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TGF (transforming growth factor)-β1 is a multifunctional cytokine that influences homoeostatic processes of various tissues. TGF-β1 expression is inhibited by androgens in the prostate gland, whereas its expression is enhanced by androgens in highly metastatic prostate cancer cells. Here, we examined regulation of human TGF-β1 promoter activity by androgen in prostate cancer cells. The full-length (−3363 to +110) promoter showed a high level of activity in response to androgen in PC3mm2 cells expressing AR (androgen receptor). Further deletion analysis revealed three distal and three proximal AREs (androgen-response elements) in the promoter. Gel-shift and footprint assays show that these AREs physically interact with the DNA-binding domain of AR. Chromatin immunoprecipitation assays revealed the androgen-dependent recruitment of AR to the ARE-containing regions of the TGF-β1 gene. More importantly, a negative ARE was detected in the TGF-β1 promoter. Both positive and negative AREs are functional in the androgen-regulated transcription of the TGF-β1 promoter. These findings imply that androgen signalling may positively or negatively regulate TGF-β1 expression in response to various signals or under different environmental conditions.
- androgen receptor
- androgen response element
- promoter
- transforming growth factor β1 (TGF-β1)
- transcriptional regulation
The AR (androgen receptor) controls physiological processes through activation and repression of specific target genes. The ligand-activated AR binds to the ARE (androgen-response element) located within target genes [1–3]. A few functional AREs have been characterized and studied in detail, including those in the human PSA (prostate-specific antigen) gene [4–6], the rat C3(1) gene (which encodes the prostatic binding protein) [7], the probasin genes [8–10], the mouse Slp (sex limited protein) gene [11,12] and the prostate stem cell antigen gene [13]. The androgen-induced repression of gene expression was found to be mediated by negative AREs. Regulation via negative AREs has been reported in the Maspin [14], prostate-specific membrane antigen [15,16] and PSA [17] genes.
TGF (transforming growth factor)-β1 inhibits the proliferation of prostate epithelial cells in the presence of physiological levels of androgens in vitro and in vivo [18–20]. Prostate tumours have been found to express higher levels of TGF-β1 than the normal prostate gland [21], and increasing levels of this growth factor are associated with cancer progression [22]. In line with this, serum levels of TGF-β1 correlate with tumour burden, metastasis, and serum PSA in prostate cancer patients [22,23]. TGF-β1 has been shown to promote prostate cancer growth by inhibiting immune responses against the tumour and stimulating angiogenesis and metastasis [23]. These observations seem to contradict indications that TGF-β1 is a negative regulator of prostate epithelial growth [18–20]. Further study revealed, however, that prostate cancer cells are less sensitive to TGF-β1 growth inhibition than normal prostatic epithelial cells because of the loss of TGF-β1 receptor expression in cancer cells [24–27]. TGF-β1 expression was suppressed by androgens in the prostate gland [28,29], whereas it was activated by androgens in hepatocellular carcinoma and prostate cancer cells [30]. A putative androgen-response sequence half-site was identified in the rat TGF-β1 promoter [30], but the mechanism for the opposing responses of the TGF-β1 gene to androgens in normal prostate and prostate cancer has not been defined.
The objective of the present study was to investigate how androgens regulate TGF-β1 expression in prostate cancer cells and to identify functional AREs in the TGF-β1 promoter. Using a series of TGF-β1 promoter deletion constructs, we identified six positive AREs and one negative ARE in the TGF-β1 promoter. We also present in vivo and in vitro evidence that androgens can directly regulate TGF-β1 expression through these AREs. This finding suggests that activation or repression of TGF-β1 expression by androgens is mediated through positive or negative AREs in its promoter.
Construction of TGF-β1 promoter reporter plasmids
A BAC (bacterial artificial chromosome) clone (RP11-662N17) that contains the human TGF-β1 locus was purchased from CHORI Bacpac Resources (Oakland, CA, U.S.A.). PCR was performed using the BAC DNA as a template to amplify human TGF-β1 promoter sequences (−3363 to +110). The PCR product was subcloned into the pGL3 luciferase reporter construct (Promega). Luciferase reporter plasmids containing various deletion mutants of the TGF-β1 promoter were constructed similarly. Point mutants of TGF-β1 AREs were generated using the QuikChange® Site-Directed Mutagenesis kit (Stratagene). All constructs were confirmed by mapping restriction enzyme sites and by DNA sequencing analysis.
Cell cultures
LNCaP, LNCaP-LN2, PC3mm2 and HT human prostate carcinoma cells were cultured in RPMI 1640 medium (Cellgro; Mediatech) with 5% (v/v) fetal bovine serum (HyClone). Phenol Red-free RPMI 1640 medium (Invitrogen) supplemented with 5% (v/v) serum (treated with dextran-coated charcoal) was used when the androgen was applied. R1881, a synthetic androgen, was purchased from New England Nuclear and dissolved in ethanol.
Transient transfection and luciferase assay
All transfection assays were performed according to the manufacturer's protocol for Lipofectamine™ or Lipofectamine™ 2000 (Invitrogen). Briefly, PC3mm2 cells (3×104) were seeded in 24-well plates 24 h before transfection. The total amounts of DNA for each transfection reaction were adjusted to 70 fmoles by addition of the pcDNA3.1 plasmid. The pRL-CMV (2.5 ng) vector that expresses the Renilla luciferase was included in each transfection reaction. The transfected cells were incubated in the absence or presence of 10 nM androgen (R1881) for 36–48 h and then harvested for the Dual-Luciferase Reporter Assay (Promega). In brief, the cells were washed with ice-cold PBS twice, and subjected to lysis in passive lysis buffer (Promega) for 15 min at room temperature (23 °C) with constant shaking. The lysates (10 μl) were used for the luciferase activity assay. The relative luciferase activity was eqivalent to the firefly luciferase activity/the Renilla luciferase activity. All transfection experiments and luciferase assays were carried out in triplicate.
ChIP (chromatin immunoprecipitation) assay
LNCaP cells were grown in the absence or presence of 10 nM androgen (R1881), cultured cells were fixed with 1% formaldehyde, and the resulting cross-linked chromatin was sheared by sonication to approx. 1 kb (Figure 1C) or 400 bp (Figure 5) fragments. These short chromatin fragments were immunoprecipitated by antigen affinity-purified AR-specific antibodies; the immunoprecipitated chromatin contained the enriched AR DNA-binding sites. The protein–DNA cross-links were reversed. Immunopurified DNA (2.5 μl) was used for the PCR reaction (30 cycles, annealing at 50 °C and extension at 72 °C for 20 s).
Gel-shift assay
The electrophoretic mobility shift assay was performed as described previously [31]. Oligos derived from TGF-β1 AREs were synthesized, annealed and labelled with [γ-32P]ATP by T4 polynucleotide kinase. In the gel-shift assays, each 20 ml reaction contained 20 mM Hepes, pH 7.9, 70 mM KCl, 1 μg of poly(dI-dC), 1 mM dithiothreitol, 0.1% NP-40 (Nonidet P40), 0.1 mg/ml BSA (bovine serum albumin) and various ARDBD (AR DNA-binding domain) proteins. The reaction mixture was incubated for 20 min at room temperature; the binding reaction was initiated by addition of the labelled probes (10000 c.p.m.) and the mixture was incubated for an additional 30 min at room temperature. The reaction mixture was loaded directly on to a 4% (37.5:1, acrylamide:bisacrylamide) non-denaturing polyacrylamide gel with 0.25×TBE [Tris/borate/EDTA (1×TBE =45 mM Tris/borate and 1 mM EDTA)] buffer and run at 150 V for 2 h at room temperature.
DNase I footprinting
DNA templates used for footprinting on the transcribed and non-transcribed strands of the TGF-β1 promoter were prepared by isolating the Pst1-XhoI fragments (−2466 to −2283 and −943 to −694) from pGL3-TGF-β1 and end-labelling with [32P] by a fill-in reaction with the Klenow enzyme. A 30 μl reaction contained 2 fmol of the 32P-labelled DNA fragment, 0.5 ng of poly[dI-dC], 5 μg of BSA, 10 mM Hepes, pH 7.9, 60 mM KCl, 0.2 mM EDTA, 4 mM MgCl2, 10% glycerol and indicated amounts of purified ARDBD. The reaction mixture was incubated for 20 min at room temperature, and then 50 μl of Ca/Mg solution (5 mM CaCl2 and 10 mM MgCl2) was added to each tube. DNase I (1 μl, 0.4 ng/ml) was added to each tube and digestion was continued at room temperature for 2 min. The reaction was terminated by adding 100 μl of stop solution (0.2 M NaCl, 30 mM EDTA, 1% sodium dodecyl sulfate and 0.1 mg/ml tRNA) and chilled on ice. After phenol/chloroform extraction and ethanol precipitation, the DNA was resuspended in 10 μl of gel loading buffer (90% formamide, 0.02% Bromophenol Blue and xylene, and 1×TBE) and loaded on an 8% polyacrylamide-urea sequencing gel. The gel was dried under vacuum and exposed to an X-ray film at −80 °C.
RNA isolation and real-time RT–PCR (reverse transcription–PCR)
Total RNAs were isolated from cultured cells using the TRIzol reagent and reverse-transcribed using the Reaction Ready First Strand cDNA Synthesis Kit (SuperArray Bioscience). The cDNA products were PCR-amplified (40 cycles of 30 s at 94 °C; 20 s at 55 °C; 30 s at 72 °C) with the RT2 real-time SYBR green PCR master mix and the gene-specific primer sets for human Nkx3.1 (PPH02267A), TGF-β1 (PPH00508A) and β-actin (PPH00073A) genes (SuperArray Bioscience) by SmartCycler II (Cepheid). Raw data processing and quantification were performed with the SmartCycler software (version 2.0C). The 2−ΔΔCT method was used to determine the relative quantification (in the presence of ligand compared with in the absence of ligand) of target gene expression [32].
Castration and androgen administration
C57BL mice (n=10) were castrated at 8 weeks of age under anaesthesia in accordance with the protocol approved by the International Animal Care and Use Committee. To castrate mice, mice were anaesthetized by intraperitoneal injection of Nembutal (45 mg/kg weight). Hairs over the surgical region were cut and the region was disinfected with 10% iodine and then with 70% ethanol. A 0.5 cm incision was made and surgical string was used to perform the ligation. Testis were then removed and the wound was closed with wound clips. The would clips were removed 5 days later. Seven days after the operation, the mice were subcutaneously injected with testosterone enanthate (3.6 ng/g body weight). The prostate glands were dissected at 0 (n=5) and 48 (n=5) h after testosterone replacement. Total RNA was isolated from prostate glands using the Trizol method and subjected to real-time RT–PCR analysis.
FLP (flipase)-mediated integration of the TGF-β1 promoter
The Wt (wild-type) and Mt (mutant) TGF-β1 promoters (−3363 to +110) were subcloned into pCM-luc. PCM-TGF-β1-luc and pcDNA5/FRT were co-transfected into HT1080 cells (HT55), which contain an FRT (FLP-recombinase target) site. The transfected cells were selected with zeocin for 2 weeks and the resistant colonies were picked up and expanded. Whole-cell lysates were prepared from cell lines and submitted for luciferase assay. HT55-TGF-β1 cells (5×104) were seeded in 24-well plates and transfected with 100 ng of the AR-expressing construct (pcDNA-AR). The transfected cells were incubated in the absence or presence of 10 nM R1881 for 36–48 h and assayed for luciferase activity.
AR directly targets the TGF-β1 gene
Expression of the TGF-β1 gene is induced by castration [29], suggesting that androgens suppress its expression. To investigate regulation of the TGF-β1 gene by androgens, we administered testosterone enanthate to castrated C57BL mice. Total RNAs were isolated from mouse prostate glands 48 h after androgen replacement. Changes in TGF-β1 gene expression induced by the androgen were then analysed by real-time RT–PCR. Expression of the TGF-β1 gene was inhibited by androgen replacement in the prostate (Figure 1A). In contrast, androgen replacement dramatically induced expression of the NKX3.1 gene, whose expression is positively regulated by the androgen [33]. Similarly, TGF-β1 expression was inhibited slightly by the androgen in the LNCaP prostate cancer cell line (Figure 1B). However, the androgen enhanced expression of the TGF-β1 gene in a variant of LNCaP (LNCaP-LN2) that was selected for increased metastatic potential by orthotopic "recycling" in mice [34]. The cells that were isolated by this procedure displayed not only increased metastasis to regional lymph nodes but also androgen-independent growth in vitro and in vivo [34,35]. In contrast, the androgen-induced expression of the NKX3.1 gene in LNCaP and LNCaP-LN2 did not change (Figure 1B).
In the ChIP assay, AR bound to the PSA and TGF-β1 promoter regions in the presence of androgen (Figure 1C, compare lane 3 with lane 4 and lane 5 with lane 6). The products amplified by PCR at the same time as the β-actin promoter, which served as a negative control, were not changed in response to the addition of androgen (Figure 1C, lanes 1 and 2). As AR directly targets the PSA promoter [36], these results indicate that AR also directly targets the TGF-β1 promoter.
TGF-β1 promoter contains both positive and negative AREs
To map AREs in the TGF-β1 gene, a genomic DNA fragment (3473 bp) that spans the 5′-flanking region of the human TGF-β1 gene was isolated from the BAC clone and was cloned into the pGL3 basic vector upstream of the luciferase gene. Transient transfection of this reporter plasmid into PC3mm2 cells resulted in an approx. 5-fold higher level of luciferase activity in the presence of androgen than in the absence of androgen (Figure 1D). The relative luciferase activities of the empty pGL3 luciferase reporter were not significantly different between cells cultured in the absence of the androgen and those cultured in the presence of the androgen. These results suggest that the cloned TGF-β1 genomic fragment contained functional AREs, which we then further mapped using a collection of deletions (Figure 1D). The deletion of nucleotides to −2112 or −999 decreased the response of the luciferase reporter to the androgen. Further deletion of nucleotides to −784 resulted in a slightly negative response to androgen. The deletion of nucleotides to −684, −575 or −394 resulted in positive responses to androgen. Further analysis with additional deletions indicated that there are positive AREs located within the regions from −2466 to −2283 and from −341 to +1, and a negative ARE within the region from −943 to −694 of the TGF-β1 promoter.
Indeed, the positive AR response was observed when the regions of −2466 to −2283 and −341 to +110 were subcloned into luciferase reporter constructs (Figures 2A and 2B). Further deletion analysis and alignments with the ARE consensus sequence [37] revealed three putative AREs in the proximal region and three within the distal region (Figure 2D). Mutation on any single putative ARE (Mt1, Mt2 or Mt3) in the distal region (−2466 to −2283) decreased the response to androgen (Figure 2A), whereas mutations on all three putative distal AREs (Mt1,2,3) completely abolished the response to the androgen. Mutation on the ARE1 (Mt1) or ARE2 (Mt2) in the proximal region (−341 to +110) decreased the response to androgen (Figure 2B), but mutation on the proximal ARE3 (Mt3) completely abolished the response to androgen. These results suggest that these putative AREs are functional.
