text:
The retroviral oncogene v-myb encodes a transcription factor (v-Myb) which is responsible for the transformation of myelomonocytic cells by avian myeloblastosis virus (AMV). v-Myb is thought to exert its biological effects by deregulating the expression of specific target genes. We have recently demonstrated that the chicken Gas41 gene, whose promoter co-localizes with an origin of DNA replication, is a bona fide Myb target gene. Because of this finding we have asked whether other Myb-regulated genes are also associated with DNA replication origins.
We show that the promoter region of the chicken adenosine receptor 2B gene (Adora2B), a known Myb-target gene, acts as a DNA replication origin. Furthermore, we have examined known replication origins for the presence of Myb binding sites. We found that the intergenic region between the genes for the minichromosome maintenance 4 protein (Mcm4) and the catalytic subunit of DNA-dependent protein kinase (Prkdc), whose human counterpart has been identified as a replication origin, contains a number of Myb binding sites. Our data show that this region also acts as an origin of replication in chicken cells. Interestingly, we found that the chicken Mcm4 gene is also Myb-regulated.
Our work identifies the chicken Mcm4 gene as a novel Myb target gene and presents evidence for the co-localization of two novel origins of DNA replication with Myb-regulated genes. Our work raises the possibility that a fraction of Myb target gene promoters is associated with DNA replication origins.
The oncogene v-myb of avian myeloblastosis virus (AMV) encodes a transcription factor which is responsible for the ability of AMV to transform cells of the myelomonocytic lineage [1,2]. v-myb is derived from the chicken c-myb gene, which plays a crucial role in the development of the hematopoietic system. c-myb is expressed in immature cells of all hematopoietic lineages and is silenced during the terminal differentiation of these cells. It is thought that c-myb is part of a genetic switch that allows hematopoietic progenitor cells to choose between alternative fates, such as proliferation, differentiation or apoptosis [2,3].
The proteins encoded by v-myb and c-myb (v-Myb and c-Myb) bind to the sequence motif PyAAC(G/T)G [4] and control the expression of a number of genes, such as mim-1 [5], the lysozyme gene [6], bcl-2 [7,8], tom-1 [9], c-kit [10], GBX2 [11], Pdcd4 [12], the genes for neutrophil elastase [13] and the A2B adenosine receptor [14] among others. Recent microarray analyses have suggested that Myb actually affects the expression of a much larger set of genes, either by direct binding or by indirect mechanisms [15].
Most of the work aimed at understanding how Myb activates the expression of its target genes has focused on the promoter regions of these genes and has provided a relatively simple picture of how Myb affects gene expression. In many cases, Myb binding sites have been identified in the promoter regions of these genes, suggesting that Myb acts primarily on the promoters of its targets. Recent in-depth analyses of some Myb target genes, however, have suggested a more complex picture in which the activation of certain target genes is mediated by cooperation of additional Myb-responsive enhancer elements with the target gene promoters. For example, the expression of the chicken mim-1 gene is controlled by interactions of Myb both with the promoter as well as with a powerful cell-specific enhancer located upstream of the gene [16]. Similarly, in case of the chicken C/EBPβ gene Myb targets the promoter as well as an enhancer located downstream of the gene [17].
Recently, another regulatory DNA element has been mapped close to a Myb-regulated gene. We have shown that chicken Gas41, whose promoter region has been known for some time to act as an origin of DNA replication [18,19], is a direct Myb target gene [20]. Other work has shown that the Myb homolog of Drosophila melanogaster is involved in the control of the replication origin responsible for chorion gene amplification [21]. These observations raised the question of whether DNA replication origins are also associated with other Myb target genes. Here, we demonstrate that the promoter region of the Myb-regulated chicken adenosine receptor 2B gene acts as an origin of DNA replication. Furthermore, we identify the chicken Mcm4 gene as a novel Myb target gene and demonstrate that the Mcm4 promoter is associated with a replication origin. Besides identifying two new DNA replication origins in chicken cells and a novel Myb-regulated gene our work suggests that a significant fraction of Myb-regulated genes might be associated with DNA replication origins.
