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The Historical Jesus Goes to Church
By (author) Arthur J. Dewey Roy W. Hoover Stephen J. Patterson
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Historical Jesus Goes to Church by Arthur J. Dewey
Book DescriptionWhat difference does scholarship on the historical Jesus make for the way we think about the meaning of Christian faith in the twenty-first century? In The Historical Jesus Goes To Church, biblical scholars--Fellows of the Jesus Seminar--speak directly to the ways in which new knowledge of the Jesus of history requires and enables us to think differently about the significance of Jesus and about the reliability and authority of the Bible. They also imagine what these new understandings imply for public worship, preaching, prayer and practice, and life in community. These articles evoke the spirit of Paul, Christianity's first theologian, who like us found himself standing at the intersection of two eras and knew that he had to let go of his past if he hoped to have a future.
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Book DetailsISBN: 9780944344613
(229mm x 159mm x 8mm)
Pages: 144
Imprint: Polebridge Press
Publisher: Polebridge Press
Publish Date: 15-Dec-2003
Country of Publication: United States
Books By Author Arthur J. Dewey
Wisdom Notes, Paperback (June 2016)
Theology at its best lends rhythm and rhyme to the raw energy of life. It improvises on this world without trying to escape to a heaven somewhere else. In this curated collection of radio commentaries and editorials, Art Dewey invites readers to remain open to new meaning as it arises from our encounters with neighbours, strangers, and friends.
Authentic Letters of Paul, Paperback (October 2010)
The Authentic Letters of Paul (1) distinguishes Pauls letters from others attributed to him in the canon, (2) disentangles component pieces of correspondence from the composite letters, (3) places the authentic letters in their chronological order and historical context, and (4) restores Pauls voice in a fresh translation from the original Greek.
Resurrection of Jesus, Paperback (January 2009)» View all books by Arthur J. Dewey
The event of Jesus' resurrection is like the event of creation: There were no eye-witnesses. So how does one make sense of the story of the resurrection - or rather stories, for not one but many reports survive from early Christianity? This work suggests that we must begin by erasing all Christian art about the resurrection from our memory.
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Russia Bonds Gain After Central Bank Ends 11-Month Easing Hiatusby and
Bank of Russia opens door for more cuts on easing inflation
Yields go back to pre-sanction levels on notes due 2027
Russia's bonds gained after policy makers cut interest rates for the first time in almost a year, sending yields toward a level last seen in 2014 before the double whammy of an oil crash and international sanctions.
Speculation the 50 basis-point reduction in borrowing costs will ease a two-year recession was stoked by the Bank of Russia's forecast the economy will swing to growth in 2017. The ruble weakened 0.4 percent to 64.5700 per dollar at 3:30 p.m. in Moscow.
"Markets see starting of the monetary easing cycle as positive for economic growth," said Vladimir Miklashevsky, senior strategist at Danske Bank A/S in Helsinki. He expects the ruble to trade in a range between 64 and 65 per dollar if oil remains at current levels.
For more on the decision, click here.
The yield on benchmark notes due 2027 dropped seven basis points to 8.59 percent, the lowest since July 11, 2014, just before the U.S. imposed sanctions on some of Russia's biggest companies over Moscow's role in Ukraine's conflict.
Further rate cuts may be in store if estimates of a slowdown in inflation are fulfilled, the Bank of Russia said in a statement. A stronger ruble and stability in the price of oil since the start of the year, the nation's biggest export earner, is containing price growth to the slowest pace in two years.
Policy makers have kept the key rate unchanged at 11 percent since July 31, even as the economy foundered in recession, in favor of sticking to inflation targets under pressure from a falling ruble.
A narrow majority of 43 economists in a Bloomberg survey correctly forecast the decision.

subset: commoncrawl


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Montrose can still challenge for the third division title according to Jim Weir, the manager, after he watched his side beat promotion rivals Stranraer 2-0 away from home to hold on to second place in the league.
Two second-half goals from John Baird, including one from the penalty spot gave Montrose the victory that extends their lead over Stranraer in the league to five points.
"It's going to be very difficult to catch East Fife who are still 13 points ahead of us. Our target at the start of the season was to make the play-offs but it would be


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text: #CWM2012 Crime Writing Tips – No. 5 #CWM2012 crime writing tip number 5 concerns suspects: **The Array of Suspects** Don't have so many that it causes confusion. Do have a decent few to keep the reader guessing. A small number is fine if plenty of twists and questionable motivation are also present. Advertisements Share this:TwitterFacebookGoogleRedditEmailPrintLike this:Like Loading... Related


