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CIDE-B [cell death-inducing DFF45 (DNA fragmentation factor 45)-like effector B] is a member of the CIDE family of apoptosis-inducing factors. The highly restricted pattern of expression of CIDE-B in the liver and spleen suggests that a mechanism exists for the tissue- and cell-specific regulation of transcription of this gene. We have analysed the promoters of the human CIDE-B gene, particularly the mechanism of cell-specific transcription. Expression of CIDE-B is driven by two promoters which are responsible for the synthesis of two types of transcript, and Sp1 and Sp3 are key regulators of basal transcription from both the upstream and the internal promoter, as indicated by EMSAs (electrophoretic mobility-shift assays) and site-directed mutagenesis. Bisulphite sequencing analysis demonstrated that the upstream promoter was hypermethylated in cells that did not express the long transcript of CIDE-B, but was hypomethylated in cells that expressed this transcript. Furthermore, methylation of this region in vitro reduced the promoter activity to ∼5% of the control. Thus methylation at CpG sites in the upstream promoter region appeared to be important for cell-specific synthesis of the long transcript. By contrast, HNF4α (hepatocyte nuclear factor-4α) bound to the internal promoter and enhanced its activity. Moreover, the short transcript of CIDE-B gene was expressed in cells which do not normally express this transcript upon introduction of exogenous HNF4α, demonstrating the involvement of HNF4α in the cell-specific synthesis of the short transcript. Thus our analysis revealed a novel mechanism for the cell-specific transcription of the human CIDE-B gene, which involves epigenetic and genetic control at separate respective promoters.
- cell death-inducing DFF45 (DNA fragmentation factor 45)-like effector (CIDE)
- cell-specific expression
- DNA methylation
- promoter
- hepatocyte nuclear factor-4 (HNF4)
- Sp1
Apoptosis or programmed cell death is an evolutionarily conserved phenomenon that removes redundant cells produced during animal development [1]. Changes in this normal process can result in the disruption of the delicate balance between cell proliferation and cell death, and can lead to a variety of diseases, including cancers, neurodegenerative disorders, autoimmune diseases and viral infections [2,3]. A novel family of cell death-inducing DFF45 (DNA fragmentation factor 45)-like effectors (CIDEs) has recently been identified, and it includes CIDE-A, CIDE-B, and CIDE-C (originally named CIDE-3) in humans, and fat-specific gene 27 (FSP27, a homologue of the human CIDE-C gene) in mice [4–6]. CIDEs share a conserved animo acid sequence similar to the CIDE-N domains in DFF40/CAD (caspase-activated nuclease) and its inhibitor [DFF45/ICAD (inhibitor of CAD)], which are two subunits of DFF complex [6–8]. Cleavage of DFF45/ICAD by caspase 3 releases DFF40/CAD from the complex and triggers DNA fragmentation and nuclear condensation [9,10]. The structure of the N-terminal domain of CIDE-B suggests that this domain might serve as a weak-interaction interface or regulatory domain [11]. Overexpression of CIDE-B results in cell death associated with the fragmentation of DNA [5]. Such CIDE-B-induced apoptosis can be inhibited by the NS2 (non-structural protein 2) of HCV (hepatitis C virus) via interaction with the C-terminal domain of CIDE-B [12]. This domain is conserved in CIDEs, and is responsible for the mitochondrial localization and dimerization of CIDE-B and CIDE-B-induced cell death [13].
According to previous reports, the expression of CIDEs is strongly tissue specific. Two cDNA variants have been reported that encode human CIDE-B [5,11]. The major short transcript of CIDE-B was detected in adult and fetal liver, whereas the long transcript was detected at lower levels in fetal liver, spleen, peripheral blood lymphocytes and bone marrow [5]. Other CIDE genes also have multiple transcripts with different levels of tissue-specific expression. For example, the major large transcript of human CIDE-C was detected in the small intestine, heart, colon and stomach, whereas a small transcript was detected at a lower level in placenta [6]. Thus the transcription of CIDE genes appears to be regulated in a strictly tissue- and cell-specific manner. However, complete analysis of the regulation of transcription of human CIDE genes has not yet been performed. Studies of the adipocyte-specific gene Fsp27, a member of the mouse CIDE family, showed that the expression of Fsp27 was regulated by C/EBP (CCAAT/enhancer-binding protein) and other C/EBP-like transcription factors [4], and that the expression of the gene was strongly induced in PPARα−/− (peroxisome-proliferator-activated receptor α−/−) mouse livers with PPARγ1 overexpression [14]. It is of interest that both C/EBP and PPARγ are critical transcription factors in adipogenesis. These observations suggest that certain tissue-specific transcription factors might be involved in the activation of CIDE genes.
In addition to the genetic regulation of gene activation that involves transcription factors, epigenetic controls provide another important mechanism for the tissue- and cell-specific expression of genes. Major epigenetic mechanisms include DNA methylation and histone modification. In mammalian cells, DNA methylation occurs predominantly at cytosine residues in the dinucleotide sequence CpG, and such methylation regulates gene expression through several distinct mechanisms. It can act directly by blocking regulatory factors from binding to their target sequences, and it can repress gene expression via the actions of MeCPs (methyl-CpG-binding proteins) (reviewed in [15]). Moreover, the apoptotic pathway can be inactivated via DNA methylation [16], and several apoptosis-associated genes (BNIP3 [17], DAPK [18], p14ARF [19], EDNRB [20], RASSF1A [21], CDH1 [22], TMS1 [23], and Apaf-1 [24]) whose expression is regulated directly or indirectly by methylation have been described. However, there are no reports, to our knowledge, of the epigenetic control of the expressions of CIDEs, and the mechanisms that regulate the specific expression of CIDE genes have not been conclusively defined.
We present here an analysis of the regulatory region of the human CIDE-B gene, and demonstrate that the cell-specific expression of two transcripts of CIDE-B is driven by upstream and internal promoters (Pu and Pi respectively) by epigenetic and genetic mechanisms respectively. Expression of the long transcript is regulated by DNA methylation of the Pu region, whereas that of the short transcript is activated by HNF4α (hepatocyte nuclear factor α), a nuclear receptor that is required for the differentiation of mammalian hepatocyte and for the normal regulation of liver metabolism [25], via interaction with its RE (response element) in the Pi. Our results also show that Sp1 and Sp3 are key regulators that are required for basal activation of both promoters.
Cell culture
All cell lines used in this study were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were maintained in our laboratory. BEL-7404 cells (human hepatocellular carcinoma), HEK-293T cells and HeLa cells were grown in DMEM (Dulbecco's modified Eagle's medium) (Gibco BRL, Grand Island, NY, U.S.A.), Jurkat cells and SMMC-7721 (human hepatocellular carcinoma) cells were maintained in RPMI 1640 medium (Gibco BRL), and HepG2 cells were maintained in MEM (minimum essential medium) (Gibco BRL). All media were supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (Gibco BRL). All the cells were cultured at 37 °C in 5% CO2.
RNA analysis
Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.). Total RNA (5 μg) was reverse transcribed using oligo(dT)18 primer and SuperScript II First-Strand Synthesis System (Invitrogen) in a reaction volume of 20 μl, according to the manufacturer's instruction. The primers used for the RT (reverse transcription)-PCR CIDE-B, HNF4α and β-actin and the PCR protocols are shown in Table 1.
CpG island predictions
The presence of a putative CpG island in proximity to the CIDE-B gene was analysed by the CpG Island Searcher program (http://www.uscnorris.com/cpgislands/cpg.cgi) [26] with the default setting (%GC>55%, ObsCpG/ExpCpG>0.65, length>500 bp).
To clone the promoter region of the human CIDE-B gene, a 1534 bp genomic fragment (from −4177 to −2644 relative to the translation start site), containing the 5′ upstream region and exon 1 of the CIDE-B gene, was amplified by PCR, using high-fidelity LA Taq polymerase (Takara, Dalian, China) and human genomic DNA from BEL-7404 cells. A 2642 bp genomic fragment (from −2642 to −1) between a CpG island and the translation start site of the CIDE-B gene was amplified in a similar manner. Various deletions of the CIDE-B promoters were created using these two fragments as templates for PCR amplifications. Also, XhoI and HindIII restriction sites were added to the 5′ end of the sense and antisense primers respectively. The amplified fragments were ligated into the XhoI–HindIII site of the pGL3-Basic vector (Promega, Madison, WI, U.S.A.) and the sequences of the products were confirmed by direct sequencing.
Site-directed mutagenesis of Pu-Sp1 (−3452/−3457) in p(−3628/−3345), Pi-Sp1 (−193/−188) in p(−204/−1), and two HNF4α REs (RE1, −142/−130; RE2, −174/−162) in p(−204/−1) was introduced by PCR, and the mutagenesis confirmed by direct sequencing. Detailed procedures and conditions and various primer sequences are available upon request from the authors.
Expression vectors for human HNF4α and DN-HNF4 (dominant-negative HNF4α) [27] were kindly provided by Dr Todd Leff (Department of Pathology, Wayne State University School of Medicine, Detroit, MI, U.S.A.).
Methylation in vitro
The CIDE-B Pu-reporter construct and the pGL3-Basic vector were methylated by incubation with SssI methylase (New England BioLabs, Beverly, MA, U.S.A.) for 3 h at 37 °C in the presence of 160 μM S-adenosylmethionine. The methylation status was verified by digestion with a methylation-sensitive enzyme, HapII, or an insensitive enzyme, MspI.
Transfections and luciferase assays
For transient transfections, BEL-7404 and HEK-293T cells were plated into 24-well plates the day before transfection at a density of 4×104 and 2×105 cells/well respectively. Cells were transfected with 0.4 μg of various luciferase constructs using Lipofectamine™ reagent (Gibco BRL) according to the manufacturer's instructions. The pRL-TK plasmid (Promega; 40 ng per sample) containing the Renilla luciferase gene driven by the herpes simplex virus thymidine kinase promoter was co-transfected with the constructs, and the luciferase activity was normalized. In addition, 0.15 μg of HNF4α or DN-HNF4α expression vector, or the empty vector, was included in co-transfection experiments. The preparation of cell lysates and measurements of luciferase activity were performed by using the Dual Reporter assay system (Promega) and Lumat LB 9507 luminometer (EG and G. Berthold, Berlin, Germany) according to the manufacturers' instructions. All transfections were performed three times in triplicate.