The chicken Mcm4 gene is a Myb target gene
The observation that the promoter of the Myb-regulated the chicken Gas41 gene [20] is associated with an origin of DNA-replication [18,19] prompted us to explore the possibility that other Myb target regions also co-localize with DNA replication origins. Ladenburger et al [22] have previously identified a replication origin located between the human Mcm4 and Prkdc genes. Both genes are separated from each other by an intergenic region of only 0.8 kb and are transcribed in opposite directions. This arrangement is conserved among humans, mice and chickens [23,24]. We noted that the corresponding chicken sequence contains a number of potential Myb binding motifs (Fig. 1). One of the sites (MBS-6) matched exactly to the consensus Myb binding motif PyAAC(G/T)G [4], while the other sites had one (MBS-4, MBS-1) or two (MBS-2, MBS-3 and MBS-5) mismatches. The human Mcm4-Prkdc intergenic region also contains 8 potential Myb binding motifs although their exact positions are not conserved between the human and the chicken sequence. To demonstrate that Myb recognizes at least some of the motifs found in the chicken sequence those sites (MBS-1, MBS-4 and MBS-6) that showed the best fit to the consensus binding motif were analyzed by electrophoretic mobility shift assays (EMSA) using recombinant v-Myb protein expressed in bacteria. As illustrated in Fig. 2A, retarded protein-DNA complexes were observed when oligonucleotides containing MBS-1, MBS-4 and MBS-6 were used, demonstrating that v-Myb is able to recognize at least three of the potential binding sites in vitro. To confirm the specificity of binding we performed a competition experiment in which binding of v-Myb to a radiolabeled oligonucleotide containing the high affinity Myb binding site A from the mim-1 promoter [5] was competed by excess of unlabeled oligonucleotides containing MBS-1, MBS-4 and MBS-6 (Fig. 2B). Binding was competed efficiently by the mim-1 A site itself but not by a control oligonucleotide containing a C/EBP binding site. Oligonucleotides containing MBS-4 and MBS-6 also competed, although less efficiently that the mim-1 A oligonucleotide. MBS-1 appeared not to compete specifically with the mim-1 A site. Taken together, this experiment confirmed that v-Myb binds specifically to binding sites 4 and 6 while binding to MBS-1 was quite weak.
Figure 1. Nucleotide sequence of the Mcm4-Prkdc intergenic region. The translational start codons of the Mcm4 and Prkdc genes are marked by arrowheads. Potential Myb binding sites are marked by boxes.
Figure 2. v-Myb binds to the Mcm4 promoter region in vitro. A. Electrophoretic mobility shift experiments were performed using bacterially expressed full-length v-Myb protein and radiolabeled oligonucleotides corresponding to Myb binding sites MBS-1, MBS-4 and MBS-6. Binding reactions were performed without Myb protein (lanes 1) or with increasing amounts of v-Myb (lanes 2 to 4). Complexes of full-length v-Myb and the oligonucleotides are marked by an arrowhead. The band at the bottom corresponds to unbound oligonucleotides. B. Radiolabeled oligonucleotide containing the Myb binding site A of the mim-1 promoter were incubated with bacterially expressed full-length v-Myb and excess of unlabeled competitor oligonucleotides, as indicated at the top. Competitors were used at 40-fold or 80-fold molar excess of unlabeled over labeled oligonucleotide, respectively. Only the upper part of the gel containing the complex of v-Myb and the radioactive oligonucleotide is shown.
To investigate whether v-Myb is bound to the Mcm4 promoter region in vivo we performed chromatin immunoprecipitation (ChIP) experiments using the v-Myb transformed myeloblast line BM2. The cells were formaldehyde-fixed and DNA-protein complexes obtained after sonication were immunoprecipitated with two different antibodies against Myb. As a control, parallel incubations with non-immune serum or with unrelated antibodies were performed. After reverse-crosslinking and purification of the immunoprecipitated DNA the samples were analyzed by quantitative real-time PCR using the primers that bind to the Mcm4-Prkdc intergenic region or control primers. The result of the ChIP experiment is illustrated in Fig. 3. We observed substantial enrichment of the Mcm4-Prkdc intergenic region in samples precipitated with the Myb-specific antibodies, whereas control immunoprecipitations with non-immune sera or unrelated antibodies showed no specific enrichment. PCR-reactions with control primers that were specific for sequences located 13 kilobasepairs upstream of Mcm4 did not show significant enrichment in any of the immunoprecipitations. Taken together, the ChIP experiment clearly showed that v-Myb was bound to the Mcm4-Prkdc intergenic region in vivo.