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5-aza-2′-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [3H]-5-aza-CdR incorporated in DNA. However, incorporated [3H]-5-aza-CdR gradually decreased when cells were incubated in [3H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [3H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as γ-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.
Citation: Chai G, Li L, Zhou W, Wu L, Zhao Y, Wang D, et al. (2008) HDAC Inhibitors Act with 5-aza-2′-Deoxycytidine to Inhibit Cell Proliferation by Suppressing Removal of Incorporated Abases in Lung Cancer Cells. PLoS ONE 3(6): e2445. doi:10.1371/journal.pone.0002445
Editor: Dong-Yan Jin, University of Hong Kong, China
Received: March 15, 2008; Accepted: May 12, 2008; Published: June 18, 2008
Copyright: © 2008 Chai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study is supported by National Natural Science Foundation of China (No.30425017, 30670417 and 30621002), and grants (2005CB522403, 2006AA02Z101, 2006CB910300 and B07001) from the Ministry of Science and Technology of China.
Competing interests: The authors have declared that no competing interests exist.
It is well known that DNA methylation is associated with histone acetylation status in regulation of gene expression [1]–[4] or cell proliferation and aging [5]. This linkage between histone status and DNA methylation was well confirmed by Cameron et al who found that several genes silenced by methylation were reactivated when treated with demethylating agent 5-aza-CdR and histone deacetylase inhibitor (HDACI) trichostatin A (TSA) together, but were not reactivated in the presence of 5-aza-CdR or TSA alone [6]. Later, several laboratories including our own extended these findings to set up a therapeutic strategy for cancer treatment, in which 5-aza-CdR acts in conjunction with depsipeptide/TSA to induce significant apoptotic cell death [7]–[10]. Since DNA methylation in the promoter region is associated with HDAC1 by a methyl-binding protein MeCP2 [11], it is believable that certain genes, if hypermethylated in their promoter region, are more tightly packed by histones and thus transcription factors access their DNA binding sites only with greater difficulty. Consequently, cell death related genes, which are silenced due to hypermethylation, could be reactivated by treatment with 5-aza-CdR; this reactivation should be strengthened by HDACI, and at the same time, cell death should be more readily observable as well.
However, 5-aza-CdR also plays an anti-neoplastic role which is methylation-independent [12]–[14]. 5-aza-CdR may act directly on mitochondria and induce apoptosis in mammalian cells [14]. A direct methylation independent evidence for the 5-aza-CdR induced cell death is that 5-aza-CdR significantly enhances expression of Apaf-1 or p19INK4d to induce cell death, however the promoter regions of these genes are totally unmethylated [12], [13]. 5-aza-CdR has also been reported to induce a synergistic effect by increasing the cisplatin bound to DNA, via a change in the topology of DNA brought about by 5-aza-CdR without its functioning as a demethylating agent [15]. These data suggest that 5-aza-CdR plays diverse roles in cells which include both demethylating functions and methylation-independent functions.
The cytotoxicity of 5-aza-CdR results from its capacity to damage DNA as 5-aza-CdR is a nucleoside analog (NA) and can be incorporated into the DNA backbone [16], [17], which in turn may induce formation of a covalent adduct between the 5-aza-CdR molecule and methyltranferases [16]. It has been demonstrated that NAs such as 5-aza-CdR or cytarabine (Ara-C), are phosphorylated into their triphosphate form and are then incorporated into DNA during replication [18]. Subsequently, the incorporated NA serve as an abase that may induce DNA damage, mutations and stalling of the DNA replication fork [19], [20]. These changes in the DNA backbone are harmful, and DNA damage sensors such as DNA-PK, p53, ATM and ATR recognize these damaged sites and repair the abnormal DNA [21], [22]. Previously, both our group and other researchers directly confirmed that 5-aza-CdR induces DNA damage and elicits p53-dependent biological reactions [23]–[25]. However, if the abnormal DNA changes induced by the incorporation of NAs are overwhelmed, or the DNA repair systems are severely inhibited, the cells will undergo apoptosis [26].
HDAC inhibitors are novel and effective anticancer agents [27], [28], which are involved in regulating many genes including activation of p53 [29], or downregulating anti-apoptotic genes [28], [30] to exert an anti-neoplastic effect. Therefore, investigation of whether or not HDACI also affects DNA repair enzymes to enhance NA-induced DNA damage is called for.
In this study, we confirm that 5-aza-CdR acts in concert with HDACI to induce a significant inhibition of cell proliferation in a methylation independent manner. 5-aza-CdR was incorporated into DNA in a dose and time dependent manner that was clearly associated with induction of DNA single-strand breaks (SSB) by 5-aza-CdR. HDAC inhibitors significantly suppressed removal of the incorporated 5-aza-CdR from DNA and thus produced a synergistic effect resulting in inhibition of cell proliferation
Materials and Methods
Cell culture and chemical treatment
Human lung cancer cell lines A549 and H719 were used in this study. Both 5-aza-CdR (dissolved in 50% acetic acid) and Ara-C (dissolved in DMSO) (both purchased from Sigma, St Louis, MO) were added fresh into the medium every 24 hrs. TSA (Sigma) was dissolved in ethanol. Depsipeptide (kindly provided by the NIH) was dissolved in DMSO.
Determination of cell proliferation by MTT assay
Equal numbers of cells (approximately 5000/well) were seeded into a 96-well plate 24 hrs prior to experimentation. Cells were treated with 5-aza-CdR, Ara-C, γ-irradiation and HDACI alone or in combination. After treatment, MTT dye solution (Sigma) was added into the 96-well plate. The corrected absorbance of each sample was calculated by comparison with the untreated control.
To determine if knockdown of DNA methyltransferases (DNMTs) is effective in DNA demethylating and reactivating silenced genes, RT-PCR was performed to detect changes in expression of p21 that is silenced due to a hypermethylation in the p21 promoter [31]. The PCR primers were as follows: p21, left primer- GGA AGA CCA TGT GGA CCT GT and right primer- GGA TTA GGG CTT CCT CTT GG; β-actin, left primer- TGG AGA AGA GCT ACG AGC TGC CTG and right primer- GTG CCG CCA GAC AGC ACT GTG TTG.
Western Blot
To identify changes in protein expression in cells treated with knockdown of DNMTs or 5-aza-CdR, cells were harvested with a scraper and then washed once with cold phosphate-buffered saline. The cells were then lysed in lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.15% Igepal CA-630, and 1.5 mM phenylmethylsulfonyl fluoride). Equal amounts of proteins (100–150 µg) were size fractionated on 7.5–12.5% SDS-PAGE. In addition, to determine the amount of the chromatin-bound RPA, cells were lysed in solution A (50 mM Tris-HCl, pH 7.8, 420 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.34 M sucrose, 10% glycerol, 1 mM Na3VO4 and protease inhibitor cocktail). To obtain nuclear extracts, cells were lysed in buffer B (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1% Triton X-100, protease inhibitor cocktail). Isolated nuclei were washed once with buffer B and further lysed in buffer A as above. To obtain chromatin-bound proteins, isolated nuclei were lysed in buffer C (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT) and further lysed in buffer A as above.
The antibodies used were anti-PARP (Santa Cruz, F-2), anti-RPA (Santa Cruz, MA34 and MA70-2), anti-γ-H2AX (Upstate, 05-636), anti-DNMT1(Santa Cruz, H-300), anti-DNMT3A (Santa Cruz, P-16), anti-DNMT3B (a gift from Dr. Xu, G, Shanghai Institute of Biological Science) and β-actin antibodies were purchased from Santa Cruz.
DNA bisulfite treatment and bisulfite sequencing
To evaluate changes in DNA methylation status after treatment with knockdown of DNMTs, DNA was treated with sodium bisulfite and purified for PCR as described previously [31]. The primers for sequencing the p21 promoter were as follows: left primer, 5′-GGG AGG AGG GAA GTG TTT TT -3′ and right primer, 5′-ACA ACT ACT CAC ACC TCA ACT-3′. The PCR products were gel extracted (Qiagen, Valencia, CA) and ligated into a pGEM-T easy vector, by using the TA cloning system (Promega, Madison, WI). Transformed bacteria DH5α were cultured overnight, and the plasmid DNA was isolated using a kit (Qiagen). At least 10 separate clones were chosen for sequence analysis.
Comet assay
The comet assay was performed as described previously [24]. In brief, frosted microscopic slides were covered with 0.5% normal melting agarose at 60°C. About 105 5-aza-CdR treated or untreated cells in PBS were mixed with an equal amount of 1% low melting agarose to form a cell suspension. After electrophoresis, slides were examined at 600× magnification and pictures were taken with a fluorescence microscope (TCS, Leica, Manheim, Germany).
Analysis of exclusion of incorporated [3H]-5-aza-CdR from DNA
The radiolabeled nucleoside analog incorporation assay was performed as described previously with modifications [32]. [3H]-5-aza-CdR was purchased from Moravek Biochemicals (MT1676, 1 mCi/ml, Brea, CA). H719 and A549 cells were treated with [3H]-5-aza-CdR at 1 µM for 48 hrs with or without depsipeptide at 0.1 µM for the final 6 hrs (from 42 to 48 hrs) and the cells were then washed with cold PBS. The cells were then replaced in [3H]-5-aza-CdR free medium and incubated for 6, 12 or 24 hrs. Cold trichloroacetic acid (TCA, 50%) was then added to the cell solution and the TCA-insoluble DNA was placed on a glass microfiber filter (Whatman, Maidstone, England). This glass filter with TCA-insoluble DNA was placed in a vial to count radioactivity with a Beckman LS 5000 Liquid Scintillation Counter.
Establishment of cell line with a stable DNMT1-deficient clone
The pCMVneo -DNMT1 antisense plasmid was kindly provided by Dr. SB Baylin [33]. The vector was transfected into H719 cells using the Qiagen Effectene Transfection kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Twenty-four hrs after the first transfection, a second transfection was performed to strengthen the efficiency of the RNAi, and then 800 µg/ml of G418 was added to select stable cells with DNMT1 antisense.
RNA interfering with DNMT3A, DNMT3B
The RNAi targeted oligonucleotide sequences are as follows: CAT CCA CTG TGA ATG ATA ATT (DNMT3A) and GAT CAA GCT CGC GAC TCT C (DNMT3B) (purchased from Shanghai GeneChem Company). The oligonucleotides for DNMT3A and DNMT3B siRNA were transfected into cells by using lipofectamine 2000 for 48 hrs.
5-aza-2′-deoxycytidine cooperates with HDACI to induce inhibition of cell proliferation by a methylation independent mechanism
It has been reported that 5-aza-CdR acts together with HDAC inhibitors to synergistically induce cell death or inhibition of cell proliferation [7]–[10]. To investigate whether the role of 5-aza-CdR involved in this synergistic inhibition of cell proliferation when combined with HDACI is epigenetic, two double knock down (DKD) clones (DKD of DNMT1/DNMT3A and DKD of DNMT1/DNMT3B) were established. To avoid inducing a difference in efficiency of transfection when cells were transfected with two vectors or two RNAi oligonucleotides, a DNMT1 deficient stable clone was first established by transfection of a vector with antisense DNMT1 cDNA into H719 cells and selected with G418. As shown in Fig. 1A, DNMT1 protein is well knocked down in this stable H719 cell line. Thereafter, DKD of DNMT1/DNMT3A, or DNMT1/DNMT3B was carried out by transfection of DNMT3A or DNMT3B RNA oligonucleotides into the DNMT1 knockdown stable cells. Figure 1B–C shows clearly that DNMT3A and DNMT3B protein levels were significantly reduced by these RNAi treatments. To evaluate whether these DKDs of DNMTs were efficient for demethylating DNA, the methylation status of the p21Waf1/Cip1 promoter was chosen for testing with bisulfite sequencing because the p21Waf1/Cip1 promoter is highly methylated in H719 cells [31]. Fig. 1D represents a schematic diagram showing the promoter region of p21Waf1/Cip1and the fragment designated for bisulfite sequencing. As shown in Fig. 1E, the p21Waf1/Cip1 promoter in H719 cells was highly methylated (51.25% of CGs in the p21Waf1/Cip1 promoter were methylated in 10 selected clones). However, methylated CGs were decreased to 23.75%, 21.5% and 12.5% when DNMT1,DNMT1/DNMT3A and DNMT1/DNMT3B were subjected to double knock down with 10 selected clones (Fig. 1E), indicating that knockdown of DNMT1 and DNMT3B together was the most efficient for DNA demethylation. This decrease in methylation of the p21Waf1/Cip1 promoter was clearly associated with expression of p21 as measured with RT-PCR (Fig. 1F).