To assay the negative ARE of the TGF-β1 promoter, we constructed a luciferase reporter (pGL3-4×ARE-E4-Luc) (Figure 2C), which includes four tandem AREs derived from the PSA gene upstream E4 core promoter with a pGL3-basic backbone. When the pGL3-4×ARE-E4-luc reporter was co-transfected with expression plasmid for AR into PC3mm2 cells, AR activated the reporter approx. 36-fold in the presence of the androgen (Figure 2C). When the TGF-β1 promoter region from −943 to −694 was inserted into the luciferase reporter between 4×ARE and E4 in both orientations, the androgen-driven activation of the luciferase activity was almost completely abolished, indicating that there is a negative ARE in this region. As a negative control, a 243 bp DNA fragment (SC) derived from the human SPDEF (GeneID: 25803) promoter (−3018 to −2775) inserted into the luciferase reporter between 4×ARE and E4 did not alter the androgen-driven transcription.
ARDBD directly interacts with the TGF-β1 AREs
To test whether ARDBD would interact with the AREs in the TGF-β1 promoter, we performed a DNase I footprinting assay. Strong footprints were observed over the region −2343 to −2311 (containing distal ARE1) (Figure 3A, lanes 5 and 6) and the region −2421 to −2341 (containing distal ARE3) (lane 11). Essentially no protection was seen over the distal ARE2-containing region (−2376 to −2354) (Figure 3A, lanes 3–6). Consistent with these observations, ARDBD bound strongly to the distal ARE1 and weakly to the distal ARE2 and ARE3 in the gel-shift assay (Figure 3B, lanes 4, 8 and 12). Mutations on the conserved nucleotides in the distal AREs (Figure 2D) abolished the ARDBD–ARE interactions (Figure 3B, lanes 2, 6 and 10). Similarly, ARDBD interacted with the proximal ARE1 and ARE3 in the gel-shift assay (Figure 3C, lanes 2 and 10), but no ARDBD–ARE2 interaction was detected (lanes 6). Thus, three distal AREs and two proximal AREs in the TGF-β1 promoter directly interact with ARDBD.
Similar analyses were performed to investigate whether ARDBD would interact with the negative AREs in the TGF-β1 promoter. No protection was observed over the negative ARE region (−694 to −943) in the footprinting assay (Figure 3A, lanes 12–16). Consistent with this observation, ARDBD failed to bind to the negative ARE in the gel-shift assay (results not shown).
Comparison of the ARE-containing regions in human TGF-β1 promoter with that in mouse and rat
Comparison of the ARE-containing regions in the human TGF-β1 promoter with published sequences for mouse (GenBank, L42456) and rat (GenBank, AF249327) TGF-β1 genes was carried out using MacVector 10.0 (MacVector). Distal ARE3 is highly conserved but distal ARE1 and ARE2 are less conserved among the three species (Figure 4B). All three AREs in the proximal region are highly conserved among the three species (Figure 4C). The negative ARE region is also conserved (Figure 4D, 249 bases, 45% identity). A previous study had identified an androgen-response sequence half site (−1932 to −1927) in the rat TGF-β1 promoter, which is conserved in the mouse TGF-β1 promoter [30]. This androgen-response sequence is not localized in the distal androgen-response region identified here.
AR is recruited onto the distal and proximal ARE-containing regions in the TGF-β1 gene
To investigate whether AR was recruited on to the positive and negative ARE-containing regions in vivo, we performed the ChIP assay with smaller genomic fragments (∼400 bp) (Figure 5A, lanes 1 and 2) and using primer pairs specific for the distal, proximal, and negative ARE-containing regions. AR bound to the distal and proximal ARE-containing regions in the presence of the androgen (Figure 5B, lanes 2 and 3, third panel). In contrast, no androgen-dependent recruitment of AR to the negative ARE-containing region was seen (Figure 5B, lane 1, third panel). As a negative control, the immunoprecipitation was performed at the same time with purified IgG (Sigma–Aldrich) (Figure 5B, fourth and fifth panels). The products amplified by PCR at the same time from the β-actin promoter, which served as another negative control, were not changed in response to the addition of androgen (Figure 5B, lane 4). These results indicate that AR directly interacted with the distal and proximal ARE-containing regions in the TGF-β1 promoter in the presence of androgen.
Positive and negative AREs in the TGF-β1 promoter are functional
Given the fact that multiple positive AREs and a negative ARE are present in the promoter of the TGF-β1 gene (Figure 4A), it is likely that regulation of TGF-β1 expression in response to androgens is complicated. To investigate the role of these identified AREs in their native promoter environment, we made point mutations on the positive AREs and deleted the negative ARE in the TGF-β1 promoter (−3363 to +110). Seven TGF-β1 promoter mutants were generated (Figure 6A). Point mutations on three distal AREs or on two proximal AREs (Figure 2D) and deletion of the negative ARE (−943 to −694) were generated separately or in combinations. The Wt and Mt TGF-β1 promoters were subcloned into the luciferase reporter (pGL3). These constructs were transiently transfected into PC3mm2 cells with the AR-expressing construct (pcDNA-AR). The transfected cells were grown in the absence or presence of androgen and submitted to the luciferase assay. Mutations on the distal AREs (M1) slightly decreased the response of the TGF-β1 promoter to the androgen (Figure 6B). In contrast, mutations on the proximal AREs (M2) completely abolished this response. Mutations on both distal and proximal AREs (M3) turned the positive response to the AR to a negative response. On the other hand, deletion of the negative ARE (M4) enhanced the positive response of the TGF-β1 promoter to the androgen. Further mutation on the distal AREs (M5) slightly decreased the positive response of the TGF-β1 promoter to the androgen, and mutation on the proximal AREs (M6) or on both distal and proximal AREs (M7) abolished both positive and negative responses of the TGF-β1 promoter to the androgen. These results indicate that the identified positive AREs and the negative ARE are functional in the TGF-β1 promoter.
Positive and negative AREs are functional in the integrated TGF-β1 promoter
The transfected promoter-reporter assay uses many copies of the construct in a single transfected cell, and the promoters, poorly integrated into the chromatin, may limit the corresponding transcription factors with respect to the exogenous construct. Yan et al. [38,39] developed a novel assay system to study the transcriptional regulation of promoters in vivo in mammalian cells. This system uses a recombinase (Flp) and site-specific recombination to facilitate integration of the promoter-luciferase sequence into a specific site in the genome of mammalian cells. Thus the activity of the integrated promoter closely mirrors expression of the endogenous gene in response to physiological cues.
We obtained the HT1080 cell line (HT55), which contains one FRT site, and integrated the Wt or Mt TGF-β1 promoter (−3363 to +110) into the FRT site in these cells (Figure 6C). As a negative control, the empty vector (pCM-luc), that did not contain the TGF-β1 promoter sequence, was also integrated into the same FRT site in HT55 cells. To determine whether the genome-integrated TGF-β1 promoter/luciferase reporters were active, the HT55-luc (lacking any TGF-β1 promoter sequence) and HT55-TGF-β1 (Wt, M3 or M4)-luc cell lines were analysed for luciferase expression. Although the luciferase activity in the HT55-luc cell line was barely detectable, HT55-TGF-β1 (Wt, M3 or M4)-luc showed robust (>50-fold) luciferase activity (results not shown), indicating that the integrated TGF-β1 promoters were active. The high luciferase activity was maintained in these cell lines over 10 passages, which contrasts sharply with that for extrachromosomal reporter plasmids, whose activity is rapidly lost.
We then determined whether the activity of the integrated TGF-β1 promoters was responsive to the androgen. Trace amounts of AR was detected in the HT cells (results not shown). We transiently transfected pcDNA-AR into these cells. The transfected cells were grown in the presence or absence of androgen for 24 h and the luciferase activity was measured. Variable responses of the integrated TGF-β1 promoter in the HT55-TGF-β1-luc cell line (Wt) to the androgen were observed. This might be due to the fact that the TGF-β1 promoter contains both positive and negative AREs and may reflect different cell densities or growth stages used for the assay. The result in Figure 6(D) shows addition of the androgen very slightly enhanced the activity of the integrated TGF-β1 promoter (Wt). Deletion of the negative ARE (M4), however, resulted in a stronger positive response of the integrated TGF-β1 promoter to the androgen. Mutation of the positive AREs (M3) turned the androgen response of the integrated TGF-β1 promoter to negative. These results imply that both positive and negative AREs are functional in the integrated TGF-β1 promoter.
Previous studies suggested that the TGF-β1 gene was negatively regulated in the prostate gland and positively regulated in prostate cancer cells by androgens. In the present study, we demonstrate that the TGF-β1 promoter contains multiple positive AREs and a negative ARE. The positive AREs interact with AR in vitro and in vivo, and both positive and negative AREs are functional in the synthetic promoters as well as in the integrated TGF-β1 promoter. These findings indicate that regulation of TGF-β1 expression in response to the androgen is complicated and could be neutral, positive or negative depending on cell types or cell environments.
Positive AREs in the TGF-β1 promoter
The following lines of evidence show that TGF-β1 expression could be positively regulated by androgens through the proximal and distal AREs in the TGF-β1 promoter. First, TGF-β1 gene expression was enhanced by the androgen in LNCaP-LN2 cells (Figure 1B). Secondly, the cloned full-length TGF-β1 promoter displayed a positive response to the androgen in PC3mm2 cells (Figure 1D). Thirdly, the distal region (−2466 to −2283) and proximal region (−341 to +110) responded to the androgen independently (Figures 2A and 2B). Fourthly, three distal AREs and two proximal AREs interacted with AR in gel-shift and footprint assays (Figure 3), and mutation on the conserved nucleotides in these AREs abolished the ARE–AR interaction (Figure 3) as well as the response of luciferase reporters to the androgen (Figures 2A and 2B). Most importantly, mutations on these AREs affected the response of the TGF-β1 promoter to the androgen (Figure 6).
The palindromic androgen-response consensus sequence (RGAACANGNTGTNCT) is involved in AR binding and positive androgen regulation [37]. The proximal ARE3 is more closely related to the consensus ARE and interacts more strongly with AR than the proximal ARE1 and ARE2. Consistent with the interacting data, mutation on the proximal ARE3, but not on proximal ARE1 or ARE2, completely abolished the response of the TGF-β1 proximal promoter (−341 to +110) to androgen. Mutation on the proximal AREs completely abolished the positive response of the TGF-β1 promoter, indicating that the proximal AREs play a critical role in the androgen-driven expression of the TGF-β1 promoter. Although the AR-binding affinity of the three distal AREs was significantly different in DNase I footprint and gel-shift assays, mutation on a single ARE decreased, but did not abolish, the response of the TGF-β1 promoter to androgen. Mutation on all three distal AREs completely abolished the response of the distal enhancer (−2466 to −2283) to androgen, but not that of the full-length TGF-β1 promoter. The organization of distal and proximal AREs in the TGF-β1 promoter is reminiscent of that in the PSA and probasin genes [4].
Negative ARE in the TGF-β1 promoter
Gene regulation via negative AREs has been described for PSA, Maspin and prostate-specific membrane antigen genes [14–17]. Our analysis revealed a negative ARE in the TGF-β1 promoter. The negative ARE strongly inhibited androgen-driven transcription in the synthetic promoter (Figure 2C), whereas deletion of the negative ARE augmented the positive response of the TGF-β1 promoter to the androgen (Figures 6B and 6D). Previous studies identified one negative ARE (XBE) in the PSA gene [17] and one in the Mapsin gene [14]. XBE contains the AR-binding site overlapped with the NF-κB (nuclear factor-κB)-binding site. AR competed with NF-κB for a DNA-binding site to negatively regulate PSA gene expression. The mechanism for the negative ARE in the Maspin gene is unknown. We did not find any DNA sequence that was significantly similar to the XBE or the Maspin negative ARE in the negative ARE region of TGF-β1 and failed to detect ARDBD interacting with the negative ARE. More interesting were the findings that the minimal sequence required for maximal inhibition activity is approx. 250 bp and that further deletion from this region gradually led to loss of its activity (results not shown). These observations suggest that the negative ARE in the TGF-β1 gene is novel and the repression by androgen is indirect. Further mapping and analysis of this negative ARE is underway.
Expression of the TGF-β1 gene is negatively regulated by androgen in the normal prostate and in prostate cancer LNCaP cells (Figures 1A and 1B). TGF-β1 expression was enhanced by the androgen, however, in a metastatic variant (LNCaP-LN2) of LNCaP cells. TGF-β1 promoter activity was enhanced in the highly metastatic prostate cancer (PC3mm2) cells. These observations suggest that there is a gradual loss of negative response to androgens and gain of positive response to androgens of the TGF-β1 gene during prostate tumourigenesis and prostate cancer progression. Consistent with this possibility is the observation that prostate tumours express higher levels of TGF-β1 than normal prostate tissue [21] and that increasing levels of this growth factor are associated with prostate cancer progression [22]. Identification of positive and negative AREs in the TGF-β1 gene would provide a basis for understanding this transition and the roles of AR and TGF-β1 in prostate tumourigenesis and progression.
We thank Kathryn Hale for critical editorial review, prior to submission, and Dr Douglas Boyd (Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, TX, U.S.A.) for the FRT-HT cell line. This work was supported by a grant (1R01 DK065156 01) to Z. W. from the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institutes of Health.
Abbreviations: AR, androgen receptor; ARDBD, AR DNA-binding domain; ARE, androgen-response element; BAC, bacterial artificial chromosome; ChIP, chromatin immunoprecipitation; FLP, flipase; FRT, FLP-recombinase target; Mt, mutant; PSA, prostate-specific antigen; RT–PCR, reverse transcription–PCR; TGF, transforming growth factor; Wt, wild-type
- © The Authors Journal compilation © 2008 Biochemical Society