Sodium bisulphite genomic sequencing
Sodium bisulphite modification of genomic DNA was performed as described previously [28]. Briefly, 2 μg of genomic DNA, which had been digested with EcoRI and EcoRV, was denatured in 0.3 M NaOH for 15 min at 37 °C and then treated with 3 M sodium bisulphite (Sigma, St. Louis, MD, U.S.A.) and 10 mM hydroquinone (Sigma) for 16 h at 50 °C. The DNA was then desalted using Wizard DNA Clean-Up System (Promega), desulphonated in 0.3 M NaOH and precipitated. The treated DNA was amplified by PCR in a 50 μl reaction volume with the bisulphite-specific primers 5′-TTTATTTGAGTTTGGAGATTTTGAT-3′ (sense) and 5′-AAATATCCCAAAACACTAACCTCTA-3′ (antisense). The purified PCR products were cloned into the pMD 18-T vector (Takara), and five clones were sequenced for each sample.
EMSAs (electrophoretic mobility shift assays) and protein analysis
HEK-293T cells were transiently transfected with 10 μg of HNF4α expression vector or empty vector pcDNA3, as described above. Nuclear extracts were prepared from BEL-7404 and HEK-293T cells as described by Gazzoli and Kolodner [29]. Nuclear extracts containing 10 μg of protein were pre-incubated in 20 μl of binding buffer (10 mM Tris/HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 50 μg/ml poly(dI-dC)·poly(dI-dC) and 4% glycerol) with or without unlabelled competitor (a 200-fold molar excess). For supershift assays, 1 μg of antibody or rabbit IgG (Sigma) was added to the pre-incubation mixture. After 30 min pre-incubation on ice, the DNA probe (20000 cpm) labelled with [α-32P]dCTP was added, and the samples were incubated at room temperature for 30 min. Reaction mixtures were separated on 4% polyacrylamide (Sp1/Sp3 assay) or 6% polyacrylamide (HNF4α assay) gels. The gel was dried and subjected to PhosphorImage analysis using a Storm 860 system and ImageQuant TL software (Amersham Biosciences, Sunnyvale, CA, U.S.A.). Antibodies used in supershift assays were rabbit polyclonal antibodies against Sp1 (sc-59X), Sp3 (sc-644) and HNF4α (sc-8987X) from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The sequences of oligonucleotides used in EMSAs were as follows (binding sites of transcription factors are underlined): Pi-Sp1, 5′-AGGACCCCATCTGGCCCCTCCCTCATCCCTCCC-3′ and 5′-AAGGGGAGGGATGAGGGAGGGGCCAGATGGGGTC-3′; and HNF4α RE1, 5′-CCAGGCAGGGGGGCCAGAGTCCAGGCTTGACTCATTC-3′ and 5′-GGGAATGAGTCAAGCCTGGACTCTGGCCCCCCTGC-3′. Mutant probes were created by changing the Sp1-binding sequence from CCTCCC to CTTTTC to create Sp1m and by changing HNF4α RE1 from GGGCCAGAGTCCA to GGGCTTTTTTCTT to create HNF4α RE1m.
For Western blotting analysis, total cell samples were harvested by applying 2×SDS Laemmli sample buffer directly on to cell monolayers after three washes with PBS. Electrophoresis, transfer of proteins on to nitrocellulose membranes and blocking of membranes were performed as described previously [30]. Antibodies specifically against HNF4α (anti-HNF4α, sc-8987X) and tubulin (Ab-4; NeoMarkers, Freemont, CA, U.S.A.) were incubated, at dilutions of 1:10000 and 1:500 respectively, with the membranes for 1 h at room temperature. Secondary antibody [goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz)] was used at a dilution of 1:2000, and incubated with membranes for 1 h at room temperature. Proteins revealed by Western blotting were visualized by chemiluminescence.
Cell-specific control of the generation of two transcripts of CIDE-B, and the structure of the CIDE-B locus
To confirm that the transcription of the CIDE-B gene is regulated in a tissue- and cell-specific manner, we investigated the expression of CIDE-B in various cell lines using the primers for CIDE-B CDS (coding sequence), the long and the short transcript of CIDE-B respectively (Table 1). RT-PCR analysis revealed that BEL-7404 and HepG2 cells expressed CIDE-B mRNA, whereas SMMC-7721, HEK-293T, HeLa and Jurkat cells did not (Figure 1A). Furthermore, both the long and the short transcript were detected in BEL-7404 cells, but the long version was not detected in HepG2 cells (Figure 1B).
The sequences corresponding to the two reported cDNAs that encode CIDE-B [5,11] were mapped to chromosome 14q11. As shown in Figure 1(C), the short transcript [5] consists of five exons (exons 3A, 4, 5, 6 and 7), whereas the long version [11] includes two additional 5′ untranslated exons (exon 1 and exon 2) and a variant exon 3 (exon 3B). Both transcripts have the same ORF (open reading frame). This region also includes two other genes located on the reverse strand of CIDE-B [31]. These genes encode the leukotriene B4 receptor (LTB4R; GenBank® accession no. D89079) and LTB4R2 (GenBank® accession no. NM_019839). The reported transcription initiation site of the LTB4R gene [32] is located only 113 bp upstream from the first exon of CIDE-B.
The CpG Island Searcher program revealed a CpG island (from −3965 to −2674) spanning the 5′ upstream area and exon 1 of the CIDE-B gene. To simplify the nomenclature, we attributed the position number +1 to the translation start site of the CIDE-B gene.
Identification of the Pu of CIDE-B
We first constructed a luciferase reporter plasmid [p(−4177/−2644)] that contained a 1534 bp genomic DNA fragment (from −4177 to −2644), spanning the 5′ upstream region and exon 1 of CIDE-B. This plasmid was used for transient transfection of BEL-7404 and HEK-293T cells. BEL-7404 cells expressed CIDE-B, whereas HEK-293T cells did not (Figure 1). The cloned fragment exhibited significant promoter activity in both cell lines, indicating that we had isolated an active Pu sequence (Figure 2A). Next, we created a series of deletion constructs to identify the minimal promoter required for activation. As shown in Figure 2(A), deletion in the 3′ region from −3345 to −3478 completely abolished the activity of the reporter gene, and allowed us to locate the minimal Pu within the 284 bp region between positions −3628 and −3345.
The minimal Pu overlapped, on the reverse strand, the LTB4R promoter. The overlapping region contains an Sp1-binding site that has been shown to bind both Sp1 and Sp3, with activation of the basal transcription of LTB4R [32]. To investigate the role of this element in the activity of the Pu, deletion and site-directed mutagenesis of the Sp1 site were performed. In both BEL-7404 and HEK-293T cells, the luciferase activities of the mutated constructs were reduced to ∼30% or even less of the wild-type activity (Figure 2B), indicating that Sp1 and Sp3 are major regulators of the expression of both CIDE-B and LTB4R.
Methylation of CpG sites in the Pu
The Pu is in a CpG island and partially overlaps the LTB4R promoter, whose activity is influenced by DNA methylation [32]. We then examined the methylation status of 32 CpG sites in the minimal Pu region by sodium bisulphite sequencing. As shown in Figure 3(B), the overall percentage of methylated CpG sites was only 10% in BEL-7404 cells, and thus this region was almost unmethylated. By contrast, in the cells in which the long transcript of CIDE-B was silenced (HepG2, SMMC-7721, HEK-293T, HeLa and Jurkat cells), there was a high degree of methylation at each CpG site examined. These results led us to the conclusion that methylation inhibits the synthesis of the long transcript of CIDE-B. Thus the effect of methylation on the Pu activity was investigated. We modified promoter constructs with SssI methylase and examined the activity of the methylated promoter. When we introduced these constructs into BEL-7404 cells, the activity of the methylated construct was only ∼5% of that of the unmethylated construct (Figure 3C). We obtained similar results after transfection of HEK-293T cells with these constructs. These observations support the conclusion that methylation of the CpG sites inhibited the activity of the Pu.
Identification of the Pi of CIDE-B
The 5′ untranslated regions of the two transcripts of CIDE-B are different, although the translation start site is identical (Figure 1C). Moreover, the transcripts have different tissue- [5] and cell-specific (Figure 1B) expression patterns. These observations suggest that the expression of the short transcript might be regulated by another cryptic promoter.
To test this hypothesis, we constructed a luciferase reporter plasmid [p(−2642/−1)] that contained the 2.6 kb region (positions −2642 to −1) between the CpG island and the translation start site, as well as several constructs that contained fragments of this region with stepwise deletions from the 5′ and 3′ side. These plasmids were used for transient transfection of BEL-7404 and HEK-293T cells. The luciferase activity of the 2.6 kb promoter construct [p(−2642/−1)] was approx. 10-fold higher than that of a promoterless control construct (pGL3-Basic) in BEL-7404 cells, whereas the activity was approx. 3-fold higher than that of the control vector in HEK-293T cells, indicating that the 2.6 kb region contains an internal promoter with significant cell-specific activity (Figure 4A). The highest promoter activity was observed in BEL-7404 cells that had been transfected with the construct p(−204/−1), which contained the region between −204 and −1. This activity was completely abolished by further deletion of the region from −204 to −149. These results suggest that the cell-specific activity of the Pi promoter is determined by the region between −204 and −1, and that the region from −204 to −149 is critical for basal promoter activity. The nucleotide sequence of the Pi region is shown in Figure 4(B). There is only one CpG dinucleotide, located at positions −8/−9. We searched for transcription factor binding sites using the MatInspector program [33] and found consensus sequences for the binding of Sp1/Sp3, HNF4α, Ap1 and CREB (cAMP-response-element-binding protein).
Sp1 and Sp3 bind to the Pi and regulate its activity
Luciferase assays demonstrated that the region between −204 and −149 represented a cis element that is important in the basal transcription of the short transcript of CIDE-B. This region includes a consensus sequence for binding of Sp1 (Figure 4B). We first performed EMSAs to determine whether Sp1 and Sp3 bind to this region. Several DNA–protein complexes were observed in nuclear extracts of both BEL-7404 and HEK-293T cells (Figure 5A, lanes 2 and 10). These complexes represented sequence-specific interactions of proteins with this region, since the addition of a 200-fold molar excess of unlabelled probe eliminated the detectable complexes (Figure 5A, lanes 3 and 11), whereas an excess of an oligonucleotide with a mutated putative Sp1-binding site (5′-CTTTTC-3′ at −193 to −188; mutation sites are indicated in bold) did not compete away the binding (Figure 5A, lanes 4 and 12). The slowest migrating DNA–protein complex was supershifted by the Sp1-specific antibody (Figure 5A, lanes 6 and 14), whereas the two rapidly migrating bands were supershifted by the Sp3-specific antibody (Figure 5A, lanes 7 and 15). No supershift was observed, however, when we used control IgG instead of specific antibodies (Figure 5A, lanes 5 and 13). Our results showed that Sp1 and Sp3 bound to the Sp1 site at −193/−188 in the Pi. We next investigated the effect of Sp1 binding on the Pi activity by site-directed mutagenesis (Figure 5B). The luciferase activity of the mutated construct was decreased to ∼14% and ∼25% of the wild-type in BEL-7404 and HEK-293T cells respectively. These results indicated that Sp1 and Sp3 bound to the Pi and regulated basal promoter activity.