(A) Representative Western blot indicates that DNMT1 protein level in H719 cells was significantly decreased in the DNMT1-deficient stable clone when compared to the non-specific oligonucleotide treated control. (B) and (C) Representative Western blots also show that DNMT3A or DNMT3B protein level was significantly decreased after RNAi treatment in DKD of DNMT1/DNMT3A cells or DKD of DNMT1/DNMT3B cells, respectively. β-actin is shown as a loading control in the lower panel. (D) A schematic diagram showing the p21Waf1/Cip1 promoter, with the black region indicating the fragment which underwent bisulfite sequencing (−233 to +2 relative to transcription start site). (E) With bisulfite sequencing, 10 clones were randomly selected and the methylation status of the p21Waf1/Cip1 promoter was determined in untreated H719 cells or in the H719 cells treated with knockdown of DNMT1, DKD of DNMT1/DNMT3A or DKD of DNMT1/DNMT3B. (F) RNA was extracted from H719 cells treated with DKD of DNMT1/DNMT3B, and RT-PCR was performed to detect expression of p21 mRNA. β-actin is shown as a loading control for the RT-PCR analysis.
An unexpected finding was that we did not observe a difference in cell proliferation when H719 cells with DKDs or TKDs (knockdown of DNMT1/3A/3B) were treated with or without depsipeptide and assayed with MTT (Fig. 2A). In contrast, cell proliferation was significantly decreased in both A549 and H719 cells which were treated with 5-aza-CdR and HDACI depsipeptide/TSA, although 5-aza-CdR alone and depsipeptide/TSA alone had little effect on inhibition of cell proliferation at the doses used (Fig. 2B–C). However, 5-aza-CdR-induced inhibition of cell proliferation was cell line dependent. For example, 5-aza-CdR showed an obvious effect in inhibiting proliferation of A549 cells (Fig. 2C), but had no effect in H719 cells (Fig. 2B). These differences in the 5-aza-CdR-induced effect on cell proliferation between A549 and H719 cells resulted from the p53 status of the treated cells [24]. A549 cells have a wild-type p53, whereas H719 cell p53 is mutated [31]. In addition, although procaine has been reported to be a demethylating agent [34], synergistic inhibition of cell proliferation was not observed when H719 cells were treated with procaine (0.5 mM for 72 hrs) and depsipeptide (0.1 µM for 6 hrs at 66–72 hrs) or TSA (1 µM for 6 hrs at 66–72 hrs) (Fig. 2D). These data suggest that the role of 5-aza-CdR in its synergistic effect with HDACI in inducing inhibition of cell proliferation may not result from its demethylating function.
(A) H719 cells with deficient DNMT1, DKD of DNMT1/DNMT3A, DKD of DNMT1/DNMT3B, or TKD (triple knockdown) of DNMT1/DNMT3A/DNMT3B were treated with or without depsipeptide at 0.1 µM for 6 hrs, and cell proliferation was tested with the MTT assay. (B) and (C) 5-aza-CdR (1 µM for 72 hrs) cooperates with HDAC inhibitor depsipeptide (0.1 µM for the final 6 hrs) or TSA (1 µM for the final 6 hrs) to synergistically induce inhibition of cell proliferation in H719 (B) or A549 cells (C). (D) H719 cells were treated with procaine (0.5 mM) for 72 hrs, and depsipeptide (0.1 µM for the final 6 hrs) or TSA (1 µM for the final 6 hrs) was then added to the treated cells. All data are the results of two separate experiments.
5-aza-CdR induces cytotoxicity by incorporation into DNA as a nucleoside analog
It is well known that 5-aza-CdR is phosphorylated to its triphosphate form in cells and then incorporated into DNA during replication [18]. In order to investigate the degree of 5-aza-CdR incorporation into cells, [3H]-5-aza-CdR was added to A549 or H719 cells for 24 hrs and DNA was then extracted with TCA precipitation. Radioactivity per µg DNA was measured. As shown in Fig. 3A–B, incorporation of [3H]-5-aza-CdR into DNA was concentration dependent in both cell lines. Incorporated radioactivity per µg DNA at 0.1 µM and 1 µM of [3H]-5-aza-CdR increased ∼2- and ∼12-fold respectively in H719 cells, and ∼10- and ∼110-fold respectively in A549 cells when compared to the radioactivity of 0.01 µM [3H]-5-aza-CdR in both cell lines.
(A) H719 or (B) A549 cells were incubated with [3H]-5-aza-CdR for 24 hrs at different concentrations and then washed with cold PBS. The cells were incubated in fresh medium for another 24 hrs, and DNA was then precipitated with cold TCA. Radioactivity was measured and relative radioactivity per µg was counted. Same amount of DNA was loaded and run at 1% of agarose gel at 100 V for 5 min as loading control, which is shown at upper panel of Fig. 3A and Fig. 3B, respectively. The results presented are from two separate experiments (mean±SD) DNA damage induced by 5-aza-CdR in H719 cells was determined by the comet assay. (C) Untreated control, (D) 5-aza-CdR at 0.1 µM for 72 hrs and (E) 5-aza-CdR at 1 µM for 72 hrs.
There was a relationship between incorporated 5-aza-CdR and DNA damage as determined with the comet assay. Figure 3C–E shows that 5-aza-CdR exhibited a dose-dependent capacity for DNA damage in A549 cells, which was demonstrated by presence of a DNA tail. Larger DNA tail area and longer DNA tail length represent greater DNA damage. Table 1 shows a statistic analysis of the comet assay comparing 5-aza-CdR treated samples to an untreated control. This data parallels the incorporated amount of 5-aza-CdR, and implies that 5-aza-CdR plays a role in cytotoxicity by incorporation into DNA.
As apoptosis can also give a positive result in the comet assay [35], we harvested 5-aza-CdR treated cells and performed flow cytometry analysis to determine whether 5-aza-CdR-induced DNA damage elicits apoptotic cell death. The cells with DNA content smaller than that of G0/G1 (region of M1 in figure 4) are considered to be apoptotic cells. As shown in Fig. 4, 5-aza-CdR did not induce apoptosis in A549 cells at 0.1 µM (Fig. 4B) or 1 µM for 72 hrs (Fig. 4C). In addition, A549 cells were treated with 5-aza-CdR at 1 µM for 48 hrs or 72 hrsand protein was extracted for Western blotting using anti-PARP (cleavage of PARP is a hallmark of apoptosis). Fig. 4D shows that 5-aza-CdR did not induce apoptotic cell death at limited doses. These data indicate that 5-aza-CdR alone at limited doses is not able to induce apoptosis and thus the positive result in the comet assay results mainly from 5-aza-CdR induced DNA damage.
(A–C) To determine whether 5-aza-CdR-induced DNA damage induces inhibition of cell proliferation via apoptotic cell death, a flow cytometry analysis was performed. Untreated A549 cells (A) or A549 cells were treated with 5-aza-CdR at 0.1 µM (B) or at 1 µM for 72 hrs (C), and the cells were then harvested for staining with Propidium Iodide (PI). DNA content with smaller than G0/G1 was considered as apoptotic DNA (area of M1). (D) A549 cells were treated with 5-aza-CdR at 1 µM for 48 or 72 hrs and protein was extracted for Western blotting using anti-PARP to detect PARP cleavage in cells treated with 5-aza-CdR. An apoptotic positive control was shown at the lane of the far right, in which there are two bands indicating the PARP (116 KD) and PARP cleavaged (85 KD).
In general, DNA damaging agents induce DNA single-strand breaks (SSB) or double-strand breaks (DSB). To determine which kind of DNA damage is induced by 5-aza-CdR, we performed Western blotting using anti-RPA (replication protein A) or γ-H2AX. Most DNA damaging agents induce cellular responses through activation of ATR (ATM and Rad3-related) [36]–[40], and the activation of ATR is largely dependent on an intermediate molecule RPA [41], [42]. Chromatin was extracted from 5-aza-CdR treated H719 cells and chromatin-bound RPA content was determined. Fig. 5A shows that chromatin-bound RPA was increased in response to increased 5-aza-CdR, indicating that 5-aza-CdR induced DNA damage may be single-strand breaks (SSB). On the other hand, γ-H2AX is considered to be a hallmark of DNA double-strand breaks (DSB) [43], and Western blotting was therefore performed using ant-γ-H2AX to rule out the possibility of 5-aza-CdR induced DSB. Fig. 5B shows that 5-aza-CdR at 1 μ did not induce DSB. However, 5-aza-CdR at higher concentration (>5 µM) induced dose-dependent DSB (data not shown). These data demonstrate that 5-aza-CdR induced DNA SSB or DSB is dosage dependent. In this study, 5-aza-CdR was almost always used at lower doses (1 µM) and thus the 5-aza-CdR induced DNA damage is SSB.
(A–B) H719 cells were treated with 5-aza-CdR (0.1–1 µM) for 48 h, and cellular extracts including the chromatin fraction (A, Chr) and soluble fraction (B, Sol) were isolated for Western blotting to detect changes in RPA70 and RPA32. The band intensity of untreated sample was set as 1. The numerical value of each sample represents the percentage of band intensity relative to that of untreated sample. (C) H719 cells were also treated with 5-aza-CdR at 1 µM for 24, 48 or 72 hrs, and protein was then extracted for Western blotting using anti-γ-H2AX. Cells were treated with doxorubicin at 1 µM for 24 hrs as a positive control (far right lane).
Depsipeptide specifically inhibits removal of incorporated 5-aza-CdR and enhances nucleoside analog induced cytotoxicity
Because DNA damage induced by low dose of 5-aza-CdR is reversible, it is particularly important to understand how HDAC inhibitor enhances 5-aza-CdR-induced toxicity. In addition, as sequential treatment of 5-aza-CdR and HDAC inhibitor is more effective in inducing cell death [44], we were led to consider whether HDACI increases uptake of 5-aza-CdR into DNA or decreases removal of incorporated 5-aza-CdR from DNA, thus enhancing 5-aza-CdR-induced cytotoxicity. First, depsipeptide (0.1 µM) and [3H]-5-aza-CdR (1 µM) were added together to A549 cells for an initial 6 hrs, and the cells were then continuously cultured in medium with [3H]-5-aza-CdR alone for another 42 hrs. Radioactivity was counted at 0–24 hrs after cells were incubated in a radioactivity-free medium. By comparing samples treated with [3H]-5-aza-CdR alone to samples treated with [3H]-5-aza-CdR plus depsipeptide, no difference in radioactivity was observed, indicating that depsipeptide does not promote incorporation of [3H]-5-aza-CdR into DNA (Fig. 6A). To test whether depsipeptide affects removal of incorporated 5-aza-CdR, a decay analysis of incorporated [3H]-5-aza-CdR was carried out in A549 cells. Cells were treated with [3H]-5-aza-CdR at 1 µM for 48 hrs, with and without depsipeptide at 0.1 µM for the final 6 hrs of treatment, and the cells were then washed and replaced in fresh medium (without experimental reagents) for 6, 12 and 24 hrs. Figure 6A shows that incorporated [3H]-5-aza-CdR gradually decreased during incubation in [3H]-5-aza-CdR free medium. Radioactivity per µg DNA at 6, 12 and 24 hrs in cells treated with [3H]-5-aza-CdR alone, for example, was decreased to 78.44±7.08%, 57.5±3.88% and 36.05±1.79% respectively, compared to the radioactivity at the beginning of incubation. This reflects removal of [3H]-5-aza-CdR from DNA. However, addition of depsipeptide significantly postponed the removal of [3H]-5-aza-CdR from DNA, and radioactivity per µg DNA at 6, 12 and 24 hrs was 80.3±13.21%, 78.71±12.72% and 82.5±20.47%, respectively, compared to the radioactivity at the beginning of incubation. This depsipeptide induced decrease in removal of [3H]-5-aza-CdR was also dependent on depsipeptide concentration. For example, the radioactivity of cells treated with [3H]-5-aza-CdR and depsipeptide at 0.05 µM, 0.1 µM and 0.2 µM showed 1.8-, 2.7- and 3.25 -fold increases respectively over that of cells treated with [3H]-5-aza-CdR alone (Fig. 6B). In addition, 5-aza-CdR induced DNA damage also gradually recovered when cells were cultured in a 5-aza-CdR free medium. The length and area of the DNA tail induced by 5-aza-CdR were shorter and smaller when cells were cultured in 5-aza-CdR free medium for 12 hrs as compared to the cells without drug-free incubation (Fig. 6C–E). This process of recovery from 5-aza-CdR induced DNA damage was significantly suppressed by depsipeptide, so that the length and area of the tails in the comet assay were longer and larger when cells were treated with 5-aza-CdR and depsipeptide together (Fig. 6F–H). The depsipeptide-induced suppression of recovery from 5-aza-CdR induced DNA damage is summarized in table 2. These data suggest that HDAC inhibitor may suppress recovery from 5-aza-CdR induced DNA damage and synergistically induce inhibition of cell proliferation.