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Can 300cc-3500cc Breast Implants Increase Size from 34A to C?
I am 25 years with no kids, 5'7" tall at 135 lbs. I am considering having breast augmentation. I am currently a 34A and would like to be a full... READ MORE
Breast Implants to Minimize Gap Between Breasts on a Wide Chest?
I have a pretty wide BWD (about 16-18 cm as far as I have measured) and am shooting for a natural 36C-D look, I am currently a small 36B with a fair... READ MORE

subset: commoncrawl


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Codelco Raises $2 Billion in Sale of Debt
Bloomberg reported that Codelco has raised $2 billion in a sale of debt to invest in copper expansion.
As quoted in the market news:
Codelco plans to invest about $26 billion this decade to modernize aging mines and offset declining output from ore with less copper content. Codelco said the yields it achieved are among the cheapest funding it has ever had.


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Did you watch the Heat/Knicks game on National TV yesterday? There was a play in the second half where LeBron James went up to catch a fast break alley oop. The only Knick back was J.R. Smith, who not wanting to get caught under an atomic dunk, went up to get the pass himself, or at the very least, disrupt the oncoming freight train of destruction.
Here's the thing though: As great of an athlete as Smith is, he's only human. Everything we've seen out of LeBron over the last month+ tells us he's not of this basketball universe.
On this particular play, LeBron couldn't finish and actually came down awkwardly on his knee. Regardless, the athletic display is incredible. Check out this larger shot:
For breaking news, rumors, exclusive content, and contests sent right to your inbox, sign up here for the Dime Email Newsletter.
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text:
Link Time!
- Jessica Lucas talks Melrose Place and good girls gone bad — Hit Fix
- Wes Craven may direct Scream IV? — Cinematical
- The best-reviewed Coen Brothers movies (and No Country For Old Men isn't number one!) — Rotten Tomatoes
- Will you watch Oprah's interview with Sarah Palin? — TresSugar
- The Biggest Loser's Jillian Michaels is getting her own show on NBC — Zap2it
- Is Guns N' Roses touring Canada next year? — Stereogum
- 10 things you never knew about The Simpsons — Flavorwire
- ABC is close to picking up a new pilot called Little Liars starring Privileged's Lucy Hale — Hollywood Reporter
Image Source: Getty


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From www.benmaller.com
Pats coach chases away Steelers trainer?
There were strange plays at Heinz Field yesterday, but nothing as out of the ordinary as Patriots coach Bill Belichick chasing Steelers trainer John Norwig away from one of his injured players, according to the POST-GAZETTE.
As tackle Matt Light lay on the turf with a serious leg injury in the second quarter, Norwig ran over to see if he could help, as opposing trainers often will do with serious injuries. Belichick walked out on the field and told Norwig, in no uncertain terms, to get away from his player. Norwig walked off the field and could be seen laughing about it with several Steelers doctors on the sideline.