Transactivation of the Pi by HNF4α
Analysis of the sequence of the Pi revealed two potential REs of HNF4α (Figure 4B), a nuclear receptor that is expressed at high level in liver. RT-PCR analysis showed high expression levels of HNF4α mRNA in HepG2 and BEL-7404 cells and very weak expression in SMMC-7721 cells, whereas there was no expression of HNF4α mRNA in HEK-293T, HeLa and Jurkat cells (Figure 6A). Given the significant activity of the Pi in liver cells, and the similar expression patterns of CIDE-B short transcript and HNF4α, we postulated that HNF4α might be involved in the synthesis of the short transcript of CIDE-B.
To test our hypothesis, the BEL-7404 and HEK-293T cells were transfected with the Pi reporter construct [p(−204/−1)] together with expression vectors for HNF4α or DN-HNF4α (Figure 6B). In BEL-7404 cells, the expression of exogenous HNF4α resulted in a 1.4-fold increase in luciferase activity, whereas expression of DN-HNF4α decreased the promoter activity to below the control (empty vector) level, suggesting that HNF4α can activate the Pi and that DN-HNF4 can repress the promoter activity by forming defective heterodimers with endogenous HNF4α [27]. More significant activation of luciferase activity by HNF4α was observed in HEK-293T cells (4-fold increase) (Figure 6B), confirming the importance of HNF4α in the activation of the Pi.
We then mutated the two putative HNF4α REs to examine their effects on the activity of the Pi promoter. As shown in Figure 6(B), mutation of RE1 reduced the luciferase activity to ∼16% of that of the wild-type and, in addition, completely abolished the HNF4α-dependent induction of the activity of the Pi. Although mutation of RE2 also reduced the luciferase activity, co-transfection of the HNF4α expression vector doubled the luciferase activity. We obtained similar results after transfection of HEK-293T cells with these same constructs. These observations indicate that RE1 does, indeed, confer responsiveness on HNF4α on the Pi.
To investigate whether HNF4α activates endogenous Pi, we introduced exogenous HNF4α into HEK-293T cells and examined the expression of CIDE-B by RT-PCR. We detected the short transcript of CIDE-B in the HNF4α-transfected cells, but not in untransfected cells and control cells (Figure 6C, upper panel), which suggested the activation of the endogenous Pi by HNF4α. No expression of the long transcript of CIDE-B was detected after introduction of exogenous HNF4α, indicating that HNF4α does not influence the activity of the Pu. The expression of exogenous HNF4α was confirmed by Western blotting as shown in Figure 6(C, lower panel).
We next investigated the binding of HNF4α to RE1 by EMSA, using a probe that encompassed this region (Figure 6D). We performed the assay using the nuclear extract of HNF4α-transfected HEK-293T cells. We observed a shifted band in the analysis of HNF4α-transfected cells, but not in that of control cells (Figure 6D, lanes 2 and 1). This binding was almost abolished in the presence of an excess of unlabelled oligonucleotide (a 200-fold molar excess) (Figure 6D, lane 3). However, an excess of mutant competitor did not eliminate the binding (Figure 6D, lane 4). Inclusion of a HNF4α-specific antibody in the reaction mixture resulted in a supershift (Figure 6D, lane 6), but control IgG did not have such an effect (Figure 6D, lane 5). These results, together with those of our transactivation studies, suggested that HNF4α binds specifically to RE1 and activates the synthesis of the short transcript of CIDE-B gene.
In the present study, we characterized two mechanisms that control the CIDE-B gene cell-specific expression. We identified two promoters of the CIDE-B gene using 5′ flanking region–luciferase fusion plasmids. The region (Pu) between −3628 and −3345 is required for the basal expression of the long transcript of CIDE-B, whereas the region (Pi) between −204 and the translation start site is required for the expression of the short transcript. An Sp1 site (−3452/−3457) in the Pu promoter with Sp1- and Sp3-binding ability has been reported previously [32]. We found that another Sp1 site (−193/−188) in the Pi region can also bind Sp1 and Sp3, as demonstrated by EMSAs (Figure 5A). The activities of Pu and Pi were greatly reduced when we introduced mutations at the Pu-Sp1 and Pi-Sp1 sites. Our results showed clearly that Sp1 and Sp3 are important for the basal expression of both the long and the short transcript of the CIDE-B gene. Sp1 and Sp3 are ubiquitous transcription factors that bind to DNA with similar specificity and affinity. It has been suggested that the relative levels of Sp1 and Sp3 can vary during cellular differentiation, thereby modulating the responses of target genes [34]. We detected significant basal activity of the Pu in HEK-293T cells which do not normally express CIDE-B. Thus it appears that Sp1 and Sp3 might not determine the cell-specific expression of the long transcript of CIDE-B. However, given the rather low activity of the Pi promoter in HEK-293T cells, we cannot exclude the possible effects of Sp1 and Sp3 on the cell-specific expression of the short transcript.
We found that the Pu-Sp1 site functions, in both directions, as an activator of the expression of the CIDE-B and LTB4R genes. A similar bidirectional Sp1-binding site has also been found in the ORF28 and ORF29 intergenic region of VZV (varicella-zoster virus) [35]. Furthermore, the tissue-specific expression pattern of the long transcript of CIDE-B is similar to that of the LTB4R gene, with both transcripts being detected in peripheral blood lymphocytes/leucocytes, spleen and bone marrow, and not in many other tissues [5,36,37]. These findings are consistent with a previous report [38] that the majority of bidirectional genes are co-expressed and regulated by their shared regulatory elements.
Bisulphite sequencing analysis revealed that CpG sites in the Pu were hypomethylated in long transcript-expressing BEL-7404 cells, but they were hypermethylated in HepG2, SMMC-7721, HEK-293T, HeLa and Jurkat cells, which do not express the long transcript of CIDE-B. This result suggested a significant correlation between the expression of the long transcript and the methylation status of the Pu. Such a correlation is strongly supported by the observation that methylation of the Pu by SssI methylase significantly reduced the promoter activity. These results demonstrate clearly that the cell-specific expression of the long transcript is primarily dependent on the methylation of the Pu region. There are two possible mechanisms by which CpG methylation can inhibit the binding of Sp1. The two methylated DNA-binding proteins MeCP1 [39] and MeCP2 [40] can compete with Sp1 for the same binding sites. Alternatively, the methylation of CpG sites can directly inhibit the binding of Sp1, as seen in a study of retinoblastoma gene expression [41]. It has been reported that Sp1 binds similarly to the methylated and the unmethylated Pu-Sp1 site [32], suggesting that MeCPs might play a role as repressors of the expression of CIDE-B long transcript and LTB4R gene.
There is only one CpG site within the 204 bp region of the Pi of the CIDE-B gene, which suggests that another regulatory mechanism, different from the epigenetic control of the Pu, might also be operative. This hypothesis is supported by results of reporter gene assays, which showed that the Pi promoter was strongly activated in BEL-7404 cells, but not in HEK-293T cells. This cell-specific activity of the Pi was consistent with the cellular expression pattern of the short transcript of CIDE-B. Moreover, we identified a functional HNF4α RE between positions −142 and −130 in the Pi. This element bound HNF4α in an EMSA, and efficiently mediated transactivation of the Pi by HNF4α in BEL-7404 cells. HNF4α is a nuclear hormone receptor that contributes to regulation of a large part of the liver transcriptome by binding directly to almost half of the actively transcribed genes [42]. Many nuclear hormone receptors interact with transcriptional co-activators with histone acetylase activity (reviewed in [43]). It has been shown that HNF4α interacts directly with the co-activator complex TRAP/SMCC/Mediator, and its function on chromatin templates is TRAP/SMCC/Mediator dependent and stimulated by the histone acetylase co-activator p300 [44]. It is reasonable to postulate that the primary function of HNF4α is to recruit co-activators to facilitate chromatin remodelling and the initiation of transcription [25]. This hypothesis is supported by our demonstration that expression of the short transcript of CIDE-B was re-activated in non-expressing HEK-293T cells upon introduction of a transgene for HNF4α. Thus our results clearly indicate that HNF4α is important for the cell-specific expression of the short transcript of CIDE-B. To our knowledge, this is the first demonstration that HNF4α can regulate the expression of an apoptotic factor.
In conclusion, we have identified two promoters of the human CIDE-B gene, which drive the expression of two transcripts of the gene respectively. Sp1 and Sp3 are key regulators of the basal transcriptional activity of both the Pu and the Pi. The expression of the long transcript is regulated by an epigenetic mechanism that involves DNA methylation of CpG sites in the Pu region. We also identified CIDE-B as a novel target for HNF4α, and demonstrated a possible role for this transcription factor in the cell-specific expression of the short transcript. Our results suggest a complex model for cell-specific transcriptional regulation of the human CIDE-B gene, which involves epigenetic and genetic control of two different respective promoters.
We thank Dr Todd Leff for kindly providing the expression vectors for human HNF4α and DN-HNF4. This work was supported by grants from the National High Technology Research and Development of China (863 Program; no. 2002BA711A02), the special funds for Major State Basic Research of China (973 Program; no. G1999053902), and the National Natural Sciences Foundation of China (no. 30571059).
Abbreviations: C/EBP, CCAAT/enhancer-binding protein; CAD, caspase-activated nuclease; DFF, DNA fragmentation factor; CIDE, cell death-inducing DFF45-like effector; EMSA, electrophoretic mobility-shift assay; FSP27, fat-specific gene 27; (DN-)HNF, (dominant negative) hepatocyte nuclear factor; LTB4R, leukotriene B4 receptor; MeCP, methyl-CpG-binding protein; ORF, open reading frame; Pi, internal promoter; PPAR, peroxisome-proliferator-activated receptor; Pu, upstream promoter; RE, response element; RT, reverse transcription
- The Biochemical Society, London