(A) H719 cells were incubated with [3H]-5-aza-CdR for 48 hrs, with presence or absence of depsipeptide at 0.1 µM for the first 6 hrs (depsipeptide+[3H]-5-aza-CdR) or last 6 hrs ([3H]-5-aza-CdR+depsipeptide) and then washed with cold PBS. The cells were replaced in fresh medium for further incubation for different intervals and the DNA was then extracted with cold TCA. Relative radioactivity of each sample at different time points was compared to the radioactivity of the cells at the beginning of the incubation. (B) H719 cells were treated with [3H]-5-aza-CdR for 48 hrs, and depsipeptide at 0.05, 0.1 and 0.2 µM was then added to the cells for the last 6 hrs. At 24 hrs after incubation, cells were harvested and radioactivity was counted. Same amount of DNA was loaded and run at 1% of agarose gel at 100 V for 5 min as loading control, which is shown at upper panel of Fig. 6A and Fig. 6B, respectively. (C–E) Comet assay shows that 5-aza-CdR induced DNA damage recovered when the treated cells were incubated in 5-aza-CdR free medium for 12 hrs. (C) Untreated control, (D) 5-aza-CdR at 1 µM for 24 hrs without incubation, and (E) 5-aza-CdR at 1 µM for 24 hrs, followed by washing for further incubation in a drug-free medium for 12 hrs. (F–H) Recovery of DNA damage induced by 5-aza-CdR was inhibited by depsipeptide treatment (0.1 µM for the final 6 hrs). (F) Depsipeptide treated cells (0.1 µM) for 6 hrs, (G) Cells were treated with 5-aza-CdR (1 µM for 24 hr) and depsipeptide (0.1 µM for last 6 hrs) without incubation, or (H) Cells treated as above and further incubated in a drug-free medium for 12 hrs.
HDAC inhibitor acted selectively with nucleotide analog to induce inhibition of cell proliferation
To determine whether HDACI selectively enhances NA-induced cytotoxicity, H719 cells were treated with Ara-C, another cytidine analog, and depsipeptide or TSA was then added to the cells to observe changes in cell proliferation assayed with MTT. It was of interest that both depsipeptide and TSA significantly enhanced Ara-C induced inhibition of cell proliferation (Fig. 7A–B). However, this synergistic inhibition of cell proliferation was not observed in the cells treated with depsipeptide and other DNA damage inducers such as γ-ray (Fig. 7C), UV (Fig. 7D), cisplatin (Fig. 7E) or etoposide (Fig. 7F). These data suggest that HDACI selectively acts in concert with NAs to induce inhibition of cell proliferation.
(A) H719 cells or (B) A549 cells were treated with Ara-C at 0.1 µM for 72 hrs in the presence or absence of HDACI (depsipeptide 0.1 µM or TSA 2 µM for the final 6 hrs). The cells were then washed and further incubated for 24 hrs for MTT assay. (C) Effect of interaction of HDACI and irradiation at 2 Gy was determined with the MTT assay. H719 cells were irradiated at 2 Gy, or treated with depsipeptide at 0.1 µM, or TSA at 2 µM for 6 hrs alone or in combination. (D) H719 cells were also treated with UV at 128 J/m2, with or without depsipeptide treatment. (E) H719 cells were treated with cisplatin at 0.1 µM for 48 hrs or (F) with etoposide at 0.2 µM for 48 hrs, and then depsipeptide at 0.1 µM was separately added to the treated cells. All data above is the result of two separate experiments.
It is clear that 5-aza-CdR is a potent demethylating agent, in that 5-aza-CdR is readily incorporated into DNA, and the nitrogen substitution for the 5-carbon in the cytosine moiety is able to confer the ability to trap DNA methyltransferase 1 [16], [28], [45]. In theory, inhibition of both DNA methylation and HDAC will increase expression of genes if they are methylated in their promoter regions, because methyl-binding proteins specifically bind to methylated DNA and recruit HDAC activity [11], [46]. In fact, the effectiveness of this combination in treatment of cancer is the subject of a number of human clinical trials by several different groups [7], [8], [47], [48]. There are reports indicating that methylation in the promoter region of caspase 8 or caspase 1 is one reason that cells develop resistance to chemotherapy [49], [50], and treatment with 5-aza-CdR could help abolish this resistance and restore chemotherapy induced cell death [49], [51]. However, in most cases of 5-aza-CdR induced apoptosis, it is generally unclear which gene or pathway leads to this effect. We tested the methylation status of several important apoptotic related genes including p53, bcl-2, bax and caspase 8, but all of these were hypomethylated in the cells tested (data not shown). In addition, in the present study, although DNMT1/DNMT3A/DNMT3B were effectively knocked down (Fig. 1A–C), depsipeptide was not able to induce a synergistic inhibition of cell proliferation (Fig. 2A). Therefore, the synergistic effects of 5-aza-CdR in combination with HDACI in inhibition of cell proliferation most likely have an alternative mechanism that is methylation-independent.
In addition to its demethylating function, 5-aza-CdR also has anti-neoplastic activity which operates by damaging DNA [24], [25], [52]. Most NAs exert their cytotoxicity through interference with DNA replication. It has been found experimentally that NAs incorporated into DNA and abases inhibit template function and chain elongation [26]. In addition, the incorporated abases competitively inhibit DNA polymerase α in opposition to the normal substrate deoxycytidine 5′-triphosphate [53]. The NAs also cause an unusual reiteration of DNA segments, which results in multiple copies of limited portions of DNA and increases the possibility of recombination, crossover, gaps and breaks [54]. 5-aza-CdR differs from physiological cytidine by the presence of a nitrogen at the 5- position of the heterocyclic ring and this substitution renders the ring chemically unstable and leads to spontaneous decomposition of its compounds in neutral solution [26]. The incorporated 5-aza-CdR or its unstable form which has a ring that breaks open inducing DNA damage, consequently may elicit many different cellular responses using different pathways to repair the resultant DNA damage. Consistent with these reports, our data demonstrate that 5-aza-CdR can induce DNA damage in a dose-dependent manner (Fig. 3C–E), and the degree of DNA damage induced by 5-aza-CdR also parallels the amount of incorporated [3H]-5-aza-CdR (Fig. 3A–B compares to Fig. 3C–E). Here it should be pointed out that 5-aza-CdR induced DNA damage is a SSB at lower doses as demonstrated by an increase in chromatin-bound RPA, and absence of formation of γ-H2AX (Fig. 5A–B). However, when 5-aza-CdR was used at a higher concentration (for example, >5 µM), formation of γ-H2AX was observed in response to 5-aza-CdR treatment (data not shown). We propose that 5-aza-CdR is randomly incorporated into DNA as a cytosine substitution, and thus higher concentrations of 5-aza-CdR result in greater incorporation of 5-aza-CdR into DNA. On the other hand, as single-strand breaks can be turn into DSBs if the replication fork is interrupted [55], [56], the possibility of co-existence of SSB and DSB is reasonable, and it may be dependent of dosage and degree of DNA damage.
However, cells have a capacity to exclude harmful abases and repair DNA damage induced by different stimuli. A chief difference from radiation- or other stimuli-induced DNA damage is that NA induced DNA damage usually is limited to an alteration in a DNA base or a nucleotide. The enzymes that are responsible for excision of incorporated 5-aza-CdR or Ara-C have not been clearly identified. However, more than 10 distinct DNA glycosylases have been found in cells which can repair abase-induced DNA damage, including enzymes specific for excision of uracil or 5-methyl-uracil [57]. There are two classes of enzymes for base excision repair: "short patch" involved enzymes and "long patch" involved enzymes [58]. The base excision repair (BER) initiated by enzymes for the "short patch" are glycosylases that include UNG, TDG, SMUG1 and MBD4. For example, uracil represents an endogenous DNA lesion that results from deamination of cytosine or incorporation of uracil instead of T during replication [59]. As a uracil DNA glycosylase, UNG, has been considered to be a key enzyme for removal of uracil in DNA [60]. However, although UNG is highly selective for uracil in DNA, it also removes certain bases that are structurally related to cytosine. These related bases include 5-fluorouracil in DNA [61], [62], 5-hydroxy-2′-deoxyuridine [63], [64] and alloxan [64]. In E. coli, for example, mutation of uracil DNA glycosylase leads to a 50-fold increase of 5-fluorouracil incorporated into DNA [65]. In human lymphoblasts, UNG was also found to remove 5-fluorouracil in vitro [61]. As early as 1980s, it was reported that 5-aza-CdR induced DNA damage results from a single-strand break and glycosylase recognizes and removes 5-aza-CdR from DNA [66]. In this study, we analyzed expression of enzymes that are mainly responsible for BER in depsipeptide treated cells, and only UNG expression was obviously inhibited (data not shown). However, UNG may be not directly involved in excising the 5-aza-CdR adduct since RNAi against UNG is not sufficient to mimic the effect of depsipeptide in its synergistic effect in inhibiting cell proliferation with 5-aza-CdR (data not shown). We also tested to see whether depsipeptide influences activities of enzymes for "long patch repair" including human apurinic/apyrimidinic endonuclease 1 (APE1) and flap endonuclease 1 (FEN1). However, depsipeptide did not inhibit expression of APE1 and FEN1 (data not shown). These data reflect the complexity of 5-aza-CdR induced DNA damage and the enzymes involved in repairing the damaged DNA.
5-aza-CdR-induced DNA damage was previously reported to come from a bulky adduct of 5-aza-CdR and DNMT1 [16]. This adduct may be excised by several enzymes that are involved in nucleotide excision repair (NER) [58]. Therefore, identification of enzymes which operate in NER is a critical step for the future.
HDAC inhibitors seem likely to be specific in their synergy with NAs to induce inhibition of cell proliferation, since no inhibition of cell proliferation after treatment with depsipeptide in cells irradiated with γ-rays or treated with other DNA damage inducers was observed (Fig. 7C–F). In support of this, HDAC inhibitor MS-275 interacts synergistically with different nucleoside analogs, but not with other DNA damaging agents to induce apoptosis in different human cancer cells [48]. These data suggest that HDACI may specifically suppress expression or activity of enzymes that are involved in repairing nucleoside analog-induced DNA damage. Therefore, to identify which enzyme(s) is responsible for repairing NA incorporation, understanding of cytosine analog-induced DNA damage is extremely critical and may be useful for designing new therapeutic strategies to treat cancer.
In conclusion, in this study we demonstrate that 5-aza-CdR exerts cytotoxicity by incorporating into DNA and inducing DNA damage. HDACI may interfere with the repair process of nucleoside analog induced DNA damage, and thus lead to a significant inhibition of cell proliferation. We believe that this novel finding will be helpful in formulating a new strategy to treat cancer more effectively in the clinic.
We greatly appreciate Drs. SB Baylin and GL Xu for providing us vectors and other reagents.
Author Contributions
Conceived and designed the experiments: GO WZ. Performed the experiments: GC LL WZ LW YZ SL YY HW YH YC YY XW DW. Analyzed the data: GO WZ. Wrote the paper: GO WZ MM.
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- 54. Canman CE, Tang H, Normolle DP, Lawrence TS, Maybaum J (1992) Variations in Patterns of DNA Damage Induced in Human Colorectal Tumor Cell by 5-Fluorodeoxyuridine: Implications for Mechanisms of Resistance and Cytotoxicity. Proceedings of the National Academy of Sciences 89: 10474–10478.
- 55. McGlynn P, Lloyd RG (2002) Recombinational repair and restart of damaged replication forks. Nat Rev Mol Cell Biol 3: 859–870.
- 56. Helleday T (2003) Pathways for mitotic homologous recombination in mammalian cells. Mutat Res 532: 103–115.
- 57. Cline SD, Hanawalt PC (2003) Who's on first in the cellular response to DNA damage? Nat Rev Mol Cell Biol 4: 361–372.
- 58. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73: 39–85.
- 59. Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362: 709–715.
- 60. Nilsen H, Rosewell I, Robins P, Skjelbred CF, Andersen S, et al. (2000) Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication. Mol Cell 5: 1059–1065.
- 61. Ingraham HA, Tseng BY, Goulian M (1980) Mechanism for exclusion of 5-fluorouracil from DNA. Cancer Res 40: 998–1001.
- 62. Lonn U, Lonn S, Nylen U, Winblad G (1990) Increased levels of 5-fluorouracil-induced DNA lesions in Bloom's syndrome. Int J Cancer 45: 494–499.
- 63. Hatahet Z, Kow YW, Purmal AA, Cunningham RP, Wallace SS (1994) New substrates for old enzymes. 5-Hydroxy-2′-deoxycytidine and 5-hydroxy-2′-deoxyuridine are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase, while 5-hydroxy-2′-deoxyuridine is a substrate for uracil DNA N-glycosylase. J Biol Chem 269: 18814–18820.
- 64. Dizdaroglu M, Karakaya A, Jaruga P, Slupphaug G, Krokan HE (1996) Novel activities of human uracil DNA N-glycosylase for cytosine-derived products of oxidative DNA damage. Nucleic Acids Res 24: 418–422.
- 65. Warner HR, Rockstroh PA (1980) Incorporation and excision of 5-fluorouracil from deoxyribonucleic acid in Escherichia coli. J Bacteriol 141: 680–686.
- 66. D'Incalci M, Covey JM, Zaharko DS, Kohn KW (1985) DNA alkali-labile sites induced by incorporation of 5-aza-2′-deoxycytidine into DNA of mouse leukemia L1210 cells. Cancer Res 45: 3197–3202.