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Adele to Perform SKYFALL at The Oscars?
Rumors of Adele's impending return have began to emerge.
Multiple sources are claiming that the Grammy winner may take the stage at the upcoming 85th Academy Awards, set to air Feb. 23. Following the announcement that the 50th anniversary of the James Bond franchise will be celebrated that night - and that the singer recorded the theme song for the lastest installment SKYFALL - she may become part of the tribute.
It also doesn't hurt that her song SKYFALL is reportedly on the short list for a 'Best Original Song' nod. Nominations will be announced on Jan. 10 - and should the 007 theme be honored, it would seem the chance of an Adele appearance would become only more likely.
Adele has recently been out of the spotlight for the good part of a year; in June 2012, she announced that she was pregnant with boyfriend Simon Koneck's child. She gave birth to a boy in October.
The success of Adele's albums has earned her numerous records in the Guinness Book of World Records. She became the first artist in history to lead the Billboard 200 concurrently with three Billboard Hot 100 number ones. Adele is also the first female artist to have three singles in the top 10 of the Billboard Hot 100 at the same time, and the first female artist to have two albums, "19" and "21" in the top five of the Billboard 200 and two singles in the top five of the Billboard Hot 100 simultaneously.


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Tuesday, November 24, 2009
Pivot3 Gets $2.0M More
Houston, Texas-based Pivot3, a developer of storage products, has raised another $2.0M in venture funding, according to a regulatory filing by the firm Monday. Details on the funding have not been released, however, the firm has previously raised funding from Interwest Partners, Lightspeed Ventures, Mesirow Financial, and Silver Creek Technology Investors. The filing indicates 10 investors were involved in the funding. More information »


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Don't miss out on a lower price on this hotel
Get an overview of this hotelRiverfront Berlin hotel in Friedrichshain with restaurant
Popular property highlights
Located in Friedrichshain, this hotel is 0.3 mi (0.5 km) from East Side Gallery and within 3 mi (5 km) of Alexanderplatz and Checkpoint Charlie Museum. Nikolaikirche and TV Tower are also within 3 mi (5 km).
Hotel Features
A restaurant, a health club, and a bar/lounge are available at this hotel. WiFi in public areas is free. Other amenities include a sauna, a conference center, and self parking.
Room Amenities
All 304 rooms offer free WiFi and free wired Internet, flat-screen TVs with cable channels, and comforts like premium bedding and pillow menus. Other amenities available to guests include 24-hour room service, first-run movies, and minibars.
Guest Highlights
- Great bathrooms and comfortable rooms
- Good location
- Good for nightlife
- Helpful staff
nhow Berlin
Hotel Amenities
Hotel Amenities
Nhow Berlin features a health club and a sauna. The hotel offers a restaurant. A bar/lounge is on site where guests can unwind with a drink. Public areas are equipped with complimentary wired and wireless Internet access.
This 4-star property offers access to a business center and meeting rooms. This Art Deco hotel also offers a terrace, tour/ticket assistance, and barbecue grills. Limited onsite parking is available on a first-come, first-served basis (surcharge).
Nhow Berlin has designated areas for smoking.
- Number of floors - 10
- Total number of rooms - 304
- Barbecue grill(s)
- Free newspapers in lobby
- Concierge services
- Free WiFi
- Free wired Internet
- Designated smoking areas
- Tours/ticket assistance
- Turkish bath/Hammam
- Area shuttle (surcharge)
- 24-hour front desk
- Laundry facilities
- Meeting rooms 10
- Parking (limited spaces)
- Terrace
- Luggage storage
- Offsite parking reservations required
- Self parking (surcharge)
- Conference center
- Steam room
- Bar/lounge
- Breakfast available (surcharge)
- Business center
- Free use of nearby fitness center
- Dry cleaning/laundry service
- Elevator/lift
- Health club
- Multilingual staff
- Restaurant
- Year Built 2010
- Sauna
Family Friendly Amenities
- Family Hotel
- Free Wi-Fi
- Laundry facilities
- Cribs/infant beds (complimentary)
Available in all rooms: Free WiFi , Free wired Internet
Available in some public areas: Free WiFi , Free wired Internet
Self parking (surcharge), Parking (limited spaces), Offsite parking reservations required
Room Amenities
- Premium bedding
- Air conditioning
- Free newspaper
- Premium TV channels
- Pay movies
- Shower/tub combination
- Iron/ironing board
- Hypo-allergenic bedding available
- In-room climate control (air conditioning)
- Minibar
- Free WiFi
- Free wired Internet
- Cable TV service
- Flat-panel TV
- Daily housekeeping
- Private bathroom
- Designer toiletries
- Hair dryer
- Free cribs/infant beds
- Desk
- In-room safe (laptop compatible)
- Room service (24 hours)
- First-run movies
- Pillow menu
Where to Eat
Full breakfasts are available for a surcharge.
Fabrics - This restaurant specializes in Modern European cuisine and serves breakfast, lunch, dinner, and light fare. Guests can order drinks at the bar and enjoy alfresco dining (weather permitting). Open select days.
Envy Bar - This bar specializes in local and international cuisine and serves dinner and light fare. Open daily.
24-hour room service is available.
Nearby Things to Do
Recreational amenities at the hotel include a health club and a sauna.
If you have requests for specific accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
- In-room accessibility
- Roll-in shower
Hotel Policies
Minimum check-in age is 18
Check-in time ends at midnight
Check-in time starts at 3 PM
Check-out time is noon
Payment types
Children and extra beds
- Children are welcome.
- Kids stay free! One child 11 years old or younger stays free when using existing bedding.
- Rollaway/extra beds are available for EUR 30 per night.
- Free cribs (infant beds)!
- Pets allowed for an extra charge of EUR 20 per pet, per night
- Dogs only
- 1 per room
You need to know
Extra-person charges may apply and vary depending on property policy.
- Deposit: EUR 50 per night
- A city tax is imposed by the city of Berlin. Business travelers with proof of business-related travel are exempt from this tax. For more details, please contact the property using the information on the reservation confirmation received after booking.
Optional extras
- Fee for full breakfast: EUR 30,90 for adults and EUR 30,90 for children (approximately)
- Self parking fee: EUR 16 per day
- Pet fee: EUR 20 per pet, per night
- Rollaway bed fee: EUR 30 per night
Hotel Name
- Berlin nhow
- nhow
- nhow Berlin
- nhow Hotel Berlin
- Nhow Berlin Hotel Berlin
We should mention
One child 11 years old or younger stays free when occupying the parent or guardian's room, using existing bedding. Only registered guests are allowed in the guestrooms.


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Wine Barrel Cuff Links
Size: .75" long x .50" high
Price: $140
These eco-friendly sterling silver cuff links are made of recycled oak wine barrels from the Napa Valley. The unique design features wood that's beautifully wine stained. (In better wineries, an oak barrel is used only once or twice; afterwards it ceases to give off its rich flavor.) A perfect environmentally friendly gift for wine-appreciating dads, husbands, boyfriends, and friends and co-workers.
Artist Bio
In 1980, when the City of New York discontinued its "Y" cutout subway tokens, Ward Wallau decided they should be recycled into cuff links. After 11 years spent petitioning the New York Transit Authority for permission to use them, he was granted an exclusive license to incorporate discontinued tokens in his jewelry. Now he also recycles outdated coins, circuit boards and SCRABBLETM tiles in his jewelry and accessories.


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Jennifer Garner and her 2-year-old daughter Violet were spotted playing at a friends house in Los Angeles. As we just reported, it's been confirmed that Jennifer is 5 months along into her second pregnancy.
Photos: Flynet
Jennifer Garner and her 2-year-old daughter Violet were spotted playing at a friends house in Los Angeles. As we just reported, it's been confirmed that Jennifer is 5 months along into her second pregnancy.
Photos: Flynet
Filed under: Jennifer Garner,Violet Affleck21 Comments »»
21 Comments on "Jennifer, Violet & Confirmed Baby Bump"
It looks like she is wearing one of those fake pregnancy pads
Wonder why ..
Ok it is a beige bra… so I was wrong .. you should be banned from saying such a remark.. Jane.. why not ban all your comments
from now on.. they are rude and offend …. so why not you..
To tell someone that they are unstable and they should be banned is rude…It was just a theory.. And there was no
conspiracy theory..Just curious..
She COULD be 5 months, but….don't people show more with their second baby?? She isn't showing that much to me.
@ Lisa – you've been warned more than once….your CAPS have got to go. It's seen as yelling and is very annoying to our viewers.
Can you tell me why capitals are considered "screaming" but making a comment about jen is considered unstable.. "screaming
capitals " what the heck is that…capitals can't scream.. Explain what screaming is?" Is it yelling because that is what screaming
Ok, this might be big mistake but I still say this once.
I don't really know why caps are considered screaming/yelling/shouting but they are. And part of netiquette is that you avoid usin them. You can read it i.e. from here: http://en.wikipedia.org/wiki/Netiquette
They clearly state that: "Another rule is to avoid typing in ALL CAPS, which is considered shouting or yelling. " You wouldn't shout all the time in real life, would you.
I haven't ever commented here, but I enjoy this site….fun to see the famous peoples kids?!
I love Jennifer Garner, she's so real. I feel really bad thought that the paparazzi caught this view of her, and that it is being published at all…really, who would want a down the shirt picture of themselves ever, and then to have it "flashed" (pun intended) all over the net? Dang!
There was some talk that Nicole Kidman was faking a pregnancy and might be wearing a pregnancy pad. That sounds ridiculous to begin with. Why on earth would anybody do that? It makes no sense. That thought never occurred to me. My sister did not show her baby bump until her sixth month. She had a six pound baby (few weeks premature) and wore her pre-pregnancy jeans home from the hospital. Go figure. Every body is different.
can't believe she is 5 months along. she doesn't look as glowing as her last pregnancy. infact, i've noticed in the past few months her face is getting slimmer – not as full and radiant as before. wonder why.
It's a beige bra!
She looks around five months pregnant to me!
I am five months pregnant with my 4th!! And I am around the same size. so I would say 5 months is about right.
In this pics Jennifer's pregnancy is definitely showing. She may indeed may be 5 months along. Come on Lisa, she does not look like she is wearing a fake pad. lol
Take a look at the picture of her bending over… that is the top
of a pregnancy pad..because if you look at the other pictures you
will see that she is not that big..
Looks like a flesh colored bra and her tummy to me, no conspiracy theory "fake pregnancy pad".
But what do I know, I don't know people who go around wearing fake pregnancy pads for the paparazzi.
Lisa Smith
Are you for real??!!! That is the most ridiculous statement I have ever heard. You need to be banned from posting comments, you are not a stable person at all, you're mental state of health should really be examined. Pathetic.
Who are these nut bags who say these celebrity pregnancies are fake? I don't think I've heard anything so ridiculous. When Jennifer takes her baby home in four months are you going to say the baby is fake too?
I still don't think she is 5 months….with this being her second baby she would be showing a lot more at 5 months. At least in my experience. Im betting she is 15 weeks or so. Im thin and I was showing way more than this at 5 months. Course..I could be wrong, but I still say she isn't that far along.
Umm…..Why are we looking down this ladies shirt???