Figure 3. Chromatin immunoprecipitation of the Mcm4 promoter region. Chromatin fragments prepared from BM2 cells were subjected to immunoprecipitation using two different Myb-specific rabbit antisera raised against the DNA-binding domain of v-Myb (anti Myb a and b). Control immunoprecipitations were performed with two different batches of normal rabbit serum (NRS a and b) or anti-Flag antibodies (anti Flag). DNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR using primers specific for the Mcm4 promoter region (-0.55 kb, left panel) or for a region approximately 13 kb upstream of the Mcm4 gene (-13 kb, right panel). The columns indicate the amount of PCR product obtained relative to the anti-Flag control reaction.
Next, we investigated whether v-Myb also affected the expression of the Mcm4 or Prkdc genes. To address this issue we made use of a cell line, designated 10.4, which stably expresses a v-Myb/estrogen receptor fusion protein [25]. Upon β-estradiol treatment of 10.4 cells the v-Myb/ER fusion protein is activated and upregulates the expression of a number of Myb target genes. 10.4 cells were cultivated for 24 hours in the presence or absence of 2 μM β-estradiol. Polyadenylated RNA from these cells was then isolated and analyzed by Northern blotting with probes specific for Mcm4 or Prkdc mRNA. As a control, a probe hybridizing to the ribosomal protein S17 mRNA was used. As shown in Fig. 4, the Mcm4 mRNA level was elevated in the estrogen-treated 10.4 cells when compared to untreated cells, suggesting that chicken Mcm4 is indeed a Myb target gene. By contrast, Prkdc was not regulated by v-Myb. 10.4 cells do not express endogenous estrogen receptor [25], suggesting that Mcm4 is not simply an estrogen-regulated gene.
Figure 4. The chicken Mcm4 gene is regulated by v-Myb. Polyadenylated RNA from 10.4 cells grown for 24 hours in the presence (+ lanes) or absence (- lanes) of 2 μM β-estradiol was analyzed by Northern blotting using probes specific for Mcm4, Prkdc or S17 mRNA.
An origin of DNA replication is located upstream of the chicken Mcm4 gene
To investigate whether the intergenic region between Mcm4 and Prkdc also acts as a DNA replication origin in chicken cells we performed nascent strand abundance assays as described by Giacca et al. [26]. The nascent strand abundance assay is a powerful and widely used method to identify and map DNA replication origins. The assay is based on the isolation and quantification of single stranded DNA 1–2 kb in length (so-called nascent DNA) which is primarily derived from replication origin regions. The method can be performed with asynchronously growing cells and does not depend on the availability of antisera against origin binding proteins (such as Orc- or MCM-proteins). Briefly, nascent DNA 1–2 kb in length was isolated from different chicken cells by sucrose gradient sedimentation. Sets of primers mapping to the intergenic region as well as to upstream and downstream sequences were used to determine the abundance of the corresponding sequences in nascent DNA using quantitative real-time PCR. BM2 cells are myeloid v-myb transformed cells that express high levels of the v-Myb protein. HD11 cells are myeloid cells that do not express v-Myb or c-Myb. DT40 is a line of B-lymphoid cells that express c-Myb. Finally, we used a subclone of the DT40 line in which the c-myb gene had been disrupted by homologous recombination [27]. As illustrated in Fig. 5 the Mcm4-Prkdc intergenic region was enriched in nascent DNA in each of these cell lines. This demonstrated that this region acts as a DNA replication origin also in chicken cells. We did not observe significant differences in the abundance of the origin region between myeloid cells expressing (BM2) or not expressing (HD11) v-Myb. Although the abundance of the origin region was higher in DT40 cells compared to myeloid cells, there was no significant difference between wild-type and c-Myb deficient DT40 cells. Thus, although Myb binds to the intergenic region and stimulates Mcm4 expression, it does not affect the activity of the replication origin.