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subset: commoncrawl


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This matching transformer is used to connect your 75ohm coaxial cable to the common 300 twin style connection on many outdoor antennas. The CM 94444 is an industry standard balun which offers excellent matching and low loss.
by chard15
on 6/30/2016
The newer CM-94444 (identified by round flat lugs on the 300 ohm wires and the nomenclature "Matching Transformer - China" does not have a linear response through the UHF band. I have used a field strength meter to compare it to other manufacturers' devices. At high VHF frequencies the CM-94444 has a loss equivalent to the others. At higher UHF frequencies (physical channel 50 is the highest in my area) the CM-94444 shows a loss of approximately 4 db. If you live in a fringe area where you will be squeezing every microvolt from your antenna, the CM-94444 may not be the best product for your needs.
by tlturner
on 7/9/2015
I used this to connect an older sd antenna to a hd tuner. The product works great, installed easily without any issues.I would recommend this product if you have an existing antenna and need to connect an 74ohm coaxial cable.
74ohm... 75ohm... Whatever it takes!
Thanks for the great review!
30-1030 - Guy Wire Clamp up to 1 1/2'' Mast
Antenna mounting hardware
30-2520 - 5' Mast Pipe - 18 Gauge - Swaged End
5' Antenna Mast Pipe
Designed for mounting all types of outdoor antennas including satellite dishes, CB and ham radio antennas, TV antennas and more. 18 gauge construction is ideal for smaller antennas, specifically UHF High Definition TV antennas, as it is light...
30-935 - 5' Mast Pipe - 16 Gauge - Swaged End
5' Antenna Mast Pipe
16 gauge corrosion-protected mast is ideal for television and communication antennas. Each section is swagged at one end allowing multiple masts to be added together.
Note: Also available in 30" Length
30-1530 - FM Dipole Antenna - Male "F" connector
FM Dipole Antenna
Dipole FM antenna is ideal for all local reception, including HD radio. Antenna includes a male "F" connector for connecting to receivers coaxial 75ohm antenna input.
33-12580 - 1x4 Component Video Distribution Amplifier / Splitter
1x4 Component Video Distribution Amplifier A/V amplified splitter is designed for distributing high definition video signals from a single source to up to four television monitors. Path is also provided for left/right unbalanced line level audio. Internal signal ...
21-8240 - Vacuum Desoldering Iron
Vacuum Desoldering Iron
Allows single-handed desoldering of connections, PC boards and components. Spring-loaded plunger and 30W heating element removes most board level components quickly and safely. Easily disassemble for fast cleaning, tip and "O" ring replace...
Great job, product arrived on time, communication was excellent, price was good. Overall very satisfied.


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Morito Garin (6 points) Edit
Those who have Morito Garin as an ancestor have a keen mind to determine someone's true loyalties. He will forsake anyone who knowingly cooperate with the Kolat, or even simply leave them alone when he has a chance to oppose them. [1]
- ↑ Secrets of the Empire, p. 245


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Los Angeles: Oscar-winning actor Eddie Redmayne is in negotiations to play the lead in "Fantastic Beasts And Where To Find Them", the upcoming prequel to superhit fantasy series "Harry Potter".
Redmayne, 33, will play Newt Scamander, who wrote "Fantastic Beasts And Where To Find Them", a textbook used by Harry Potter and his friends when they attended Hogwarts School of Witchcraft and Wizardry.
Eddie Redmayne
JK Rowling, the author of "Harry Potter", has progressed through the first screenplay. The new film series takes place 70 years before the "Harry Potter" films.
That franchise was hugely important to Warner Bros, which is eager to get it back. Frequent "Potter" helmer David Yates will direct and David Heyman will be back to produce the film.


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Sittin on go, Bryan white
#----------------------------------PLEASE NOTE---------------------------------# #This file is the author's own work and represents their interpretation of the # #song. You may only use this file for private study, scholarship, or research. # #------------------------------------------------------------------------------# # Date: 11/5/97; 1:55:19 PM From: Garth Mccrady <firstname.lastname@example.com> Subject: Sittin on go by Brian White Sittin' on Go as recorded by Bryan White Written by Josh Leo,and Rick Bowles. This song is kind of tricky, but if you play along with the disc, it is not that difficult Keep on pickin' Capo second fret Intro is in (G) (G) I'm waiting on the green light to make you mine But your heart's just taking it's own sweet time You want to be sure if you'll sink or swim Well grab a hold of me and just jump on in (C) Your playin it safe (D) but baby I'll wait Chorus E A D A Ooh whenever you're ready D A E A I could be yours in a second or so E A D A Ooh baby come and get me D E A My heart's sittin on go E A D A Ooh it's off to the races D A E A All you've gotta do is let me know E A D A Ooh I'm ready and waiting D E A My heart's sittin on go G You oughta give in now instead of resisting Cause I'm not above a little arm twisting I'm betting it's only a matter of time Before you and me are standing at the finish line C You're having your doubts D But you'll come around (repeat chorus) D E A my heart's sittin' on go D E A my heart's sittin' on go
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Congratulations on your acceptance to Hogwarts School of Witchcraft and Wizardry!
Once you are sorted into your House, you can participate in the Hogwarts House Cup competition! There are a few different ways that you can earn points for your House and help them to win.
4 Ways to Earn Hogwarts House Points:
*Read books! Each book is worth 10 House points.
*Attending Programs: For each library program you attend, you will receive 20 House points. You will receive 40 points for attending the kickoff program starring wizard rock band Draco and the Malfoys.
*Volunteering at the library: For every hour you volunteer at the library, you will receive 20 House points.
**BONUS POINTS: You will receive 5 House points for sharing something on social media about the Hogwarts summer reading program. You can post pictures from a program you attend, a picture of your acceptance letter, the latest book you are reading, etc. Just be sure to tag Guilford Free Library in it and use the hashtag #GFLHouseCup.
It's easy to participate! Use the log given to you to keep track of each book you read between June 19 and August 25. Come in anytime during the summer to have your log checked and added to your House's total points.
Participating in the House Cup automatically enters you into a raffle for a chance to win some Harry Potter swag. The members of the winning House of the House Cup competition will be invited to attend an ice cream party at the end of the summer, so help your team out by reading as many books and attending as many programs as you can!
Sign up at the reference desk starting Monday, June 19! Enjoy a summer full of Harry Potter inspired programming!
Live Wizard Rock Concert with
Draco and the Malfoys sing songs about the Harry Potter universe from Draco Malfoy's point of view.
Registration required. Please sign up online, over the phone, or in person at the reference desk.
You will earn 40 HOUSE POINTS (that's double the points!) for attending this event.


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Account Information
Log In to Your Account
Welcome Back
Log into your account to:
- View your order history and saved orders
- Update account information
- View or add to your wish list
Thanks for returning to Bell Pine Art Farm. For customers who haven't registered or logged in since before 2015, you can login with the email used previously to place an order as your username, along with the password: passage. Once logged in you can change the username and password if you choose. As always, please contact us if you have questions or I can help in any way.


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Researchers Figure Out a Way to Keep Apples Fresh for Weeks
But would Granny Smith approve?
But would Granny Smith approve?
These technologies were either wonderfully weird, woefully misguided, or just behind the times.
Eight-inch, 5 1/4-inch, 3 1/2-inch — floppy disks are the most iconic symbols of old-school computing. But how do they actually work?
Welcome to the year 2014.
Users of 16GB iPhones, listen up. Sandisk is releasing a new line of cases that boost your iPhone's storage capacity. Read more >>
The new My Passport portable hard drive might be one of the most flexible computer accessories you can buy.
Think of how much extra data you can hide in your Rubik's Cube now.
Of course, helium is not cheap — and neither is the technology.
Just how well does an SSD hold up compared to a rugged HDD, when attacked with a machete and generally maltreated? We found out and have the video evidence to prove it. See more >>
Unless you've somehow found a way to earn a living by torrenting and hoarding movies and TV shows, you probably don't need it.
Meaning you don't need to make room on an already crowded extension lead for yet another massive wall wart.
The attractive advancement could overcome what's know as the "magnetic recording trilemma".
Turning Rey's Star Wars Speeder into a USB flash drive is a great marketing idea, right? Putting a paltry 4GB of storage in it? Not so. It can't even hold the HD digital copy of The Force Awakens you're so desperate to download. Read more >>
The designed-for-enterprise drive will be available this summer.
It'll soon be time to toss out your dusty old memory cards.
No lost promises here as the mammoth drives are shipped to special customers today.


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Hatim Theme song MP3
The theme song of Hatim,a serial ...
Star Plus Drama " Hatim " - Opening Theme MP3
Drama - Hatim Cast - Rahil Azam, Ragvendra Kiku, Nirmal Pandey, Pooja Ghai Rawal, Tom Alter, Jaya Bhattacharya The series is based on the legendary ...
Hatim's theme song on flute MP3
Hatim - Theme ( ReMAKE by Talha ) [MP3 Download] MP3
The Indian Television Series Hatim - Theme by Talha. MP3 Download link http://mtalha.net/music/
HATIM theme on flute. MP3
It's so melodious and easy to play(Childhood memories).
IPL MUSIC tone on acoustic guitar by Hatim MP3
Tab is available at www.tabandchord.com my fb page is www.facebook.com/guitarist.hatim.
Hatim Tai MP3
Movie:- Hatim Tai (1990) Starcast:- Directed by:- Produced by:- Music by:- Laxmikant Pyarelal The movie story deals with a baby boy being born into the ...
Randy Orton Theme Song 2012 HD MP3
subscribe . -follow me on twitter : https://twitter.com/hatimserhani -facebook : https://www.facebook.com/hatim.serhani.
Mere Haath Mein - Full Song | Fanaa | Aamir Khan | Kajol MP3
Release Date: 26 May 2006 ▻ Buy from iTunes: http://goo.gl/cUjm7s Watch Full Movie: ▻ Google Play - http://goo.gl/RjOxZQ ▻ YouTube - http://goo.gl/5RpFom ...
The Avengers Theme Song MP3
From Marvel comes The Avengers' Theme Feel free to comment.
Hitman: Agent 47 Official Trailer #1 (2015) - Rupert Friend, Zachary Quinto Movie HD MP3
Subscribe to TRAILERS: http://bit.ly/sxaw6h Subscribe to COMING SOON: http://bit.ly/H2vZUn Like us on FACEBOOK: http://goo.gl/dHs73 Follow us on ...
new song.....by HATIM MP3
new singer .....plz give a comment about him and the song.... http://www.facebook.com/hatim.ghozali http://www.facebook.com/mencapai.evolusi.
Life OK Launches New Serial The Adventures of Hatim - Exclusive MP3
Life OK launched it's new show the Adventures Of Hatim. This show is produced by Nikhil Sinha who's popular show Mahadev is doing wonders for Life OK .