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Town Hall Meeting Come and Be Part of the Conversation about Heroin, Opiates & Recovery
May 17, 2016 at 6:00 pm
Morning Star Fellowship Church
For more information...
The Bucks County Juvenile Court functions as both a judicial and an administrative agency primarily with respect to children under age 18, who are charged with juvenile delinquency for violating the criminal law.
As a judicial agency, the Court determines questions of fact and law based on evidentiary hearings. As an administrative agency, the Court is responsible for providing custodial services and treatment including probation supervision and placement to children under the jurisdiction of this agency. All children arrested for committing felonies and misdemeanors are processed through the office.
Twenty-four hour a day probation services are required for intake and for decisions concerning detention. To carry out its work, the Court employs a professional staff of 37 Juvenile Probation Officers and managerial, 9 clerical and 4 Victim Witness, and 9 fiscal personnel.
Office hours:
8:30 am through 5:00 pm
Monday through Friday


text:
- News
Europe's banks poised to return to acquisition trail
Lenders seek new sources of growth as regulations bite into earnings
European banks, still nursing their wounds from the continent's financial crisis, are starting to look for opportunities to expand internationally through acquisitions.
Bank executives in Spain and Italy, two of the countries hit hardest by Europe's economic woes, say they are scouting for potential targets abroad. And in France, central bank officials have suggested privately that lenders look at possible acquisitions in Southern...
To Read the Full Story, Start Your Free Trial or Sign In


text:
StartUpHire is the place to find Developer jobs in and near Mojave. Review the list of Developer Mojave career opportunities to the left. These Developer jobs represent some of the most rewarding careers available.
You can refine your job search for Developer career opportunities in Mojave by adding a job title, company name, or keyword to the search box above … or click the "Advanced Search" link for more options.
Don't see the employment opportunity you're looking for? Check back soon for more Developer jobs in Mojave, or sign up for an email alert (link below) to receive periodic updates on new Developer jobs in Mojave, CA.
Demand for Developer Professionals in Mojave, CA
Developer Jobs in Minneapolis, MN
Try fewer or different keywords.


text: I appear to have suddenly lost print contrast in my process - and I'm at a total loss. My enlarger is an Ilford Multigrade 500 head on a D5500 chassis. My standard print developer is an "Easy 130" which I prepare by adding 20 g sodium sulfite, 45 g glycin and 2.5 g BZT to a gallon of Dektol stock. I dilute 1+2 and develop prints for 3 minutes at 21 deg. C. Suddenly, both Grades 2 and 2½ are printing at Grade 1 contrast. I tried a working strength at 1+1 and extended development to 5 minutes - still Grade 1. Next I redid the safelight test by covering a piece of paper exposed to a threshold level of light with a coin for 7 minutes - no issue. I then changed both lamps in the enlarger - no difference! Next, I exposed a series of prints in my D 5 with a dichroic head at various degrees of magenta filtration and found the following: 25M - Grade 1; 75M - Grade 2½ and 100M - Grade 3. I then repeated this series with a newly mixed batch of straight Dektol 1+2 with 3 minutes development. Again - no difference. In fact a Grade 1 exposure of 60 Y gave me an ISO range of 195 which is a Grade 00 ! I had used some older paper originally, (only 6 months old, mind you.) Just in case I bought a new box of MGIVRC Glossy which I received only a couple of days before the final tests. The grades were determined by printing a 4x5 Stouffer 31 step wedge. I can't think what else to try. It must be in the process somewhere because this low contrast problem occurs across two separate enlargers. I really don't want to start over-developing my film just to attain mid-range contrast on a "Grade 2½" paper. It's probably something stupid that I've overlooked - but I'm damned if I can think what it is? H-E-L-P Thanks a lot Bob


text:
Worthington Teen To Be Tried As Adult In Kidnapping, Assault Case
A Worthington student, with a history of drug abuse and unruly behavior, will be tried as an adult for a violent crime, which left a girl nearly dead.
On Friday, Judge Dana Preisse decided Joseph Lyons, 18, should be tried as an adult even though he was 17 at the time of the alleged crime.
The victim's mother, who asked not to be identified for her daughter's protection, says Lyons was a friend of her daughter's at a Worthington High school.
Last July, Lyons and her daughter were sitting in his car in her neighborhood.
"Started beating her out of the blue and she grabbed his hands and asked him, 'what are you doing?'" said the mother. "He grabbed her by the neck and then put another hand inside her mouth and lacerated her throat and suffocated her."
The pictures are horrendous. She passed out from the beating. Police say Lyons threw her in the trunk but she came to inside and frantically called her mother.
"It was really horrible. It was like a nightmare," said the mother, "I answered the phone and she was screaming desperately."
Her daughter ended up at Alum Creek park. She told her mom she was punching the inside of the car and Lyons finally just let her go.
"She still has nightmares, sometimes she screams in the middle of the night," said the mother.
As her daughter deals with the mental anguish, the family is learning more about Lyons' past. Several offenses for drugs and violence, but never given serious punishment. This mom is relieved he'll be tried as an adult this time.
"Somebody else maybe wouldn't be so lucky as she was," said the mother.
Lyons sat in court in tears, while the defense attorney tried to convince the judge that he needs to be tried in the juvenile system.
"He's savable not because he comes from a perfect family. But he comes from a family, a good family that cares, and a family that supports him," said defense attorney, William Ireland.
Lyons is now being held on a $300,000 cash surety bond. He faces charges of kidnapping, felonious assault, and aggravated robbery.


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Police are seeking public assistance to find those responsible for attacking a deaf and mute man in his 60s, who was violently assaulted and had his electric scooter stolen.
At 9.30pm on Sunday, the 64-year-old man who, was at home in Patterson Drive in Swan View, noticed his electric scooter had been stolen from his back patio.
He went to the park opposite his home to look for the scooter as it had been stolen before and abandoned there.
In the park he was approached by three young males who came from the direction of a nearby lake.
Two of the males pushed and shoved him before one of them then hit him in the face with a bottle.
The victim fell to the ground before running to his home.
He suffered a laceration and swelling to his cheek.
The victim's scooter was later found in the lake, partially submerged and damaged.
The three young males were described as being between 15 and 18 years old.
One was wearing a red T-shirt and a baseball cap and was of skinny build with a thin moustache.
He has provided a composite image of the main offender.
Anyone with information is urged to call Crime Stoppers on 1800 333 000.


text:
For Online Computer Support, Ask a Computer Technician
Are you referring to your Internet Explorer favorites?
Let us know
Could you right click the start button and choose Explore, Then navgate to C:\Documents and Settings\yourusername\Favorites andyu should ee all of your favourates listed.
From there you can delete, Copy or move them to a different machine.
Let us know if you need more info.
That is the default folder for all of your favorites, If they are not there then I assume something else is happening so could you tell me what prompted this search for them?
Did they disappear from your IE window?
If you could open your Internet Explorer then click Favorites to open the drop down window, Find your favourite in the window and right click it, Then choose send to, Desktop (create shortcut) nd repeat those steps for each one you wish to have an icon for.
Let us know how you get on.


text:
Bongo Canvas Tote Bags are an Idakoos specialtiy! Idakoos has many things to offer. Whether you want to get a Bongo Canvas Tote Bag for a special occasion, give a Bongo Tote Bag as a present or need to represent with a funny Custom Bongo Bag during Holidays or Family and Friends get-togethers, Idakoos is sure to have the right Bongo Canvas Tote Bag for you! Even if it turns out you can't find the right Custom Bongo Bag with the perfect design for your mother, father or children (or friends!); Idakoos offers all of its Bongo Canvas Tote Bags as Custom Bongo Tote Bags. Personalize any Bongo Canvas Tote Bag the way you want to ensure the perfect gift.
Showing 1 - 24 of 83 Canvas Tote Bag


text:
The fiscal year 2012 budget process has been anything but typical or predictable. While fiscal year 2012 starts in just a few weeks on October 1, 2011, the annual appropriations process is far from complete and funding for federal education programs has not yet been finalized. Nevertheless, congressional action in the months that have led up to the start of fiscal year 2012 will have important effects on education funding levels in the appropriations process as well as for other programs, such as student loans and education tax benefits.
This brief by the New America Foundation's Federal Education Budget Project explains how the congressional budget process affects education funding, with special emphasis on the unique circumstances of the fiscal year 2012 budget process. It provides a summary and analysis of the 2012 budget process to date, relevant points of the Budget Control Act of 2011, and little-understood loopholes like advance appropriations.
Read the full issue brief here.