Figure 5. Replication origin activity of the Mcm4-Prkdc intergenic region in chicken cells. A. Schematic illustration of the Mcm4-Prkdc intergenic region. The black boxes and the arrows denote the transcriptional start sites of both genes. The positions of primer pairs used in the PCR analysis are shown below. The scale at the top is in kilobases. B. Nascent strand abundance assay. For quantification, real-time PCR was performed using the primer pairs indicated at the bottom. The columns show the average abundance of the corresponding sequences in nascent strand preparations of BM2 (black columns) or HD11 (white columns) cells relative to the sequences amplified with the -0.5 kb primer pair. Error bars mark the standard deviations. C. Nascent strand abundance analysis of the Mcm4 promoter region using wild-type (black columns) or c-myb deficient (white columns) DT40 cells.
Identification of an origin of DNA-replication in the promoter region of the chicken adenosine receptor 2B gene
We also examined the promoters of two known Myb-regulated genes, the mim-1 and adenosine 2B receptor genes, for replication origin activity. Both genes are direct Myb targets and their promoters have been analyzed in detail [5,14,28-30]. Nascent DNA from different cell lines was analyzed using sets of primers spanning a region from -5 kb to +5 kb around the promoter in case of the mim-1 gene and from -3.2 kb to +4.8 kb around the promoter of the A2B receptor gene. We did not observe enrichment of mim-1 promoter sequences in preparations of nascent DNA, indicating the mim-1 promoter does not coincide with a DNA replication origin (data not shown). By contrast, the promoter region of the A2B receptor gene was enriched in nascent DNA, suggesting that an origin of DNA-replication is located at the 5' end of the A2B receptor gene (Fig. 6). As in the case of the Mcm4 replication origin we did not observe significant differences in origin activity of the promoter region of the A2B receptor gene between the different cell lines.
Figure 6. Replication origin activity of the promoter region of the chicken adenosine receptor 2B gene. A. Schematic illustration of the chicken A2B receptor gene. The black boxes show the two exons of the gene. The transcriptional start site is marked by an arrow. The position of primer pairs used in the PCR analysis is shown below. The scale at the top is in kilobases. B. Nascent strand abundance assay of the adenosine receptor 2B promoter region using the PCR primer pairs indicated at the bottom. The columns show the average abundance of the corresponding sequences in nascent strand preparations of BM2 (black columns) or HD11 (white columns) cells relative to the sequences amplified with the -3.9 kb primer pair. Error bars mark the standard deviations. C. Nascent strand abundance analysis of the promoter region using wild-type (black columns) or c-myb deficient (white columns) DT40 cells.
The identification of the chicken Gas41 gene, whose promoter region functions as a DNA replication origin, as a bona fide Myb target gene [20] has raised the question whether the co-localization of a Myb-regulated promoter with a replication origin is just a rare coincidence or a more common phenomenon? To address this question we used two different strategies: i.) We examined the promoter regions of known Myb-regulated genes for replication origin activity using the nascent strand abundance assay. ii.) We investigated the activity of v-Myb at the Mcm4-Prkdc intergenic region, which harbors several potential Myb binding sites and is associated with a replication origin in human cells [22].
Analysis of the adenosine receptor 2B gene, a known Myb target gene [14,30], showed that the sequences around the 5' end of the gene were enriched in preparations of nascent DNA. This observation strongly suggested that the promoter of the A2B receptor co-localizes with a DNA replication origin. A further case of such co-localization was revealed by the analysis of the Mcm4-Prkdc intergenic region. Our data showed that v-Myb binds to this region both in vitro and in vivo and stimulates transcription of the chicken Mcm4 gene. Furthermore, we demonstrated that the Mcm4-Prkdc intergenic region is a replication origin also in chicken cells. Thus, chicken Mcm4 is a novel v-Myb target gene whose promoter region is also associated with a DNA replication origin. As these examples show, Myb-regulated genes and replication origins co-localize in some cases. However, it is also evident from the limited number of genes that have been analyzed so far that not every Myb target gene promoter is associated with a replication origin. For example, the promoter region of the mim-1 gene does not possess replication origin activity.