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Launchpad CRM Opportunity Analysis
Launchpad CRM's Opportunity Analysis tools have been developed through a deep understanding that sales forecasting, budgeting, and business planning are critical management activities for all companies.
Sales forecasts and analyses are not just for the benefit of the Sales and Marketing Department but are a means to help Senior and Executive Management Teams make informed decisions.
Looking to the future to help make decisions is challenging, so methods which can add a greater degree of budgeting accuracy are always welcomed. The opportunity sales funnel and forecasting analysis tools provided by Launchpad CRM bring many benefits to companies:
- Delivers an increased Sales and Marketing ROI by minimising the overhead spend on analysis and enabling focussed resource expenditure on the activities most likely to contribute to sales
- Provides rapid visibility of the health of the Sales Funnel and Sales Forecasts
- Improves business budgeting accuracy through real-time analysis of which opportunities are on-schedule for conversion (and which aren't!)
- Identifies which opportunities are qualified and on-schedule for closing and which need management focus to improveme performance in the marketing and sales cycle
- Improves communication and accountability between responsible marketing and sales personnel
- Allows analysis of sales process effectiveness
- Results in more highly qualified leads and improves Pwin performance and overall sales growth.
Interactive forecasting and analysis of opportunities is a key feature of Launchpad CRM. Unlike a static reporting engine, Launchpad CRM provides live graphical charts that immediately reflect new and updated opportunities against dynamic filters.
Analysis and Forecasting of Opportunities
Launchpad CRM provides the following analysis modules;
- Forecasting
- Sales Funnel
- Comparative Analysis
- Roadmap
Each module can optionally be filtered on any, or all, of the following;
- View horizon - constrain how far into the future should the analysis look
- Teams and users - specify which user, or team, the analysis should consider
- Funnel stage - set the minimum maturity that should be included in the analysis
- Certainty - factor the opportunity values by either Probability of Win or Owner Confidence
The Forecast module provides a graphical and tabular depiction of opportunities on a timeline.
The data is presented by both value and also mapped to funnel stage. Funnel stage mapping provides an immediate indication as to where attention might need to be focused; For example, an opportunity due for imminent conversion that is still high in the funnel stage is a cause for concern.
Sales Funnel
The Sales Funnel analysis depicts opportunities in bar-chart format comparing total funnel stage to number of opportunities, as well as in a traditional funnel style with the exception that the width of the funnel is sized according to the total value of opportunities in it.
Sizing the funnel width by value immediately draws attention to whether your sales team are identifying and incorporating new opportunities fast enough, and equally whether they are being pushed through the funnel efficiently and effectively.
A company's sales funnel is the lifeblood of the business. You need a healthy funnel if sales and growth targets are to be met. If the sales funnel is the 'wrong' shape it becomes immediately apparent when using this graphical analysis feature of Launchpad CRM.
Comparative Analysis
Use the comparative analysis module to review performance against a previous period; either month, quarter or year.
The roadmap module depicts opportunities grouped by product on a timeline graph; a great marketing tool for watching customer trends and directing focus as needed to boost under-performing product lines.
Launchpad CRM - Pain-free CRM for business growth
Whether you are prospecting, qualifying, proposing or closing, Launchpad CRM delivers pain-free operation and allows Sales and Marketing teams to concentrate on the critical task of generating new opportunities and closing them to realise sales growth.
Click here to Take a look at the other features embedded within Launchpad CRM. Most of them are only available as premium plug-ins - or are simply not available - in other CRM tools.


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Discussion in 'Fan Zone' started by The30YardSlant, Sep 11, 2005.
For the next 3 hours, I'm a DA Bears biggest fan
Da Bears
Gooooooooooooooo Bears
Yeah, they probably don't have a chance in hell but I'll be rooting for them anyways.
Starting Orton at QB right?
Well at least it's not Hutch.
Da Bears!
RIP Chris...
I'll be watching the Bengals too cause I have Palmer on my fantasy team so am hoping for a breakout year from him.
Imagine if they should pull a shocker today.......
The wheels would come off the bandwagon so fast it would make Eagles fans gasp
Hey, you never know. Odds are that foreskin D is going to eat that rookie qb alive. BUT, Portis is nursing a bum knee, Ramsey's probably good for at least one fumble and/or int., and the Bears D is pretty good. It's going to be interesting to see how Benson does today.
I know I'll have my fingers and toes crossed.


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Mohamed Helmy reveals bizarre clash with Shikabala
Former Zamalek head coach Mohamed Helmy spoke about a previous weird clash...
The game started with Zamalek looking to redeem themselves after their previous loss to Entag El-Harby and they did come out of the blocks quickly.
Zamalek XI: Ahmed El-Shennawy; Omar Gaber, Mohamed Koffi, Ali Gabr, Hamada Tolba; Tarek Hamed, Moruf Youssef, Mostafa Fathi, Ayman Hefny, Mahmoud Kahraba; Mayuka.
Arab Contractors XI: Mahmoud Aboul-Soud; Mohamed Samir, Fathi Mabrouk, Ahmed Ali, Ahmed Moudi; Ali Rabo, Mohamed Abou-Sheashaa, Karim Mostafa, Omar Gamal; Mohamed Fadl, Ahmed Ali.
It was Zamalek's playmaker Ayman Hefny who got the champions first shot on after great work; he cut in from the wing but could only hit his shot wide from the goal.
The Whites came back soon after in the 10th minute. Back into the starting lineup after a run of games on the bench, Mostafa Fathi weaved his way through Contractors' players from the half-way line but couldn't get a shot on time.
The warning signs were there and it was all Zamalek. That was until former Whites' player Omar Gamal should have done better from close range after Ahmed Ali put him through, but his shot went straight at El-Shennawy.
It looked to be going into half-time goalless but Ayman Hefny's corner found Mayuka, whose poor shot turned into an assist for Mohamed Koffi. The Burkinabé defender headed the ball into the corner to give Zamalek the lead in the 44th minute.
Zamalek doubled their lead in the first minute of injury time. Ayman Hefny showed brilliant skill in the box until he was taken down, but everyone stopped except Mayuka who turned and shot, to score the Whites' second goal.
This game threatened to be a goal-fest as Zamalek reached the Contractors goal numerous times during the second half, but without really finding the last touch.
Ten minutes into the second half, a great team move should have finished with a Fathi pass to Mayuka to finish into an empty net. However, the young Egyptian winger shot tamely to the strikers' frustration.
The Contractors did have one sole chance to get things back on track in the second half. Ahmed Ali did great to get past two Zamalek players, but El-Shennawy denied former Al Ahly player Mohamed Fadl from reducing the deficit in the 72nd minute.
Substitute Shikabala, who came on to replace Hefny, looked lively late on. However, after some great skill, his shot hit closer to the corner flag than to the goal itself.
The win took Zamalek to 41 points, six points behind leaders Al Ahly and also gave Scottish manager Mcleish his first win as Zamalek boss. Struggling Contractors remained on 19 points in the 15th place, four points above the relegation zone.


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Swifter Than Life Itself was an Assassin Slayer technique which taught that throughout Medinaat al-Salaam, whispered stories tell of the swiftness of the cruel blades of the Assassins. [1]
- ↑ Legend of the Burning Sands Roleplaying Game, p. 167
|This article is a stub. That means that it has been started, but is incomplete. You can help by adding to the information here.


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Phew! We're finally done setting up!
We started with a pile of hopefully will work together stuff and lo and behold! It worked!
Thats not the finished booth but I didn't get pics of that tonight. You'll see those really soon!
I'm super proud of the girls and all the awesome work they've put in! I couldn't have done it without them!
I'll have some better stuff for you soon. Its 10pm home time and I've got to find dinner 🙂
If you're at the show, booth #1408 is where you'll find us, stop by!
It is looking great Kristy, well done to you and the rest of the Odd Girls 🙂 xx
Thanks Rene'!
looking good..


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Washington Wizards center Marcin Gortat might have a real talent for spying.
The 6'11 big man on Wednesday night proved that he is sneaky and also has the ability to mix in well with the enemy without raising any suspicion.
During a timeout in the Wizards' 118-92 blowout win over the Celtics, Gortat quietly slipped inside a Celtics team huddle and then just casually walked away when it was over.
So convincing he was that none of the C's seemed to notice that there is a sixth guy with them in a white – not green – jersey.
Of course, the man nicknamed the Polish Hammer isn't the first to pull off such a stunt. Before him there was the Celts' very own Rajon Rondo, who tried slipping into a Miami Heat huddle a few years ago but wasn't as successful as Gortat was just now.
The Los Angeles Lakers' Steve Blake did something similar last year in a game against the Brooklyn Nets. So close to the coach he was that he may have actually learned the play the Nets were about to run.
By the way, when Gortat was later approached by reporters for a comment on his huddle invasion, he said:
Gortat on joining #Celtics huddle: "Rajon Rondo was kind enough to let me in. For a second I felt like a Celtic." #WizardsTalk #NBA — J. Michael (@JMichaelCSN)
April 3, 2014