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Holiday How-To: Thanksgiving Crafts and Pumpkin Recipes
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Latest Comments
"The Hushabye Shell Afghan is a beautiful blanket that would be a great shower gift. The pattern is an easy shell stitch and the colts are great for a boy or girl. I am sure that it will keep baby nice and warm. It is made with a nice four ply yarn and should work up fast. This one to save for a later date."
"The wavy rainbow baby blanket is one of the cutest that I have seen. The colors are so bright that a baby will enjoy looking at it. The blanket could also be worked up in other colors that would be great for that special little one. this is not for a beginner, so if this is your first try at crocheting you may not want to try it. Looking forward to working on this."
"The Springtime Lace Afghan Pattern is so cute. It will make a great baby blanket for that special baby. This could be made in many different colors that will really be pretty. The pattern reads really easy and a beginner could do this.I am looking forward to to this project in the very near future."
"he Cape Cod Afghan is really a cute pattern. It would be a great one for beginners. The pattern only calls for two skeins of yarn. The pattern reads really easy. This could also be used as a baby blanket and would look great in one of those baby colors. This is one pattern that I will definitely save for a later project down the road."
Top 10 Thanksgiving Decorations to Make, Part 2
By: Caley Walsh
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Recently, I published Part 1 of the Top 10 Thanksgiving Decorations to Make. While living aboard for several years, I always missed the quintessential American holiday of Thanksgiving. Therefore, I am a big advocate for honoring Thanksgiving as much as the other end-of-year holidays. As a craft editor, I've been gathering the best Thanksgiving decorations to make this holiday.
Turkey craft are an obvious choice for the best Thanksgiving decorations to make, but they also offer a surprisingly amount of variety. Turkey crafts range from easy and fun crafts for kids to more elegant crafts, and all are great for Thanksgiving decorating. With a coat of paint and some craft foam feathers, you can recycle un-carved pumpkins from Halloween into Thanksgiving turkeys. You can also use foam balls for the basic body shape of a turkey. Use a smaller foam ball or egg-shaped foam shape for the head.
For something easy and adorable, take two brown pom-poms, one slightly smaller than the first, in yarn or wool roving. Glue the smaller pom-pom to the top as the head and add feathers to the back in orange, red and yellow felt. Add eyes, beak and waddle to the head pom-pom. Pom-poms are easy to make with a pom-pom maker or cardboard circles. Place each of the pom-pom turkeys on your guests plates.
More turkey Thanksgiving decorations to make:
- Turkey Bead Pendants- Create beaded turkeys with this tutorial for fun and elegant wine charms. Guests can take home as a party favor.
- Turkey Piñata - A turkey piñata is surprisingly easy to create with balloons, papier-mâché (newspaper and glue) and paint. Kids can help decorate the piñata before Thanksgiving and it will provide the perfect activity on the holiday.
- Natural Turkey Decoration- This turkey decoration is made with natural items like pinecones, nuts and beans. You likely already have all the materials in your house.
- Decorative Turkey- This felt turkey is adorable and easy to create with felt. The kids can also help with this craft.
Table Linens
Update boring table linens with a bit of Thanksgiving crafting. Use stencils and fabric paint to easy add autumn leaves, cornucopias or other harvest images to a plain tablecloth. You can even use permanent fabric dye, stencils and a paintbrush to create a lasting Thanksgiving tablecloth. Use freezer paper to create your own stencil from images on the web.
Try your hand at embroidery or cross-stitch with a leaf or other design in the corner of yours placemats or napkins. You can use an embroidery or cross-stitch chart to make any needlework easier. Create embroidered napkin rings with a ring of embroidered felt or fabric glued over a cardboard tube (toilet paper tubes cut into 1-inch segments for example). Embroider the felt with a simple acorn shape.
- Flower Bouquet Felt Block- Follow this technique to add hand-embroidered felt shapes to fabric or felt. You could use this technique to embellish your table linens with leaves and other harvest shapes.
Burlap is also a fantastic material for Thanksgiving, with its rustic, harvest appearance. Use looser-weave burlap picked up from your local garden center. You can make a long table runner to extend the length of your dining room table and/or individual placemats. You can certainly sew the edges neat and clean, or simply cut straight and let the edges fray for that rustic look.
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Julie's Reviews > Ten Things We Did
Ten Things We Did (and Probably Shouldn't Have)
by Sarah Mlynowski (Goodreads Author)
by Sarah Mlynowski (Goodreads Author)
Julie's review
bookshelves: chick-lit, young-adult
Mar 20, 2012
bookshelves: chick-lit, young-adult
Read from June 25 to July 02, 2012
I really enjoyed reading about all of the crazy things April got herself into and out of. I didn't want it to end!
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My daughter made this chicken wreath for me,she did a good job for her first wreath and I love it!
Chicken wreath
Rockcreekfarm Shawnee, OK Dec 11, 2012
Very nice.
porkpal Richmond, TX Dec 11, 2012
What a cute idea!
CajuninKy Biggs, KY(Zone 6a) Dec 11, 2012
How fitting.
terri_emory Alba, TX(Zone 8a) Dec 14, 2012
Massively cool!
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Duration: 9min 56sec Views: 58 046 Submitted: 1 year ago
Description: Cosplay Babes welcomes Charlotte Fox, in her first and ONLY porn scene! Yuffie Yulan cosplays as Kukaku Shiba the leader of the Shiba Clan.
Categories: Cosplay Babes Channel Amateur Cunnilingus German Group Sex HD Videos Interracial Matures Old+Young Swingers Teens Threesomes Bleach Cosplay Babes