text:
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Womens Classic Cluster Necklace
Give any outfit a subtle touch of glamour with this women's long cluster necklace. With a dangling tassel and an array of pendants and gems on a diamante loop, attached to a silver rope chain, this necklace is the perfect finishing touch for your evening attire.
- Fabric: METAL
- Info
- Delivery
- Reviews
Product Code: 94728-SILVER
Need Help? Call us: 029 2027 0944
8.30am– 5.00pm GMT (Monday – Thursday)
8.30am – 4.00pm GMT (Friday)
UK Standard Delivery £3.99 (3-5 working days Mon-Sat, excl. Sundays & public holidays*)
UK Standard Delivery Over £50 FREE (3-5 working days Mon-Sat, excl. Sundays & public holidays*)
UK Next Day Express Delivery £4.99 (orders placed by 3.00pm will be delivered next working day Mon-Sat, excl. Sundays & public holidays*)
UK Pre Noon Express Delivery £5.99 (orders placed by 3.00pm (Mon-Thur) will be delivered before noon the next working day, excl. Saturdays, Sundays & public holidays*)
*Full delivery information can be found Here
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text:
"We wanted the freshest salad possible for our patrons, so we decided to let our customers harvest their own salad."
Kawaihae Harbor, Big Island, HI (PRWEB) April 30, 2014
The Blue Dragon Restaurant on the Big Island of Hawaii, winner of numerous dining awards, has launched what they are calling the "Living Salad" or the Freshest Salad in the World.
Thirty miles north of Kailua-Kona, the Blue Dragon's Chef Noah Hester is the mastermind behind this 21st century salad. With the farm at table concept becoming so popular, Hester thought why not take it a step further. "We wanted the freshest salad possible for our patrons, so we decided to let our customers harvest their own salad," he commented in a recent interview.
Hester went on to explain that a distinctive collection of living greens is delivered right to the table while still growing in sustainable wooden boxes. Small scissors and a seasonal vinaigrette accompany each salad. The salads are also served with baby organic heirloom vegetables.
Gary Cass from Turnip Wagon Farms grows the salads. He worked closely with Hester to make his vision a reality. Cass commented, "The Living Salad is a mix of micro and baby greens, which include upland cress, red Russian kale, Chia, Simpson lettuce, and Mizouna. We carefully plant the seeds in beautiful boxes containing organic soil and naturally grow the salads to maximize flavor and nutrients simultaneously."
Delphina Dorrance, co-owner of the Blue Dragon likens the Living Salad having a pristine farm garden at your table. She said, "Did you know that from the time your vegetables are picked, to the time you buy them at the grocery store, anywhere from a few days to weeks have passed. It is well documented that from the moment salad is cut the leafy greens begin to lose vitamin C and other nutrients. Lettuce is especially vulnerable to damage and may lose up to 50% or more of its nutritional value very quickly. At our restaurant you're actually enjoying the freshest salad possible."
Co-founder, Bennett Dorrance, carry on saying, "Our customers love this salad! Kale, one of the items in the Living Salad, is a very popular salad green that combines a wonderfully distinctive taste with high levels of antioxidant vitamins A, C and K, calcium and phytonutrients. And the dressing is delicious."
Continuing the ongoing wellness trends and healthy consumer preferences, the Blue Dragon's new super-greens salad combines freshness, maximum flavor and nutrient density. Chef Noah says it is his intention to raise the question, "How fresh is your salad?"
Ranked high on social media such as FaceBook and sites like Trip Advisor, the Blue Dragon is the recipient of numerous awards, including North Hawaii's Best: Chef, Live Music, Night Life, Dining Atmosphere, Family Entertainment, and Bartender, as voted by the readers of The North Hawaii News. The Blue Dragon feature fresh coastal cuisine, locally caught fish and daily picked produce from their own organic farm.
For more information or to schedule an interview, please email Jennifer Hackleman at bluedragonmanager(at)gmail(dot)com, visit http://www.bluedragonrestaurant.com, or call 808-882-7771.


text:
How long has it been since SMU beat Rice in Houston?
Ronald Reagan was in the White House.
A gallon of gas was 89 cents.
Top Gun was a box-office hit.
The top song on the Billboard chart: That's What Friends Are For by Stevie Wonder, Dionne Warwick, Elton John and Gladys Knight.
You get the picture.
The last time SMU beat the Owls on the road was Sept. 13, 1986, a streak of 10 straight losses.
A look at the Owls' winning streak in the series:
Aug. 29, 2008
|Rice 56, SMU 27
Comment: Chase Clement threw six TDs, including three to Jarett Dillard, to spoil June Jones' SMU debut in the season opener.
Nov. 25, 2006
|Rice 31, SMU 27
Comment: Jarett Dillard had three TD catches as the Owls secured their first bowl bid since 1961.
Oct. 9, 2004
|Rice 44, SMU 10
Comment: Ed Bailey rushed for 158 yards and four TDs.
Oct. 26, 2002
|Rice 27, SMU 15
Comment: Greg Henderson replaced injured QB Kyle Herm and led the Owls to the go-ahead scoring drive in the third quarter.
Nov. 4, 2000
|Rice 43, SMU 14
Comment: Dan Dawson had two interceptions as the Owls scored the most points against the Ponies since a 41-10 win in 1960.
Sept. 5, 1998
|Rice 23, SMU 17 (OT)
Comment: Clint Leschber atoned for an earlier fumble with the game-winning TD in overtime.
Oct. 19, 1996
|Rice 35, SMU 17
Comment: Quarterback Chad Nelson ran for 197 of the Owls' 440 rushing yards and scored two touchdowns.
Nov. 5, 1994
|Rice 17, SMU 10
Comment: With SMU faced with fourth-and-goal from the Rice 10 with four seconds remaining, the Owls stopped Jacques Smith at the 1 to preserve the victory.
Oct. 10, 1992
|Rice 28, SMU 13
Comment: Trevor Cobb rushed for 210 yards and two touchdowns and the Owls defense forced five turnovers.
Nov. 10, 1990
|Rice 30, SMU 28
Comment: Quarterback Donald Hollas passed for 238 yards and two touchdowns as the Owls recorded their first five-win season since 1980.
Follow me on Twitter at Chronicle_Owls.


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Please change the store drop-down. This store doesn't deliver to United States
Eroica Chianti jersey.
Warning: Last items in stock!
Availability date:
Eroica look and modern performance, two apparently conflicting elements which blend perfectly into a single jersey. The Chianti Jersey is a replica of the Gaiole wool jersey, inspired by the small village of Gaiole in Chianti, where the Eroica adventure began. Made with Energy Microsense, a 100% polyester fabric, the jersey feature an ergonomic cut, being slightly longer at the back, plus full covered zip and triple back pocket to store everything you need.


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Need help with dhcp.conf file for diskless nodes
I currently have a 2 node distcc distrubuted compiling cluster setup. It works fine. Now I am interested in moving the clients to be diskless. This totural should be all I will need to get my setup going... but... I am having a really hard time with the dhcp.conf file.
I am using etherboot with floppys to boot my clients.
http://pastebin.com/594913 << that is my basic dhcp.conf file for setting up my diskless nodes for http://www.gentoo.org/doc/en/diskless-howto.xml but why is my client not getting dhcped when booting from a floppy? is there somethign wrong in this?
its funny because this will dhcp a normal computer fine.. and no its not the client because I have tested it on my currently running ltsp server..
thanks for your help
|All times are GMT -5. The time now is 03:07 AM.


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Pacific West Capital and LISA: Active Partners in the Life Settlement Industry
More than 100 companies across the United States rely on LISA to be their voice within the secondary insurance market. LISA, or the Life Insurance Settlement Association, acts as the trade body for brokers, providers, financing entities and service firms. The association urges the structured development of critical regulations and promotes integrity and competence within its membership ranks.
Pacific West Capital Group, a Los Angeles-based senior settlement facilitator, has been a member of LISA since 2006. As one of the industry's pioneers, Pacific West Capital has carved out a niche for trusted investments that deliver fixed returns over a mid- to long-range period of time. For a $20,000 minimum investment, qualified California residents can purchase fractional interest in the proceeds of policy that meets stringent underwriting standards.
Pacific West Capital, along with all other LISA members, must comply with a code of ethics and with standards of professional conduct. These provisions include the following:
- Placing client interests above personal interests
- Honestly portraying life settlement transactions and making disclosures promptly
- Maintaining client privacy and transaction confidentiality
- Improving the knowledge and professional capabilities of themselves and others
- Acting ethically and complying with all laws and regulations
Along with these professional principles, Pacific West Capital Group reviews cite the firm's friendliness and personal service. Clients such as M.W. from Long Beach and A.H. from Fresno applaud CEO Andrew Calhoun's attention to their financial well being. Since the beginning, this type of customer satisfaction has been LISA's goal.
Related Posts:
- Unavailable, please contact us for more information.


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Feb. 25, 11:43 a.m., North Avalon Drive: Police received a report that a resident's car was keyed overnight. The resident also passed on possible suspect information to police.
Bike theft
Feb. 17, McKenzie Park: A Menlo Park resident who stopped to use the restroom at McKenzie Park during an afternoon ride reported that his bicycle, valued at an estimated $8,000, had been stolen while he was in the bathroom.
Police spokesman Sgt. John Korges said the victim had not locked up his yellow and gold Colnago road bike.
One dead in Mtn. View car wreck
Mountain View police discovered a charred body in the wreckage of a burned vehicle early Feb. 24 on eastbound Central Expressway just east of Bernardo Avenue.
According to police, officers arrived at approximately 1 a.m. to find the car engulfed in flames. Police found one deceased person inside the car after the fire was extinguished.
Preliminary investigation leads officers to believe the 2007 BMW left the roadway and hit a tree before catching fire.
Thus far, no witnesses have been located. The identity and gender of the deceased driver are unknown.
Investigators ask witnesses to contact the Mountain View police at 903-6344.