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Hampstead Village, London. Famous for its beautiful and much-loved Heath - a piece of quiet countryside in a vast metropolis. Living on the edge of the Heath is American widow, Emily Walters (Diane Keaton), who can't quite focus on the things that need attention, like her lovely old apartment, her finances and even her son. Despite encouragement from her close friend Fiona, she doesn't want to admit that she's drifting through life without much thought to her future. One day while looking out across the Heath from her attic window, Emily spies a ramshackle hut, which appears to be inhabited by an unkempt man. She witnesses him being attacked by a group of professional thugs. Shocked, she calls the police and watches, through her binoculars, as help arrives. The next day she ventures into the woods in search of the man. Donald Horner (Brendan Gleeson) has lived quietly and harmoniously on the edge of the Heath for 17 years but now his lifestyle is under threat - his home is the target of property developers who've started using heavy-handed tactics to remove him. When Emily introduces herself, he is naturally suspicious and politely thanks her but rebuffs her attention. Despite his gruff exterior, there is something gentle and alluring about Donald and when Emily discovers that Fiona is leading a community initiative in support of the developers, she bravely steps up to take Donald's side in the escalating battle, determined to defend the emotional and physical livelihood of this quiet but unusual man - who has grown to trust her and who - despite his situation, just might be the man to sweep her off her feet.
Despite the title the Wishbone family are far from happy. In an attempt to reconnect as a family Mum, Emma, plans a fun night out. However her plan backfires when an evil witch curses them and they're all turned into Monsters.
United by their renegade spirit and a determination to win against substantial odds, these riders take on the international circuit. The film offers unique insights into the first five years of their journey, bearing witness to the ethos of the team as embodied by all ¬タモ from the strongest to most embattled members. Out of a culture that embraces a deeply human approach to sport, unlikely champions are born, and seemingly improbable team and personal goals are achieved.
Four friends and a pastor's wife set out on a trip to Las Vegas to win souls for Jesus. But when they arrive, they become tempted to sin.
Tulip Fever is set in 17th century Amsterdam and follows a married woman who begins a passionate affair with an artist hired to paint her portrait during the height of "tulip mania". The lovers gamble on the booming market for tulip bulbs as a way to raise money to run away together.
The story revolves around a scrappy young dog who fights to save a town from becoming the litter box of a nefarious feline warlord.
Set entirely within the confines of a police van, Clash dramatizes the ongoing political unrest in Egypt two years after the Arab Spring. It's 2013 and mass protests have led to the ouster of the ruling Muslim Brotherhood by the army. A series of arrests finds rival demonstrators trapped in a paddywagon together under the threat of impossibly stifling heat. From this simple setup, Mohamed Diab weaves a white-knuckled tale of resistance and shared humanity that artfully evokes the political fault lines in the Arab world's most populous nation.
Emerging from a NY subway on her way home from college, Lucy (Brittany Snow, Pitch Perfect) discovers her neighborhood of Bushwick engulfed in utter chaos. Trying to escape the violence, Lucy seeks refuge in the basement of Stupe (Dave Bautista, Guardians of the Galaxy), a former Marine on his way out of town to find his family. As the unlikely pair navigate through a hail of gunfire and lethal explosions, they learn they are in the middle of a civil war as Texas attempts to secede from the US. With the clash between local residents and the militia escalating, Lucy and Stupe must rely on each other in an impossible race to get out of the city and survive another day.
Paris, 1884. An orphaned girl arrives in Paris from Brittany. Flicie Milliner is 11 and has no money but one big, passionate dream: to become a dancer. With nothing left to lose, Flicie takes a big risk: she 'borrows' a spoiled brats identity and enters the Opera Ballet School. But how long can she be someone else? Mentored by the tough and mysterious cleaner, Odette , Flicie learns that talent is not enough - it takes hard work to be better than her ruthless, conniving fellow students. That and friendship. Felicie's inventive, exhausting and charismatic best friend Victor also has a dream: becoming a famous inventor. Together, they both encourage each other to reach for the stars.
Set against the backdrop of 1960s San Francisco, BIRTH OF THE DRAGON is a modern take on the classic movies that Bruce Lee was known for. It takes its inspiration from the epic and still controversial showdown between an up-and-coming Bruce Lee and kung fu master Wong Jack Man - a battle that gave birth to a legend.
Tales of an Immoral Couple centers on Lucio and Martina, two former lovers whose passionate romance began while attending a strict Catholic high school in Mexico. Twenty-five five years later, they unexpectedly run into each other in the city of San Miguel de Allende and despite still sharing an undiminished love for one another they both pretend to be in happy marriages - even if it's all lies. As they try to maintain their facades, a comedy of errors ensues that reveals the true reasons why the couple ended up separating in the first place.
Trained from an early age by rigorous, perfectionist Professor Bojinski, Polina is a promising classic dancer. She is just about to join the prestigious Bolcho Ballet when she discovers contemporary dance. That throws everything into question on a profound level. Polina leaves it all behind and moves to France to work with famous choreographer Liria Elsaj. Despite her determination and hard work to the point of obsession, Polina just can't seem to break through. So she moves to Anvers in search of work - and a new life.
From the producers of the THE RING and THE GRUDGE and based on the award-winning short by Lars Klevberg, comes the next iconic and bold new vision in horror: POLAROID. High school loner Bird Fitcher has no idea what dark secrets are tied to the mysterious Polaroid vintage camera she stumbles upon, but it doesn't take long to discover that those who have their picture taken meet a tragic end.
BEACH RATS centers on Frankie, an aimless youth on the outer edges of Brooklyn, struggling to escape his bleak home life and navigating questions of self-identity. Frankie spends his summer balancing his time between his delinquent friends, a potential new girlfriend, and older men he meets online. As Frankie's competing desires begin to consume him, his decisions leave him hurtling towards irreparable consequences.
In the spring of 1980, a teenager is gunned down in the streets of Flatbush, Brooklyn. The police pressure a child witness to identify a suspect. As a result, Colin Warner, an 18-year-old kid from nearby Crown Heights, is wrongfully convicted of murder and sentenced to life in prison. Colin's childhood friend Carl 'KC' King devotes his life to fighting for Colin's freedom. He works on appeals, takes loans for lawyer fees and becomes a legal courier to learn the court system. This incredible true story is adapted from the acclaimed This American Life segment by writer/director Matt Ruskin, with Lakeith Stanfield playing Colin Warner and Nnamdi Asomugha as Carl King.
Based on the inspiring true story of a salesman-turned-pastor, the tiny church he was ordered to shut down, and a group of refugees from Southeast Asia. Together, they risked everything to plant seeds for a future that might just save them all.
This film follows the story of Gaurav, who is content with his "same stuff, different day" routine, and dreaming about settling down with the girl of his dreams, Kavya, and having his "happily ever after". Kavya, on the other hand, prefers someone more risky and adventurous - perhaps someone more like Rishi! When a case of mistaken identity rocks Gaurav's life, he stands to lose everything he has worked so hard for! What follows next is a roller-coaster ride, complete with action, romance and laughter, taking the viewers on an unpredictable and hilarious thrill ride.
GOTTI follows infamous crime boss John Gotti's rise to become the "Teflon Don" of the Gambino Crime Family in New York City. Spanning three decades and recounted by his son John Jr., GOTTI examines Gotti's tumultuous life as he and his wife attempt to hold the family together amongst tragedy and multiple prison sentences.
Bloody revenge is at the heart of this stylish, kinetic action-thriller that gives a welcome shot of adrenaline to the classic femme fatale story. Honed from childhood into a merciless killing machine by a criminal organization, assassin Sook-hee is recruited as a sleeper agent with the promise of freedom after ten years of service - and she jumps at the chance for a normal life. But soon enough, secrets from her past destroy everything she's worked for, and now nobody can stand in her way as she embarks on a roaring rampage of revenge.
Actors Sidharth Malhotra and Jacqueline Fernandez, who are paired together in a film for the first time, on Monday revealed that their upcoming movie Reload has been renamed A Gentleman - Sundar, Susheel, Risky.
Five women veterans who have endured unimaginable trauma in service create a shared sisterhood to help the rising number of stranded homeless women veterans by entering into a competition that unexpectedly catalyzes moving events in their own lives to bring them in a quest for healing and hope.
After being defeated by humans centuries ago, the Winged Tribe has lost their ability to fly. Seeking vengeance, a royal descendant of the tribe has begun searching for the magical Naga Pearls, which he plans to use to destroy the humans. When the legendary pearls fall into - and out of - the hands of Heiyu, a wily human street punk, he must join a team of unlikely heroes as they race to prevent the destruction of their people in this magical fantasy adventure.
Emma, a brilliant but awkward app designer, moves to San Francisco after a massive betrayal by her boyfriend with only her beloved pets, an adorable cat and an energetic mutt, in tow. One night, drawn by the light of a Supermoon, Emma's pets escape into the night and are transformed into full-grown men, forcing Emma to reconsider her outlook on dating and hilariously work out her trust issues.
Biographical drama starring Jack Lowden as Morrissey lead singer of the band The Smiths and Jessica Brown Findlay as artist and musician Linder Sterling. This movie tells the story of Morrissey's early life, and it was written and directed by Mark Gill.
A young American couple, lost in the Thai countryside, find themselves haunted by an evil spirit. Desperate for a solution, the couple spirals deeper into a surreal supernatural world leading them into a frenzied, terrifying confrontation with pure evil.
Dee Wallace headlines as Diane, a matriarch presiding over the gathering of her squabbling grown children on Christmas Day. Tensions give way to terror when a deformed stranger appears at the door with vengeance on his mind. Diane must protect her family as limbs are severed and secrets are brought into the light.
This is a story of a brave man who fought and won against all odds: Sarpanch Rupinder Singh Gandhi was a prolific sportsman, daring leader and true friend. This story shows how circumstances and situations convert him into "something else" when he and his friends make decisions that end up making them outlaws.
Based on the famous Japanese manga written by Tsugumi Ohba and Takeshi Obata, Death Note follows a high school student who comes across a supernatural notebook, realizing it holds within it a great power; if the owner inscribes someone's name into it while picturing their face, he or she will die. Intoxicated with his new godlike abilities, the young man begins to kill those he deems unworthy of life.
Fathom Events, and Mayweather Promotions t present the highly-anticipated showdown, Mayweather vs. McGregor, LIVE in movie theatres nationwide on Saturday, August 26. Witness the super-fight that no one believed was possible when legendary undefeated world champion Floyd Mayweather battles UFC superstar Conor McGregor in the most anticipated sporting event of the year. Sparks will fly as the two biggest names in their sport take center stage for a once in a lifetime sports spectacle. Don't miss this epic match for the ages LIVE on the big screen!
Nagasaki, early 20th century. The geisha Cio-Cio-San, called Madame Butterfly, has married an American naval officer, Pinkerton, who deserts her shortly after the wedding. Butterfly bears his son and patiently awaits her husband's return. One day, the American consul receives a letter from Pinkerton in which he says he has an American wife (his marriage in Japan not being legally recognized in America) and is returning to fetch his child. Pinkerton's ship finally arrives and he sets out to visit Butterfly with his wife Kate. Filled with remorse, however, he is unable to face Butterfly and leaves Kate alone with her. Butterfly tells Kate that she will give Pinkerton his son if he comes to fetch him. Rejecting a life of disgrace, she kills herself with her father's sword. Pinkerton arrives to find her dead.
Fathom Events and GKIDS are partnering to bring Hayao Miyazaki's Castle in the Sky to select cinemas nationwide for a special three-day event on August 27, 28 & 30 as part of Studio Ghibli Fest 2017. From the legendary director comes the story of a young girl who falls out of the sky and into the arms of young Pazu. Together they search for a floating island, the site of a long-dead civilization promising much to those who can unlock its secrets. Dubbed in English on August 27 and 30, subtitled on August 28, this special three-day event will also include an exclusive edition of GKIDS MINIFEST, an ongoing festival of award-winning short animated films from around the world!


