in the absence of a major oxidative burst, (iii) the oxidative burst plays a role in limiting the spread of cell death, and (iv) RPH1 is a positive regulator of the P. brassicae -induced oxidative burst and enhanced expression of defence-related genes. Surprisingly, RPH1 encodes an evolutionary highly conserved chloroplast protein, indicating a function of this organelle in activation of a subset of immune reactions in response to P. brassicae. The disease resistance-related role of RPH1 was not limited to the Arabidopsis model system. Silencing of the potato homolog StRPH1 in a resistant potato cultivar caused susceptibility to the late blight pathogen Phytophthora infestans. [source] Local DNA features affect RNA-directed transcriptional gene silencing and DNA methylationTHE PLANT JOURNAL, Issue 1 2008Ute Fischer Summary Transcription of a nopaline synthase promoter (pNOS) inverted repeat provides an RNA signal that can trigger transcriptional gene silencing and methylation of pNOS promoters in trans. The degree of silencing is influenced by the local DNA features close to the target promoter integration sites. Among 26 transgenic Arabidopsis thaliana lines harbouring single copies of a T-DNA including a pNOS- NPTII reporter gene at different chromosomal loci, NPTII RNA levels showed limited variation. When challenged by the silencer transgene providing the pNOS RNA signal, reduction of the NPTII RNA levels in the F1 generation varied by more than 100-fold, ranging from no reduction to reduction to <1% of the non-silenced level. Silencing was generally correlated with proportional DNA methylation in the pNOS region, except for one target transgene showing substantial DNA methylation without adequate silencing. Silencing was progressive through generations. Differences in the degree of silencing among the target transgenes were transmitted at least to the F3 generation, and seemed to be influenced by transgene-flanking sequences. Apparently, close-by repeats promoted, whereas close-by functional genes diminished, the response to the silencing signal. [source] Endogenous isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicumTHE PLANT JOURNAL, Issue 2 2006Senthil Subramanian Summary Legume iso/flavonoids have been implicated in the nodulation process, but questions remain as to their specific role(s), and no unequivocal evidence exists showing that these compounds are essential for nodulation. Two hypotheses suggest that the primary role of iso/flavonoids is their ability to induce rhizobial nod gene expression and/or their ability to modulate internal root auxin concentrations. The present work provides direct, genetic evidence that isoflavones are essential for nodulation of soybean roots because of their ability to induce the nodulation genes of Bradyrhizobium japonicum. Expression of isoflavone synthase (IFS), a key enzyme in the biosynthesis of isoflavones, is specifically induced by B. japonicum. When IFS was silenced using RNA interference in soybean hairy root composite plants, these plants had severely reduced nodulation. Surprisingly, pre-treatment of B. japonicum or exogenous application to the root system of either of the major soybean isoflavones, daidzein or genistein, failed to restore normal nodulation. Silencing of chalcone reductase led to very low levels of daidzein and increased levels of genistein, but did not affect nodulation, suggesting that the endogenous production of genistein was sufficient to support nodulation. Consistent with a role for isoflavones as endogenous regulators of auxin transport in soybean roots, silencing of IFS resulted in altered auxin-inducible gene expression and auxin transport. However, use of a genistein-hypersensitive B. japonicum strain or purified B. japonicum Nod signals rescued normal nodulation in IFS-silenced roots, indicating that the ability of isoflavones to modulate auxin transport is not essential to nodulation. 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