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Guardian Focus podcast
With nearly 1 billion people around the world already going hungry each day and dozens of countries facing food shortages in the near future, food security has become an urgent issue
In this week's Focus podcast Martin Wainwright looks at the likely effects of George Osborne's comprehensive spending review on the lives of people in his hometown
Our team of experts discusses the winners and losers in the government's comprehensive spending review
Podcast Guardian Focus podcast: Why do record numbers of young people have sexually transmitted infections?
Rosie Swash investigates the reasons for the UK's high level of sexually transmitted diseases among 15 to 24-year-olds
This week's Guardian Focus podcast looks at state investment in the arts, as Britain's creative industries prepare for large-scale government funding cuts
In the wake of the UN millennium development goals summit, we look at the challenge of meeting the needs of the world's poorest and ask what should happen to global development after 2015
Pakistan has been hit by its worst floods since 1929 in the week President Zardari chose to visit Britain. We discuss the realtionship between the two countries and the ability of the Pakistani state to deal with the numerous crises it faces


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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
I recently got a speeding ticket on the 408. I have never in my life gotten a ticket. I was told to go to court and plead no contest so I don't get the points. I have a court date next week and have no idea what do or say to them.
No contest simply means you are not contesting the charges (you are not admitting guilt, but you are not going to fight the charges and will accept a sentence).
The above answer is for general information only. It is not "legal advise" but analysis, and different lawyers may analyze this matter differently, especially if there are additional facts not reflected in the question. I am not your attorney until retained by a written retainer agreement signed by both of us. I am only licensed in California. See also avvo.com terms and conditions item 9, incorporated as if it was reprinted here.
I agree with the other attorney, you are not admitting guilt, you are simply stating you do not want to contest the charges anymore. Unless you were speeding significantly you should be fine. If you are still unsure, you should hire an experienced ticket attorney to help with your case.
Good luck.
Entering a no contest plea does not automatically mean you will not get points on your driver's license record. To avoid points request that the judge or hearing officer withholds adjudication of guilt.
Good luck
Legal disclaimer: The answers given are limited to the facts as given and presumed by the answer itself. Without seeing actual written documentation or having a conference to more fully explore the issues, this short answer has only limited application. Make sure to seek legal counsel and provide all documentation to get assistance in making informed legal choices.
Pleading no contest will not mean that you do not get a point, it is as my colleagues have said not admitting guilt, but it is treated the same way as a guilty plea by criminal courts. You can plead no contest and ask for traffic school, that will keep you from getting a point on your driving record.
The information provided is not intended as legal advice and does not establish an attorney client relationship. If you are in the greater Sacramento area and wish a further consultation please contact me at (916)594-9442.
I would recommend speaking to an experienced traffic lawyer before you enter any plea. As stated by other responses, stating no contest does not ensure you will not receive points or other sanctions I'm sure you are hoping to avoid. There may be legal defenses to your ticket that you could explore in court. Additionally, depending on the speed of the ticket you may be facing additional consquences outside of just a fine and points. Before you head to court speak with an experienced lawyer.
Post a free question on our public forum.Ask a Question
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Sports & Recreation, ProfessionalFollow
Grantland's Bill Simmons discusses sports and pop culture with celebrities and media personalities.
November 19th, 2014
Episode 38 of 112 episodes
'Saturday Night Live' executive producer Lorne Michaels joins Bill Simmons to discuss the show's ups and downs over the last 40 seasons.
You'll need to sign in to write a comment.
Welcome to the daily audio workout for your head.