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Computerized coin bitcoin commenced the new year by hopping above $1,000. Without precedent for a long time late on Sunday. Having beated all national bank-issued money related structures with a 125 percent move in 2016.
Bitcoin – an electronic "cryptocurrency" that has no central power. Depending rather on a large number of PCs over the world. That affirm trades and add new bitcoins to the system skiped 2.5 percent to $1,022. On the Europe-based Bitstamp exchange, its most astonishing since December 2013.
Despite the fact that the advanced money has generally been profoundly unstable. A ten times increment in its esteem in two months in late. 2013 took it to above $1,100, before a hack on the Tokyo-based Mt. Gox trade saw it dive to under $400 in the next weeks. It has in the previous two years been more steady.
Its greatest day by day moves in 2016 were around 10 percent. Still exceptionally unpredictable contrasts and fiat monetary standards. Yet particularly lower than the exchanging of 2013. Which saw every day value swings of as much as 40 percent.
Bitcoin may helps in the previous year by expanded request in China. On the back of a 7 percent yearly fall in the estimation of the yuan in 2016. The Chinese coin's weakest appearing in more than 20 years. Information indicates most bitcoin exchanging is done in China.
Bitcoin utilizes to move cash over the globe rapidly and secretly. And does not fall under the domain of any power. Making it appealing to those needing to get around capital controls, for example, China's.
It is likewise may interest those stressed over an absence of supply of money. For example, in India, where Prime Minister Narendra Modi expelled high-group monetary certificates from flow in November.
"The creating war on cash, and capital controls, is making bitcoin take after a practical. If high danger, elective," said Paul Gordon. A board individual from the UK Digital Currency Association and kindred supporter of Quantave. A firm hoping to make it less complex for institutional theorists to get to cutting edge coin exchanges.
In spite of the way that bitcoin is still some way off the amazing high of $1,163. That it came to on the Bitstamp exchange late 2013. There are currently more bitcoins available for use – 12.5 adds to the framework at regular intervals. Its aggregate worth is at a record-high above $16 billion. Putting its esteem at around the same as that of a normal FTSE 100 organization.


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Orange and Almond Macaroons
Be the first to review
READY IN: 55mins
Recipe by Cat R.
From the Sainsbury seasonal selection of recipes. I haven't tried this yet but am looking forward to. If anyone finds out how many eggs makes 75ml of white, share your discovery (or I will when I get round to it)!
See Discussion:
Ingredients Nutrition
- 200 g caster sugar
- 30 g granulated sugar
- 100 g ground almonds
- 15 g rice flour (it should be ground rice, not sure if this is the same as rice flour)
- 75 ml egg whites
- 1 small orange, zest of (or clementine)
- sliced almonds (for decorating the tops)
Directions See How It's Made
- Preheat the oven to 350F, 180C, GM4.
- Sift the sugars, almonds and ground rice into a bowl.
- Add the egg white and orange zest, beat thoroughly with an electric beater for 5 minutes.
- Allow the mixture to stand for 10 minutes, then continue beating for a further 5 minutes.
- Pile the mixture into a piping bag, fitted with a 2.5cm plain piping nozzle.
- Pipe the mixture onto bakewell or rice paper. Press a flaked almond onto the top of each macaroon.
- Bake until light brown (20-25 min).


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Category: upcoming
Haptic Synapses at SubZERO 2011 flyer
Here's the flyer for us playing at SubZERO 2011!! As long as the rain waits until Saturday to show up, it's going to be crazy awesome! Be there! Thanks again to Matthew Gamez for the great photo!
Haptic Synapses live at 2011 SubZERO festival
We will be playing throughout the night at the 2011 SubZERO street festival, June 3rd, 2011. Check out the website for more info: http://www.subzerofestival.com/
upcoming: Sidescroller 4
We are playing a live show at Sidescroller 4! Wewt. You are invited!! Learn to Play Too and Sidescroller present an evening of techno music and video games to make you think. * live electronic music performance from Haptic Synapses * independent video games * enlightenment * Learn to Play Game Challenge awards It all takes place at the Euphrat Read More …


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(This page covers the phenomenon of financial inflation in the United Kingdom of Great Britain and Northern Ireland and its successor states)
During the Thatcher government, inflation remained low as an apparent result of government policy and the confidence of the markets in the government. At the end of the Thatcher term in May 1984, the Retail Price Index stood at 88.97. Inflation rose considerably as soon as Denis Healey won the election, and by December 1989 the RPI was at 197.97. During the second Caroline decade, the most important event was the Third World War, leading to almost immediate scarcity of oil in the West and a sudden spike in inflation which was however masked by the fact that the then National Governments in the Federated Kingdom were able to control the economy very closely. Hence the inflation did not noticeable take place until rationing ended and the economy returned to normal in 1997. The National Governments warned that inflation would suddenly rise at the end of their term in order that no party could gain political capital from the event when it happened. By December 1999, the RPI reached 670.73. In the following decade, inflation stayed at the previous rate and by the end of 2009 it was 2272.46.
Influence on British currency
The increased circulation of banknotes led to the faster deterioration of the paper and by June 1986 the two pound coin was introduced, at first as a commemorative coin for the Commonwealth Games but immediately afterward as general currency. The five pound coin was introduced in 1993 in each of the new nations with the break-up of the UK. Several of the successor states then introduced Tyvek banknotes for higher denominations from early 1995.
With the exception of Grand Duchy of Cornwall, every nation had completely withdrawn cash by 2008, though it continued to be used in the black economy.


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International Shipping SPM
Special Priority Mail (SPM) is a service that provides the fastest and best performance, using a perfectly organized international network.
- Guaranteed delivery on the next working day to the most important destinations in Europe and the USA.
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Submit your shipments to an ELTA or ELTA Courier shop of your area or call us at 11120 (local charge from landline) and 21006073005 and we will come to pick them up at your door.


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Boogey and PayBac (also known as The Lost and Found) are releasing a 15 track album on the 3rd of march called "FaceOff" and have decided to give their fans a sort of preview of what they can expect on the album.
Both rappers bring their own unique brand of hip-hip to two different instrumentals off Kanye West's The Life Of Pablo.
Along with the Freestyle they have also given us the Tracklist to the album.
Enjoy the Song and dont forget to get FaceOff by The Lost & Found on the 3rd of March.
DOWNLOAD Boogey & PayBac – "No more Parties/30 Hours"
See track list below
Also Download
Submit your comment
3 total comments on this postSubmit yours
Because there is Boogey in this, i'm downloading it
Boogey can hold his own anywhere in the world!!


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Just a drive from the shack to the east coast via the mountains in my Spyker D8 "Paris-to-Peking". [Disclaimer] This isn't meant to be a show of my driving skill. It's more of a demo of what to expect in a free ride.
atari, test drive, test drive unlimited 2, tdu, tdu2, tdu 2


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In Ruby, Sapphire and Emerald, Golducks can be found in the Safari Zone by surfing. They are quite rare.
In Pokemon Fire Red you can find them in the Cerulean Cave, the Berry Forest (Surfing), Seafoam Island, and Cape Brink.
It might be easier to get a Psyduck in all those games, and evolve it to Golduck at level 33.
Psyducks and Golducks are not available in Leaf Green, Colosseum and Pokemon XD.


text: Understandably, few customers want to do business with companies that have been in bankruptcy, fearing their financing will not be safe. Clients may also be pressing for improved pricing, Marshall says, which could put pressure on CIT's margins. The lender must also transition its board and management team. It announced seven new directors on its 13 member board, leaving open the question of the strategic direction of the company, Marshall says. But ultimately, both Gallo and Marshall say CIT will be able to rebound and makeover its image. -- Reported by Jeanine Poggi in New York. Follow TheStreet.com on Twitter and become a fan on Facebook.


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This article, Land of Lizards, is the property of Na'Jorne.
The Land of Lizards is one of the more weaker countries but holds a military power of multiple Kekkei Genkai users. Even those who have special abilities that are known to be taboo, such as Ice Release of the Yuki Clan. At the current moment, the Daimyo is the Head Ninja of Karasugakure. Those who are born here
have no certain affinity to a certain nature because the land had been populated by people around the Ninja World. However, those who learn the Summoning Technique will normally have an affinity to the Lizards.
The land is located between Takigakure's unknown land and the Land of Sound. Its only big land mark that doesn't include Karasugakure is the Mountains' Graveyard which has been cleaned out and had gotten rid of the secret hideout to show that they had no affiliations with Madara Uchiha.


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Articles tagged with: language
In June 2011, many Gmail accounts were compromised by hackers. Passwords of many US officials, political activists and officials in different countries were stolen.
Ever since then, Google had been working on it's Two Step Authentication method. This method has enhanced the security of Google account holders. This method requires password based authentication along with a verification code which is sent to the user either through voice, call/text or is generated on the user's smartphone device via a Google app from which one is accessing his/her account.
In the tech industry, suing rival companies is not a big deal so its no surprise that Oracle sued Google.
The internet is full of translator websites, however, none are excellent and few are accurate. Ase Deliri is attempting to solve this by creating the Silo universal translator. The site offers support for 880 language/ dialect combinations, including sign language, and offers you the ability to search across all dialects within a language.
Silo's content is user-generated, so people have to register and upload information. Also, the translations are phrase-based, as opposed to translating individual words like most current online translators which often come up with interesting and inaccurate results. Users will be able to tag phrases with descriptions such as "insult" or "local proverb."


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Stop Worrying About Balding, Because There Is Finally a Hair Loss Secret That Is Actually Permanent!...
One critical appearance tag of our is the hair. The thick pitch-black hair adds intriguing glamor. The Brazilian blonde curls are sure to get most out these modern flappers. It can be distinguished to see hair thinning, which can be common among many individuals across different age periods and both gender. There are a variety of facial treatments that happen to be an excellent source of demand including the indulgence facials using gold extracts filled with antioxidants, specific facials treating aging, wrinkles, tanned skin, etc. These kinds of facials need to be undergone at least two times per month and so are best if done at leading celebrity facial salons in Dubai. What best than being able to pamper yourself from a hectic month full of workloads.
Relaxing and rejuvenating oneself, is everyone's right. One of the best solutions to relax the mind, soul, and the body is if you recruit a soothing hair treatment done in hairlineink. Good hair days are difficult ahead, however, when they are doing, it might raise the morale to a degree. The way nice hair looks impacts your style and how you look in public. Hair treatments should not be put through trail and errors such that when you find yourself at the most efficient hair spas, you'll certainly require a good job done. Only take a bit off at a time and don't forget to hold checking the mirror to see how you're progressing. Curly hair will spring back when you've to make the grade so ensure you don't make the grade short.
Be careful you don't cut yourself. Using a beard trimmer or male organ hair shaver now is easier while you don't worry about injuring yourself or trimming hair too short. The strip of tresses is separated into smaller units which can be planted in the bald area. The follicular unit extraction (FUE) strategy is more technical because it involves the removal of person hair roots that may be pulled out or obtained from a place of great growth to an area of baldness. The plantation can also be done individually, and so the surgeon should be extremely skillful and careful. All the ways of hair restoration are lucky and still have been providing accomplishment to millions.