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- You can find this game in :
This Game in Truck Games Categories. This Game in Truck Games Categories.
- How to play :
Drag and rotate objects into place to build a sturdy bridge for the train to cross! Get the train to the tunnel to complete a level.You must use arrow keys or A-S-D-W keys for to rotate objects.and click to signal for to start the train at the left top corner.
Good Luck
- Tags :


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KVIE, UC Davis NewsWatch Highlight King Hall Outreach Program
UC Davis NewsWatch, a broadcast segment that covers the latest news at UC Davis and airs on KVIE PBS Channel 6, highlights the King Hall Outreach Program (KHOP) in its latest installment. The segment also appeared on the UC Davis home page.
KHOP is a unique program that helps high-potential college students who are either economically disadvantaged or part of the first generation of their families to attend college to prepare for the law school admissions process. The NewsWatch segment includes interviews with Dean Kevin R. Johnson, Professor Emeritus Cruz Reynoso, and KHOP students Gorev Ahuja and Sydnie Reyes.
The UC Davis NewsWatch segment featuring KHOP was broadcast on KVIE on August 20, 23, 25, and 26, with possible future airdates to be announced.
UC Davis NewsWatch
KVIE video on YouTube


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I'm on Sverve!
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Tag Archives: what do I want to be when I grow up
A new direction?
So, I started a new job recently. For a variety of reasons, I didn't want to stay at my previous job, and decided to look for something new. I found it, or it found me, whatever, and I thought perhaps … Continue reading
Posted in Faith, Parenting, Uncategorized, Work Tagged balance, family, life, what do I want to be when I grow up, work 2 Comments


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Catching Up With The Band Perry
GRAMMY-nominated country trio the Band Perry recently visited The Recording Academy's headquarters in Santa Monica, Calif., to participate in an exclusive GRAMMY.com interview. The band discussed how they discovered they had been nominated for the 2011 Best New Artist GRAMMY at "The GRAMMY Nominations Concert Live!!" and performing on the special, recording their 2013 sophomore album, Pioneer, and plans for their forthcoming third studio album.
"We were kind of in the belly of the arena [at Nokia Theatre L.A. Live], [in the dressing room] doing hair and makeup," said Kimberly Perry regarding the band's first GRAMMY nomination. "And we were watching the TV feed and our name was called and we had a mini shout fest. I'm surprised they didn't hear us on TV."
Hailing from Mobile, Ala., the Band Perry comprises siblings Kimberly Perry (vocals/guitar/piano), Reid Perry (bass) and Neil Perry (drums/mandolin/accordion). After singing together and performing separately in various bands throughout their childhood, the Perry's formed the trio in summer 2005. In 2008 the Band Perry met Garth Brooks' manager Bob Doyle, with whom they worked on recordings that caught the attention of label executives Scott Borchetta and Jimmy Harnen at Republic Nashville.
In 2010 the band released their self-titled debut album, which peaked at No. 4 on the Billboard 200 and featured the Top 20 hit "If I Die Young," which earned Kimberly Perry a GRAMMY nomination for Best Country Song. The Band Perry released Pioneer in 2013, which peaked at No. 1 on Billboard's Top Country Albums chart and No. 2 on the Billboard 200 on the strength of the hits "Chainsaw," "Don't Let Me Be Lonely" and "Done."
With plans underway to record their third studio album, the Band Perry are currently on tour throughout the United States, with dates scheduled through October.


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Directors who dodge company debts by restarting a failed business via a new company – known as "phoenix" activity – are set to face tougher laws. Draft laws released yesterday make directors personally liable for debts where the new company has a similar name. A second bill will give the corporate regulator additional powers relating to phoenix companies. The laws follow through on a federal government election promise. Submissions are due on January 24 and February 29.
Katie Walsh
About 15,000 tax agents risk operating illegally unless they register with the Tax Practitioners Board in the next month. Business activity statement agents, who help businesses file GST returns, have until January 29 to renew their registration with the Tax Practitioners Board and continue to operate legally.
Katie Walsh
Receivers have been appointed to Birchwood Consolidated, developer of the 26-level Equus development in Perth. The project was completed in July and includes 138 apartments, 48 commercial suites and 30 retail spaces in the central business district. Craig Shepard from KordaMentha said it would start a new sales campaign. He said Equus had been hurt by higher costs and subdued demand but it was high-quality property. Ben Wilmot
The federal government has received a report on overhauling the school funding system, but will not release it until the new year. Funding review chairman
A stevedore killed on a Sydney wharf last year had, along with other senior colleagues, followed unsafe work practices, a report has found. The 49-year-old Patrick employee was killed on the night of March 28, 2010, when two shipping containers crushed him at Port Botany. A final report by the Australian Transport Safety Bureau said the victim had been seen to follow some unsafe work practices, such as working under a container. AAP
An audit office report warned yesterday that a program to reform the Defence Department's information technology systems was behind schedule. The report said that a failure to address the program's shortcomings could also frustrate efforts by Defence to achieve the federal government's overall $20 billion 10-year savings program because IT systems' reform underpinned the entire effort. John Kerin
The Australian Competition and Consumer Commission has started public consultation on NBN Co's special access undertaking, which was lodged on December 5. The undertaking outlines the pricing plans of NBN Co, which is building the government's national broadband network, until 2040. The regulator will release another consultation paper in February.
John McDuling
Philip Morris yesterday became the third tobacco giant to file a legal challenge against new Australian laws that will force tobacco products to be sold in dull, plain packaging from late next year.
The Australian Financial Review


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What: Third annual 707 Cannabis College Expo
When: Noon to 7 p.m. Saturday
Where: Mateel Community Center, Redway
Admission: $10
Southern Humboldt's Third annual "State of the Herb" medical-marijuana forum hosted by the 707 Cannabis College will happen high on the hill at the Mateel in Redway on Saturday. It's an event where folks can "come meet faculty, listen to expert speakers, watch demos on hemp spinning, shop from local vendors or just come and enjoy the all-day Q&A with Kevin Jodrey (Cultivation Director for the Humboldt Patient Resource Center)," according to a news release. There will also be performances by comedian Sherry Glaser and the Bada Bling! Burlesque.
What: Third annual Jefferson State Old Time Revue
When: 8 p.m. Saturday
Where: Arcata Playhouse, 1251 Ninth St.
Admission: $10 (advance), $12 (door), all ages
Missing Link Records will present the Third annual Jefferson State Old Time Revue on Saturday at 8 p.m. at the Arcata Playhouse. Performers this year include Los Angeles country-blues singer-songwriter Frank Fairfield, Olympia alt-folk quartet The Blackberry Bushes Stringband, Arcata bluegrass sextet The No Good Redwood Ramblers, Oregon dance-folk quintet The Sour Mash Hug Band, Oregon string quartet The Wainwright Brothers and New York alt-folk duo Country Mouse, City Mouse. Advance tickets are available for $10 at Missing Link Records in Arcata. Beer and wine will be available for those 21 and over with an ID at the all-ages show.
What: Mojo Child (Doors tribute)
When: 9 p.m. Saturday
Where: Blue Lake Casino, 777 Casino Way
Admission: Free, 21+
Doors fans can "Break On Through (To The Other Side)" of Humboldt on Saturday in the Wave Lounge. Mojo Child will cover the late legend Jim Morrison and his band all night -- it'll be a Roadhouse Blue(s) Lake Casino.
What: Go 'Merica
When: 8 p.m. Saturday
Where: Six Rivers Brewery, 1300 Central Ave., McKinleyville
Admission: $7, 21+
This Saturday night, Six Rivers Brewery in McKinleyville will host Go 'Merica -- a fun party with DJs, dancing, burlesque, live classic rock and country, games and stand-up comedy. All active and retired armed forces personnel can get in for half price with a military ID at the party put on by the Va Va Voom Burlesque Vixens, DJ ItchieFingaz and the Brewery.


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A playful circus theme is at the core of the Cirque family of bentwood stools designed by Martino Gamper for Wiener GTV Design. The bent element, which is the brand's signature trait, is surprisingly and unexpectedly inserted in the base of the stool, forming the outline of the seat in the form of a chain comprising two or three wooden rings that loop around the full length of the stool legs. This striking styling effect blends in seamlessly with the functionality of the seat, which is available in three heights and with or without backrest.
Made in Austria by GTV.
Low backless- Height 18 1/2" Diameter 17 3/4"
Tall backless - H 30 3/4" Diameter 17 3/4"
Tall with back - H 38 1/4" Diameter 17 3/4" Seat Height 30 3/4"
Materials: Bentwood stool lacquered finish. White and black lacquered finish.
No Sales Tax
Free shipping on U.S. orders
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We'll keep you posted about new designs, special offers, stories, and more. We promise it will be worth your while!


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Salt Lake City Mayor Rocky Anderson is in Australia this week to speak about climate change. He is a featured speaker at the Business and Sustainability Summit 2005 in Melbourne. An agenda says the mayor will speak about "driving change - the benefits of leading on sustainability and the battle against climate change." He will give the presentation he made in Buenos Aires, Argentina, last December. There, he spoke about Salt Lake City's work on reducing greenhouse-gas emissions at a U.N. conference on the Kyoto Protocol. One of the organizers of the Australian conference heard Anderson speak in Argentina and invited him to Melbourne. This week's conference addresses the impact of climate change on Australia's development. Environment Business Australia, which represents the environment and sustainability industry, organized the summit. Duncan Moench, Anderson's spokesman, said Environment Business Australia is paying for Anderson's flight. The mayor is taking three days off to travel in Australia and will pay for that himself. - Heather May
Rocky at Australia climate panel
Published October 25, 2005 1:06 am
This is an archived article that was published on sltrib.com in 2005, and information in the article may be outdated. It is provided only for personal research purposes and may not be reprinted.
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Apple Computer Inc. defended itself in a London court Thursday, saying it did not bite into the music company's territory covered by a 1991 agreement.
The computer-maker launched its defense in the second day of a trial involving two companies, which share the word "Apple" and similar logos. Apple Computer said its agreement does not prohibit data transfers.
The music company, formed by The Beatles, is suing the computer company for the third time in trademark-related suits. It withdrew the first two in exchange for cash settlements estimated at over $26 million. As part of the settlement, the companies drew a line in the sand for their prospective businesses and markets.
Now the music company, Apple Corps, claims the computer company crossed the line by distributing and selling songs through iTunes, which uses the apple logo. iPods are sold in Apple Stores, which display a monochromatic, bitten apple logo. Apple Corps, with its shiny green Granny logo, is trying to stop the sales and recoup damages.
The trial is expected to continue next week.