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Kovilan had written 11 novels 12 collections of short stories besides articles and a play. His novels includes, Thakarnna Hridayangal (1946), A minus B(1959), Tharavadu (1963), Ezhamedangal (1965), Thazhvarakal (1969), Thottangal (1970) and Thattakam (1995) among others.
He received Ezhuthachan Puraskarom in 2006, which is the highest literary honour provided by Kerala government. He got the honour for his outstanding contribution to Malayalam literature.
He also won Kerala Sahitya Academy award in 1972 and 1977, besides the Kendra Sahitya Academy award in 1998 and Vayalar award in 1999. He was also a member of the Kendra Sahitya Academy.
Kovilan, whose real name was V V Ayyappan, was born on July 9, 1923 at Kandanissery in Guruvayur. He had worked in the Royal Indian Navy from 1943 to 1968 in the Corps of Signals. He is survived by two daughters and a son.
OneIndia News


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7 Breakers Way Korora NSW 2450
Email Agent
"Breakers Way With Self Contained Residence"7 Breakers Way, Korora
Taking in an elevated position in one of Korora's most sought after streets, this high quality brand new three bedroom home with media room plus a large one bedroom self-contained residence offers great value and options galore.
You will be impressed when you walk through the modern main residence featuring stunning hardwood timber floors, high ceilings, open plan design, designer kitchen with stone bench tops and quality appliances. The open plan and light and bright living area flows seamlessly to the large covered alfresco area, where outdoor dining and entertaining can be enjoyed. A second living area makes the ideal media/games room or lounge/parents retreat.
The self contained residence downstairs has separate direct access, separate living area, open plan kitchenette, main bathroom & built-ins. This residence can be used for extended family and friends.
Extras include gas hot water and two Daiken reverse cycle air-conditioning units.
The double garage is complemented by an additional workshop area. The grounds have been extensively landscaped front and back with low-maintenance in mind.
All this ultra-modern living comes with the convenience of being within a ten minute drive of Coffs Harbour, Jetty café strip and beautiful local beaches.
Completion early 2017
- Absolutely brand new 4 bedroom quality residence
- Exclusive Breakers Way location within a 5 minute walk to Korora beach
- 3 bedroom residence plus a self contained 1 bedroom residence
- Open plan design, light & bright, high ceilings, separate media room & designer kitchen with quality appliances
- Beautiful polished hardwood timber floors, 2x air conditioning systems, gas hot water & fully landscaped grounds
Inspection Times
- No inspections scheduled
- Contact Agent
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Florent & Mundey Real Estate Coffs Harbour
Coffs Harbour NSW, 2450
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Real Estate For Sale in Korora, NSW 2450 priced between $670,000 and $1,240,000


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Hotfrog NewZealand provides information regarding Oliver Smith. Oliver Smith provides Weddings services. Contact them on +64 21 714 014 or by visiting them on their website http://www.oliversmith.co.nz.
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With Instructables you can share what you make with the world, and tap into an ever-growing community of creative experts.
Handmade rabbit hutch
this stinks
How to spoil your hermit crabs: Food Edition
Crab Cakes
Turn Tri Meshes to Quads in One Step
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Magical Fairy Garden on a Fairy Budget
Cut T-Shirt with Bows on Back
DIY Hair Bows (12 patterns)
Chocolate Peanut Butter Candy
Kids Super Sci-Fi Rocket fueled Jet Pack made for pennies!
Creeps (Spicy Marshmallow Creepers)
Peel Off Window Art
DIY Nigiri Sushi Plush
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Pocket-sized Watercolor Altoids Tin
Makedo Fun Basketball Ring
Minecraft Instructables Robot
How to make String Hearts
Join 2 million + to receive instant DIY inspiration in your inbox.
© 2016 Autodesk, Inc.


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To make people aware about the effects that the consumption of snuff , the British National Health System developed an application that lets you see how your face will look like in ten to twenty years if you keep the habit of smoking.
The "Smoking Time Machine" , the name of this free application for iOS and Android , take a photo of your face, align it to a template and shows you how your face will look.
Kate Norman, spokesman for the project said the idea is that the younger population awareness , with something tangible, the way in which the damage this habit. "It's something very close and difficult to ignore," he told the "Daily Mail" .


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First Seventh Day Baptist Church Of Alfred Ny
5 Church St, Alfred, NY 14802
Contact: G Douglas Clarke Treas
Our Mission+ Edit Mission Statement
Add a mission statement for "First Seventh Day Baptist Church Of Alfred Ny." What do they do and what makes them unique?
Start a Conversation
First Seventh Day Baptist Church Of Alfred Ny is a Subordinate Corporation. Its Group Exemption Number (3649) indicates that it is a member of the First Seventh Day Baptist Church Of Alfred Ny. For Tax purposes Contributions are deductible. The organization is a Church 170(b)(1)(A)(i)
Financial information in-depth
Form 990 documents
We do not have Form 990 documents for this nonprofit yet. Check back periodically.
Assets and Revenues reported None
- Asset Amount:
- Income Amount:
- Form 990 Revenue Amount:
Organization Information
- NTEE Code:
- Religion-Related
- Christianity
- Ruling Date:
- July 1, 2000
- Group Exemption Number:
- 3649
- Subsection Code:
- Affiliation Code:
- Subordinate
- Classification Code:
- Deductibility Code:
- Contributions are deductible.
- Foundation Code:
- Church 170(b)(1)(A)(i)
- Activity Code:
- Organization Code:
- Corporation
- Exempt Organization Status Code:
- Unconditional Exemption
- Advanced Ruling Expiration Date:
- Filing Regulation Code:
- 990 - Not required to file (church)
- Filing Regulation Code:
- No 990-PF return
- Asset Code:
- 0
- Income Code:
- 0
- Account Period:
- December
This is my nonprofit
Become accredited today and earn the trust of your donors.Get Started


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Here is a tally of our adoptions for this year:
Cats = 2Dogs = 1Rabbits = 0Guinea Pigs = 0Hamsters = 0Gerbils = 0Rats = 0Mice = 0Ferrets = 0Pot Bellied Pigs = 0
If you have a friend looking for a pet, encourage them to look at our list of adoptable pets. Thanks!
Here is a tally of our adoptions last year:
Cats = 31Dogs = 15Rabbits = 3Guinea Pigs = 4Hamsters = 0Gerbils = 0Rats = 6Mice = 2Ferrets = 2Pot Bellied Pigs = 0
Robyn's Nest is a non-profit animal rescue and adoption organization located at 1291 W. Market St., Germantown, Ohio. It is operated by Robyn McGeorge, a registered veterinary technician. She graduated with an associate of applied science in veterinary technology in 1994 from Raymond Walters College, University of Cincinnati.
If you are interested in adopting from Robyn's Nest you may come in to the rescue, or check our website for available animals to adopt. Some animals live in foster homes, so if you would like to meet one of them, let us know and we will have the foster parent contact you to set up a meeting. Adoption applications are available on our website or at the rescue. We do check veterinary references to see if your current or past pets have been well cared for. We like to see that basic veterinary care has been provided including spay/neuter, vaccinations, heartworm testing and prevention.
Cocoa and Hershey were groomed by Becky at Robyn's
Nest. They love their haircuts and they are so happy to
have helped save the lives of homeless animals with
Robyn's Nest has moved to Germantown as of Sept 1st, 2015. Our new building is much larger, brighter and cheerier!
The cats and small animals have separate rooms, so cat hair will be less of a problem.
50 percent of the money you spend on grooming at Robyn's Nest goes to help homeless animals! We need our grooming clients in order to keep helping the animals. Please tell your friends and family about our wonderful groomers, Victoria and Becky. They are very gentle with the animals and makes them look great!
Robyn's Nest Animal Rescue
dogs, cats, small animals
247-9272 ~1291 W. Market St., Germantown, OH 45327
Day, Evening & Sat. appts.
CAT BOARDING at rescue
PET SITTING avail. in your home
Mon. 9-5 ~Tues. 2-8 ~Wed. 9-5 ~Thurs. 2-8 ~
Robyn's Nest is NON-PROFIT
Proceeds go to rescue homeless animals
Please stop in and see us!
We could use any of the following items. Any donation would be greatly appreciated!
Thanks to everyone who donated shoes . We have collected 2500 pr. of shoes and they will be picked up this week. We will be receiving $1,000.00 from Woof Trax. The money will help keep our rescue animals warm this winter, as well as fed and in good health! What a blessing. Please continue to bring your old wearable shoes,( boots, slippers, heels, sandals, anything) , they can be childrens or adults, to Robyn's Nest in Germantown.
Wyatt has been adopted to a couple who recently lost their min pin at 14 yrs of age. He will have a fenced yard and a creek to play in!
Mini is a small pit and she had been looking for a home for 2 years before finding her special person. Not only does Mini have a Mom, Julia, now she has a new life, traveling the country! Mini and her Mom drive a truck all over the USA. Mini loves to ride in the truck and she is great company for her Mom.
©2016 Robyn's Nest Inc. — All Rights Reserved.
Robyn's Nest is a registered member charity (#99710) of the Miami Valley Combined Federal Campaign.