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Carnival Ecstasy - Central America/Panama Canal: Carnival Ecstasy Cruise Review by FBristol
Overall Member Rating
Carnival Ecstasy - Central America/Panama Canal
Destination: Panama Canal & Central America
Embarkation: Los Angeles
The food was wear we were most surprised,very good to excellent on most nights. We eat in the dining room late seating for all or breakfast and diners, lunch was on deck with the usual fare.Our wait staff was good on par with some of the better restaurants in our area.The Ecstasy in most part was clean, some of the high traffic area carpet was stained in hallways but overall she was in good shape for a ship that gets this much traffic every week. We were told she carries 2200 to 2400 every week, year round. The only thing that we had More trouble with was the fact that we booked the lowest grade inside cabin category 1A. It had one lower and a fold down bunk bed. At 62 the bunk bed is not the thing to do, I new this going in, but thought I would give it a try considering what we paid for this cruise (cheep).I tried to get a up grade at the embarkation but was told the ship was full.
By the way embarkation went very fast and was by far the best we have been on on the 5 cruises we have taken in the last 20years. Our cabin E183 was good size, clean, and was in an excellent location on E deck, but No more bunk beds for us. Ports of call we did our own thing as we normally do when we cruise, a little walking through some shops and then back on board. We have been to the ports of call before on RCCL.
Over all our cruise was very good for what we paid, we got a very low price on this cruise and we were pleased for the four days we spent with Carnival. FB,ABQ,NM Less
Published 09/21/04
Cabin review: 4AE183 InteriorInterior (4A) Reviews >>
Read Cruise Critic's Carnival Ecstasy Review >>
First time on Sirena
Not the best from Princess
A Great Cruise On a Fantastic ...
Amazing Vacation
Bland, old ship; boring food, ...
Good time, but not what I was ...
Very fun and comfortable
Fun Ship Liberty
Decrepit ship, food failures, ...
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I'm 34 but look 24 without my beard, late 20's with my beard. My question is, when you meet a person your same age and they look that age, do you view them as older. Not maturity wise, just older because they look older than you. I typically view a person that's 34 and looks 34 as older. I have to remind myself sometimes that we are the same age.
Most Helpful Girl
Yeah lmao. One of my classmates is the same age as me but she looks so much older and I think of her as an older, more mature person.00|00|0


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We Are One - The War of 1812: The Battles for St. Michaels, Maryland
"Welcome to August 10, 1813, in the little town of St. Michaels, Maryland. On this hot, dark morning, the feared British Navy attacked the town of three hundred, defended by local militia. This book tells the story of that attack and a subsequent attempt on August 26. We also look at the life of citizens at that time and the lasting impact of this war on the Chesapeake Bay.
Unfortunately, few records survive and little exists in contemporary images from the time. As with all wars, legends grow and are passed among generations. It is not our intention to pass judgment on any of the wonderful stories that have come down to us through the past two centuries. Rather, we celebrate the truth that is behind all legends and enjoy..." (From the Preface)
A Project of the 1813 Book Editorial Board. Published by the Commissioners of St. Michaels, Maryland.
2013, Walda DuPriest-Brandt, Ed., 60 pp, Paperback


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Green and gold hillsides are laid out in every direction from this charming home. Surrounded by beautiful private gated grounds with a pool tucked into a small olive grove, the villa is immaculate inside and out, tastefully finished in comfortable country style.
High summer days will see you heading for the impressive 14 x 7m private swimming pool or sitting in the shade on one of the three covered terraces, iPad, novel or guidebook in hand as you contemplate a day of exploration or well-deserved relaxation. If you visit in the glorious golden days of autumn, the cosy beamed living room with open fire is the perfect place to unwind in the evenings and revisit the day's adventures over a glass of fine local wine.
Umbria is sometimes regarded as Tuscany's quieter cousin, full of off-the-beaten-track gems and a good choice for those who feel they have 'done' Tuscany and are looking for an equally beautiful alternative. Whatever fortunate circumstance brings you here, Villa Donatella is an enchanting base to enjoy the cuisine, culture and character of Umbria.
The local area
The nearest town is Umbertide, 13km away, which dates back to pre-Roman times and deserves at least half a day to explore its castle, museum and several churches. A little further is the 16th- century Citta di Castello, nestled on the banks of the Tiber in all its Baroque, Gothic and Renaissance splendour.
Gubbio, 42km away, has been described as one of the most beautiful medieval towns in Italy; an appealing mix of restaurants and bars with impressive historic buildings such as the Palazzo dei Consoli and Palazzo Pretorio. The walled city of Perugia lies 45km to the south, home to the Umbrian National gallery and a vibrant place to visit year round thanks to its two universities. For a complete change of scene and pace, Lake Trasimeno is around 30km from the villa and a popular spot for boat trips and bird watching.


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Andrew Feinstein, Edinburgh Book Festival, 13 August 2012
Andrew Feinstein is a former member of the African National Congress and the South African Parliament, who fell out with the ANC leadership over corruption linked to a gigantic arms deal South Africa signed. His most recent book is The Shadow World: Inside the Global Arms Trade and that is what he was discussing in Edinburgh. the chair of the session described the book as hugely impressive in terms of its research and its writing, and hugely depressive in terms of the story it told. Feinstein felt a book lke this was needed because there had been no global survey for the layman of the arms trade since Anthony Sampson wrote The Arms Bazaar in 1977. He wanted to look not only at the black market in weapons but at the official inter-government transfers: and in all his researches he failed to find a single arms deal which did not involve corruption or bribery to some extent.
He was asked why there had been so much concentration on small arms. He felt that this was because after WW2 the hitherto unified anti-arms-trade movement had fragmented into "opposition to nuclear weapons" and "the rest". Non-nuclear campaigners had picked specific targets because that made for easier camoagning, whether it be landmines, cluster munitions, small arms or whatever. Also, the various agencies such as Oxfam and Amnesty International were seeing every day the end results of the trade in small arms. But as his research had shown, the downstream trade in small arms in Africa and elsewhere could not exist without the upstream "official" deals between governments which bring the weapons into the marketplace to begin with. This is why he feels that only a comprehansive global arms treaty will address the issue. Unfortunately, the USA (whose weapons purchases are roughly equal to those of the rest of the world) has just scuppered the arms treaty under consideration at the United Nations. Feinstein is no pacifist, and believes there is a place for the arms trade to permit legimitate defence, but he believes that it needs to be tightly regulated.
He took a question from a British Aerospace apologist who advanced the view that the bribes paid in the al-Yamamah deal were "Saudi's money anyway", just paid to "manage the deal". Feinstein pointed out that £1 billion was spent solely to persuade the Saudi Air Force to buy the BAe/Saab aircraft instead of the Italian one the air force wanted. This wasn't "management": this was a pure bribe. He also reminded the BAe stooge that British taxpayers were very heavily involved in such deals. Various British companies had sold arms to the Indonesian regime which were used in its genocide in East Timor. When the Indonesian economy collapsed and all its debts were written off, Britain's Export Credit Guarantee Department paid the whole cost of those weapons: which is to say, the British taxpayer picked up the tab for Indonesian genocide.
Andrew Feinstein believes that three measures would cut out around 85% of the corruption which currently surrounds the arms trade:
1) Oblige companies to list all intermediaries used in a deal; to list what they did, and how much they were paid for doing it.
2) Ban the consideration of "economic offsets" (where a company promises to invest a similar amount to its earnings from the deal into the country's economy - you buy our fighters, we'll build your hospitals, so to speak) from procurement decision-making. (The WTO already bans it for every kind of international trade except arms deals.)
3) Force companies to use only banks in their own or the buyer's countries (hence ruling out the use of tax havens and making money laundering more difficult).
These big arms dealers all have powerful political protection. As one of them put it to Feinstein, "If they wanted me in prison would I be talking freely to you now?" Remember that the British government's Serious Fraud Office was investigating the al-Yamamah deal until it was instructed to abandon its investigation by none other than Tony Blair. Powerful pals indeed.
Feinstein was fascinating, engaging, wholly convincing and highly articulate. I will definitely read his book, and recommend it to anyone else interested in current affairs.


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With First Tutors: Music you can search for a Corwen oboe tutor. Oboe tuition are the perfect method to start a stimulating new hobby, acquire a new skill and attain confidence with oboe. Use our service to find Corwen oboe teachers and select which best suits your requirements so you can enjoy your oboe lessons in Corwen today!
This is the best place to find Corwen oboe tutors in the UK, helping you find Corwen oboe tuition close to you and Corwen teachers to assist with any instrument, for example, piano, violin or guitar. We can help you find Corwen oboe lessons or a Corwen oboe tutor to prepare for oboe exams and improve your oboe skills! Our search will order Corwen oboe teachers in your area by their lesson charges and proximity to you.
If you are a oboe teacher and would like to offer your oboe lessons in Corwen or throughout the UK, please register with First Tutors: Music or learn about us here. Becoming a Corwen oboe tutor is a brilliant way to improve your own knowledge by giving oboe lessons while helping others, all in your local Corwen area!


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Install the BEAST KODI
If you want to install the very latest Beast Wizard then here is how, you may already know how to do it, but also may run in to problems, here is a foolproof way and hopefully you won't have any issues. If you do have even the smallest of problems please do not hesitate to get hold of me on here or on twitter, all my handles are @cssc0der.
Here are a few screenshots of the gorgeous looking build before you install the Beast KODI
As you can see from the Images their are some new sections that have been added and it all just works, this also does come with a free 5 day Vader Streams trial which is well worth it just for that. It comes with a live EPG and you can record too.
How to Install The Beast Kodi:
- You must first register here or Register here When the page opens add a username, email address & passwordand then click Register
- After the registration notification appears select log in
- Enter the email & password you used in the first step and click Log in
- In the next screen you should see your username and email. If they are correct click on Verify Here
- If you get a message saying your address has now been verified you can now move on to installing the build
- Open Kodi
- Select SYSTEM > File Manager
- Select Add Source
- Select None
- Type the following EXACTLY http://thebeastkodi.com/repo or http://wizard.place/repo and select Done
- Highlight the box underneath Enter a name for this media Source, type beast & Select OK
- Go back to your Home Screen
- Select SYSTEM
- Select Add-Ons
- Select Install from zip file
- Select beast
- Select Download_this_Beast.zip
- Wait for Wizard enabled notification
- Go back to your Home Screen
- Select PROGRAMS
- Highlight – DO NOT SELECT – The Beast Wizard
- Open the Context Menu (C on your keyboard, right click with a mouse, long press on touch screen devices or Menu button on your remote) and select Add-on settings
- Add the Email and Password you created at the beginning of the guide and select OK
- Select The Beast Wizard
- Select the build you wish to install from this wizard
- The Beast will now start to download
- After the download completes the build will extract
- After extraction if you are on an Android/Amazon device unplug and plug back in and the Beast will be installed and ready to use. If on any other type of device select OK
- Select OK again
You have not just install the Beast KODI