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One Last Question, and I Promise I'll Quit...
View Single Post
Senior Member
Join Date: Nov 2009
Location: White City (tad north of Medford) Oar-E-Gone
Posts: 10,475
Re: One Last Question, and I Promise I'll Quit...
Originally Posted by
I've been asked this question countless times over the last 35 years, and in light of the newer technologies out there, today's inquiry made me wonder if I was seriously out of step with the world. A friend wondered why I still have so many books and storage boxes of magazines scattered around my office when there are electronic readers like the "Kindle" or "Nook" available. My answer has never varied and remains the same... I prefer a collection of books or magazines that I can lay my hands on.
Your thoughts? Magazines and old-fashioned books, or the electronic readers?
Jerry, aka hairwing530
Bringing this initial postage back to the top. Excellent question, but I think it may be a question of 'age.' Time in Grade?
The feel of 'paper' in my hand is a good thing. You can post 'anything' on the Internet, but to get it in print? Another whole game.
Sit, read, come back to same time later, contimplate, agree/agree to disagree. Wonderful stuff. You're mind isn't fully engaged? You're 'dead,' you just haven't stopped breathing yet.
Fun post up in another thread: 'You've only got 6,000 posts here, but 16,000 posts' over there.' Had to laugh, but my work day (PST) starts AT 0100 TO 0200 my time. Two lap tops working to follow 'the markets.'
One set for 'fun time,' the other for 'really serious shxt.'
#2 is the mental coffee break?
I suspect so.
When wealth is lost, nothing is lost; when health is lost, something is lost; when character is lost, all is lost. - Billy Graham"
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Too poor to own an aircraft? Don't worry, a new app will let you hire a private jet
JetMe is a new app for iOS (Android coming soon) that wants to be like Uber or Airbnb, except instead of letting you book a ride or a place to stay, it lets you book a private jet.
I know what you're thinking -- why book a private jet when you can just use your own aircraft -- but believe it or not, there are some people who don't have their own planes (because they prefer to travel by yacht, for example). For those people, JetMe might be the perfect solution.
In all seriousness, there are benefits to booking a private jet, including avoiding other people, and the hassle of getting to the gate, and the cost of private jet flights are often on par with business class airline tickets.
The free app lets travelers search for, book, and pay for private jet flights (the full price is shown in the app) and there are no membership fees, costs for facilities, managers, brokerage and handling companies, and non-automated order processing costs to worry about.
Private jets often immediately return to their point of origin in order to avoid expensive airport parking fees, and JetMe aims to take advantage of these low load or "empty leg" flights, something it describes as a "win-win for travelers and jet owners".
At the moment, JetMe only allows you to book flights from the San Francisco Bay Area to Greater Los Angeles Area, but more cities will be added shortly.
"You don't have to be a rock star to fly like one," says Yuriy Romanyukha, CEO, JetMe. "When the idea for JetMe struck, the other founders and I sealed ourselves in our apartment and worked tirelessly, the whole time envisioning how we could deliver affordable luxury air travel experiences better than any service out there so far. Twelve hours later, the sun was coming up, and we had our prototype. Now we're open for business, with plans to expand across the United States and beyond, including back to our native Ukraine, JetMe's birthplace".
If you can't afford to hire a private jet by yourself, there are options for sharing the flight with other passengers and saving up to 50 percent of the cost.
iPhone owners can grab the app now. Android and web versions will be available shortly.


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Our direct-measurement model has been adopted by over 80,000 publishers for a variety of reasons – because of its accuracy, near-real time reporting capability, and flexibility – and today we directly-measure more than 10 million unique web properties (sites, blogs, videos, widgets, etc.).
We can generate audience characterizations for virtually any sized audience and while many innovative modeling techniques allow us to accomplish this, nothing is more important to us than the industry's participation and the growing base of directly-measured media consumption data.
This week, we reached an important milestone – over 1 trillion measured events since we launched just two years ago.
1 trillion data records. It's a lot of information and requires a very sophisticated global data collection and processing infrastructure. Uptime is key. We're committed to this platform and to delivering real-time audience based content and ad decisioning solutions to the market.
We want to thank the whole marketplace for participation. Every day more of you join and it's this collective participation that allows us to provide the services that we do. Thank you, and looking forward to the next trillion!


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|YDS: 5.5 French: 4b Ewbanks: 13 UIAA: IV+ ZA: 11 British: MS 4a [details]
|Page Views:
|Submitted By:
|Doug Hemken on Jul 5, 2007
|Comments on Can Can
|Add Comment
|Show which comments —
By Paul Huebner
From: Portage, WI
Aug 11, 2007
rating: 5.5 4b 13 IV+ 11 MS 4a
|Another great lead that's easy to protect. After setting pro for up and downward pull before starting off, the 1st piece I use is a #1 tricam (pinky) in a small pocket just before heading off left for the alcove below the slightly, overhanging fist-size crack. Moving up and into the crack (a couple of moves) is the crux of the climb. Down climb is off of the back side of the summit into the large gap between the Frigate and the main bluff (see unnamed walk off). From there a short traverse to north takes you to easy ledges (steps) to get back down to your pack.
By Chicagio
Jul 17, 2011
rating: 5.6 4c 14 V 12 S 4b
|If you approach this from the northwest side (previous commenter refers to this as the walk-off, for leaders), it is easy enough climbing that you can set up top rope. A threaded sling and a #6 & #10 hex will do. The little roof move is a blast! Don't disturb any frogs when threading!
By Scott Greenhalgh
From: New Hartford, Iowa
Jun 1, 2014
|There is a fun variation going through the right crack out of the roof in the 5.7 to 5.8 range.


text:
|February 7, 2012, 05:50 PM
Senior Member
Join Date: November 2, 2005
Posts: 1,164
Refit Barrel - STUMPED!
What are the main fixes to the problem of a firearm which fails to lock back on the last round fired from the magazine?
What benefit would refitting the barrel have in correcting this program?
|February 7, 2012, 05:54 PM
Senior Member
Join Date: February 28, 2008
Location: Michigan
Posts: 2,303
It would be a great help if you told every one the Rifle involved.
Make, model, type of acton, caliber, size of magazine.
And why do you want to refit the barrel?
|February 7, 2012, 08:00 PM
Senior Member
Join Date: September 19, 2008
Posts: 4,678
[What are the main fixes to the problem of a firearm which fails to lock back on the last round fired from the magazine?]
Causes, not fixes:
A worn locking edge on a vertical magazine actuated slide lock.
A worn/broken stop latch.
[What benefit would refitting the barrel have in correcting this program? ] - None.
|February 7, 2012, 09:16 PM
Join Date: March 17, 1999
Posts: 24,136
The OP just said "firearm", which could include any gun with a magazine - pistol, rifle, or shotgun - including many that were never made to lock open on empty.
I think we need to wait for pictures or at least a better description of the "firearm."
Jim K
|February 7, 2012, 11:57 PM
Senior Member
Join Date: November 2, 2005
Posts: 1,164
The firearm in question is a Springfield Armory EMP 9mm 1911 pistol. It fails to lock back the slide on the last round fired. It makes no difference who shoots it. It malfunctions with both 115 Gr. and 124 Gr. FMJ ammo. It fails with all three magazines which came with the gun. The first trip back to SA the barrel was refitted and the slide stop was tuned. They machined off some of the back of the slide stop. The gun continued to malfunction. It went back to S.A. and is in the process of being returned. They again, refitted the barrel and did extensive test firing.
So beside the original question of what good would refitting the barrel do to correct this program? If it continues when returned are their other possible fixes?
|February 8, 2012, 04:19 AM
Senior Member
Join Date: August 30, 2009
Location: Northern AZ
Posts: 7,172
I can't think of any other reason besides magazine followers that aren't hitting the hold open, unless the barrel link is causing a slight downward twist of the slide stop. (I am assuming that there is a barrel link and not a cam lock arrangement.) The only other thing, although far fetched, is that the recoil spring is not allowing the slide to travel back far enough for the slide stop to engage.
|February 8, 2012, 12:32 PM
Senior Member
Join Date: December 29, 2004
Posts: 3,342
Check if the magazine follower is catching the slide stop since that is how the 'last shot lock' operates.
See if the follower is catching when you hand cycle the slide (good test with dummy rounds).
Look down into the rejection port as you cycle the slide by hand.
If the mags have a lot of use on them they may need new springs.
You can also try different magazines.
If someone messed with the slide catch it may need to be replaced.
|February 9, 2012, 06:27 AM
Senior Member
Join Date: November 13, 2006
Posts: 5,181
Maybe:Hotter ammo,firmer hold,lube,any rubber buffers added?.45 slide stop in a 9mm.
Q:Is it real easy to engage the slide stop manually?Smetimes a little detent is drilled in the slide stop,and it can be too much detent real easy.
Empty mag inserted,pull the slide full back,does it engage?
Weak mag spring?Try a new Wolfe +5%
|February 11, 2012, 02:50 PM
Senior Member
Join Date: November 2, 2005
Posts: 1,164
UPDATE: After a second trip to Springfield Armory for repair, (on their dime) the second "barrel refit" appears to have corrected the problem. Went to the range and shot 113 rounds of mixed 147 gr. fp, 115 gr. fmj and 115 gr. RNL. All worked perfectly with the exception that twice the slide failed to lock back on the 147 gr. FP. The OAL of the 147 Gr. cartridges are shorter than the other ammo I shot whether that had anything to do with it I don't know.
|February 13, 2012, 05:52 AM
Senior Member
Join Date: August 30, 2009
Location: Northern AZ
Posts: 7,172
A so-called "barrel refit" would make sense regarding my thoughts that the swinging link was exerting too much pressure on the slide stop.
The resulting friction/torsion when the barrel is swinging down out of engagement with the locking lugs in the slide would cause the slide stop to rotate down.
|February 22, 2012, 08:38 AM
Junior Member
Join Date: February 19, 2012
Posts: 3
Refit Barrel-stumped
Check the lever spring, My Walther broke this very small spring and slide would not stay back on last round. I man. new spring, problem solved...
|February 22, 2012, 08:53 AM
Senior Member
Join Date: January 27, 2010
Location: Norfolk, VA
Posts: 2,905
I had the same problem with my brand-new EMP .40 S&W. Eight mags, and none would lock the slide back reliably. I contacted SA, and they sent me a new slide stop pin with a different profile (I think they called it an "A-cut profile") that fixed the problem immediately and completely.
Glad they got yours taken care of as well!
|February 22, 2012, 08:53 AM
Senior Member
Join Date: April 10, 2001
Location: A Place Worse than California
Posts: 782
That little spring can get away from you too during a strip cleaning. I always have a few extra around.
"It was people who upheld their duties to their office, the constitution, and the public by opposing Hitler who were called traitors"
"...a historian asked what had happened to the German people for them to accept a criminal government. Unfortunately, nothing needed to happen. In nations across the world people accept government crime."
"In democracies as well as dictatorships, subordinates illegally obey their rulers. Subordinates who remain true to their oaths of office by opposing their rulers are rare."
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