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Big M Loop is a 11.8 mile loop trail located near Manistee, Michigan. The trail is rated as moderate and primarily used for hiking, nature trips, and mountain biking.
mountain biking
nature trips
hiking4 months ago
13 miles on the Big M today. lovely well marked trail... we did 1-9 to 32 down to 29, across the NCT and back to 23 and then home to 4,3,2,1
1 year ago
my gf skipped leg day and ruined the fun :/
trail running4 years ago
One of my favorite places to run. There is a good variety of terrain.
Deb W.
trail running5 years ago
Big M is one of my favorite places to train in MI. Plenty of hills and well marked trails. You can get a 25 mile loop if you incorporate the outer loop with some of the inner loops. Nice woods and even wildlife.


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Print out this $1/1 Post Pebbles Cereal Coupon to score a Post Cereal deal that will be starting on 8/18 at Walgreens. You will want to print this coupon now if you want to do this deal as I'm guessing it will disappear fast!
Here's the Walgreens Deal:
Post Pebbles Cereal — $1.75 (starting 8/18)
$1/1 Post Pebbles Cereal Coupon
Final Price: $0.75/each
You should be able to print this coupon twice.
Thanks Thrifty Jinxy!
Find a complete list of Walgreens Weekly Deals.


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Microsoft Office University 2011 for Mac
Guide price: £70
Last updated on 30th Sep 2012
Tags: microsoft
Microsoft Office University 2011 for Mac
Microsoft Office Mac University 2011 Mac English DVD Active enrolled university students are eligible to use the Office University product The end user must verifiy University Status using wwwofficecomverify before product can be used W6L00001 ML
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Shared medical office
Find Shared medical office space on rent or sublease. Clineeds a platform helps you to provide Sublease Medical office space on rent.Further more info call (331)642-0547 or visit http://goo.gl/TN5eHX


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"Sea Trail" Sunset Beach Sports & Outdoors Tip by etfromnc
Sunset Beach Sports & Outdoors: 2 reviews and 0 photos
Golf, meetings, vacations, and even real estate investment opportunities can be yours at the Sea Trail Conference Center and Golf Resort. Three separate courses deigned by three different course designers (Rees Jones, Dan Maples, and Willard Byrd) offer a beautiful array of challenges.
You can take a virtual tour of their courses at their website.
Their overnight accomodations are mostly one to four bedroom villas and start at around $300 per night. They do have planned activities for children and a fitness center which includes a swimming pool.
Address: 211 Clubhouse Rd, Sunset Beach 28468
othercontact: 888-261-5944
Phone: 888-229-5747
Theme: Golfing
Website: http://www.seatrail.com
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Style: 39181672 Color Code:
Printed mini dress with side cutout accents and an open back with an adjustable tie. Button closures on the sides with a high neck and overlay detailing. Full skirt and hidden side zip closure.
New Romantics
Dreamy fabrics, slubby textures, and ethereal sheer details come together to create New Romantics, a collection of pieces that are hand touched and hopelessly romantic
too complicated!
I bought this dress because I liked the print, the colors, and the criss cross top. However, when it came in, I literally tried for about 1 hour to figure out how to put it on correctly. After I couldn't figure it out, I enlisted the help of a girlfriend. She could not figure it out either. One of my boobs was literally just hanging out of the dress (and I have small boobs, like an A/B cup). I later asked my boyfriend to help me figure it out, and he couldn't do it either! The dress was either sewn incorrectly or is just too much of a jigsaw puzzle. Needless to say, I returned it...


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This book describes the development of systems of magnetic resonance imaging using the higher magnetic field strength of 3 tesla, in comparison to the current gold standard of 1.5 tesla. These new systems of MRI make it possible to perform with high spatial, temporal and contrast resolution not only morphological examinations but also functional studies on spectroscopy, diffusion, perfusion, and cortical activation, thus helping research and providing an important tool for routine diagnostic activity. At the same time the new systems offer unparalleled sensitivity and specificity in the numerous conditions of neuroradiological interest. Table of contents Techniques and Semeiotics: High-field MRI and safety: I. Installation.- High-field MRI and safety: II. Utilization.- 3.0 T MRI diagnostic features: comparison with lower magnetic field.- Standard 3.0 T MR imaging.- 3.0 T MR angiography.- 3.0 T MR spectroscopy.- 3.0 T diffusion studies.- Imaging nervous pathways with MR tractography. 3.0 T perfusion studies.- High field strength functional MRI.- Recent developments and prospects.- 3.0 T brain MRI: a pictorial overview of the most interesting sequences.- Application: High-field neuroimaging in traumatic brain injury.- 3.0 T imging of ischemic stroke.- High field strength MRI (3.0 T or more) in white matter disases.- High field neuroimaging in Parkinson's disease.- High field 3.0 T imgaging of Alzheimer diesease.- 3.0 T imaging of Alzheimer disease.- 3.0 T imaging of brain tumours.- fMRI activation paradigms: a presurgical tool for mapping brain function.- 3.O T fMRI in psychiatry
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Titolo: High Field Brain MRI ( Magnetic resonance imaging )
Autori: Salvolini - Scarabino
Editore: Springer Verlag
Volume: Unico
Edizione: 2006
Lingua: Inglese
Finitura: Copertina rigida
Pagine: 240


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Egyptian customs regulations are very strict on paper, but they are often unevenly enforced. If importing items, be sure that all paperwork is in order and prepare for delays when obtaining permission from the proper government authorities. If entering as a visitor or other private individual, prepare to go through custom formalities.
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Duty Free
Very few items are allowed into Egypt duty free. Diplomatic, oil company and aid project personnel are allowed to bring in household and personal items duty free. Visitors are allowed to bring valuable personal items into Egypt with out paying duties but may have to prove that they are exiting Egypt with the same personal items to avoid being charged when leaving the country. Some electronics, such as photographic and video equipment and computer accessories, can be dutiable in any quantity.
Restricted Items
Many items will be allowed into Egypt only after customs officials collect high tariffs. Alcohol, for example, is subjected to a 3,000% duty and a 100% sales tax. Items deemed new by customs officials are subjected to duties determined by the customs officials. Telecommunications equipment and media may be screened by censors and held by customs in order to obtain approval for entry or import from the Ministry of Culture. Similarly, animals and seeds must be approved by the Ministry of Agriculture to enter the country.
Prohibited Items
Egypt prohibits anything that could be deemed pornographic or anti-Islamic. Furthermore, no weapons (firearms and ammunition, swords, daggers and explosives) are allowed into the country. Gold and silver are very heavily restricted; anything not for obvious personal use or not in very small quantities is not allowed. Antiquities more than 100 years old are not allowed out of the country, except in certain circumstances requiring special permission, certification and documentation.
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Hotels in Pünderich
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Ferien-Weingut RockenbachHauptstr. 7, 56862, Pünderich / MoselThis 3-star guest house is set on the outskirts of picturesque Pünderich, surrounded by its own beautiful vineyards a few steps from the River Moselle. The large terrace and barbecue area have a scenic view of Marienburg Castle.Rates: EUR 40.00 - 60.00Stars: ★★★Rooms: 6
Gästehaus Kühn's WeinschenkeMarienburgerstr. 20, 56862, PünderichGästehaus Kühn's Weinschenke is in the centre of historic Pünderich 100 metres from the banks of the Moselle River. Guests may enjoy wine tasting accompanied by local dishes in the rustic cellar.Rates: EUR 38.00 - 70.00Stars: Rooms: 9
Vias FerienwohnungRathausstraße 47, 56862, PünderichThis apartment in Pünderich offers a scenic view of the Moselle River and Marienburg Vias Ferienwohnung provides access to a covered roof terrace, offering 2 bedrooms and a spacious living room.Rates: EUR 60.00 - 0.00Stars: Rooms: 1
Zum Ritterstutz-Bougainville, 56862, PãnderichZum Ritterstutz-Bougainville is located in Pãnderich, 14 km from Hahn Airport in Frankfurt and offers free parking. The accommodation will provide you with balcony and CD-player. The kitchen is fully equipped, including a dishwasher.Rates: EUR 71.50 - 0.00Stars: Rooms: 1