In the light of the co-localization of replication origins with several promoters containing clusters of Myb binding sites we wondered how frequently such clusters occur in the chicken genome. We screened approximately three million basepairs of randomly selected chicken genomic sequences for the presence of clusters of Myb binding motifs (AACG or AACTG) that are similar to those found at the MCM4, Adora2b and Gas41 replication origins. While on the average one such motif is present per 200 base pairs we found that clusters of at least six of such sites within stretches of 250 base pairs occur only once per approximately 25 kb. Although the number of such binding motif clusters is higher than the estimated number of replication origins in the chicken genome (one replication origin per approximately 100 kb, as extrapolated from mammalian cells [31,32]), it appears unlikely that the concurrence of clusters of Myb binding motifs and DNA replication origins reported here is purely accidental. It will therefore be interesting to examine such clusters of Myb binding motifs for their potential role as replication origins.
The functional consequences, if any, of such co-localization are unclear at present. We have not observed that v-Myb or c-Myb influence the activity of the replication origins identified here. v-Myb or c-Myb, therefore, appear not to be involved in the control of origin activity. Recent work on the Myb homolog (Dm-Myb) of Drosophila melanogaster has provided evidence that Dm-Myb performs a non-transcriptional role in the control of the replication origin responsible for chorion gene amplification [21]. Furthermore, it has been shown that some of the phenotypic consequences of mutation of Dm-Myb can be compensated by B-Myb, but not by c-Myb or A-Myb [33]. It is therefore conceivable that B-Myb plays a role at the replication origins identified here. Another interesting possibility is that rather than being regulated by Myb the presence of a replication origin might affect the activation of the associated gene by Myb. For example, compacted chromatin structures will be opened during replication thereby providing transcription factors access to the DNA. It is therefore possible that the association with a replication origin imposes an additional level of control onto the adjacent gene, which would be expected to be cell cycle dependent. It is presently not known whether the Mcm4 and Adora2B genes are expressed in a cell cycle dependent manner. However, in this respect it is interesting to note that the phenotypic changes of myeloid cells transformed by a temperature-sensitive version of v-Myb that accompany activation of v-Myb by down-shifting the cells from the non-permissive to the permissive temperature were not observed when DNA replication was inhibited [34]. This observation is consistent with the idea that the activation of at least some Myb-regulated genes depends on DNA replication. Clearly, further work is necessary until we can discern which of these possibilities, if any, is right.
Independent of a possible role of DNA replication origins in Myb-inducible gene expression our work is also of interest because we have identified two novel replication origins in chicken cells. So far, only very few replication origins are known in chicken cells, such as the origins located in the alpha and beta globin gene clusters [35,36] and between the lysozyme and Gas41 genes [18,19]. It is also interesting to note that the origin in the Mcm4-Prkdc intergenic region has been conserved between between chickens and humans.
Another interesting aspect of our work is the identification of the chicken Mcm4 gene as a Myb-regulated gene. MCM4, together with other MCM proteins, forms the hexameric MCM2-7 complex which functions as helicase during the S-phase of the cell cycle. The MCM2-7 complex is loaded onto the DNA prior to the initiation of DNA replication and constitutes an essential part of the pre-replication complex [for reviews see: [37-39]]. In addition to acting as a helicase in concert with the other MCM proteins forming the MCM2-7 complex there is evidence that MCM4 also performs a specific regulatory role. MCM4 is extensively phosphorylated when cells are exposed to inhibitors of DNA synthesis or to UV irradiation, leading to a block of DNA fork progression [40,41], suggesting that MCM4 plays a role in the DNA replication block checkpoint system. In the normal course of the cell cycle MCM4 is phosphorylated by Cdk2, leading to the inhibition of the helicase function of the MCM complex [42,43]. Blockage of this phosphorylation leads to reinitiation of replication and genomic instability. These observations suggest that MCM4 is a key component of the MCM complex whose manipulation can influence the control of DNA replication and genomic stability. Interestingly, elevated Mcm4 mRNA levels have been found in certain tumors, suggesting that increased Mcm4 expression can contribute to the malignant properties of the tumor cells [44,45]. Recently, a hypomorph allele of mouse Mcm4 was identified and shown to cause chromosomal instability [46]. Thus, Mcm4 is an interesting novel Myb target gene that provides a possible link between Myb, DNA-replication and checkpoint control.
We have identified the chicken Mcm4 gene as a novel Myb-regulated gene and shown that the Mcm4 promoter region is associated with an origin of DNA replication. Similarly, our work shows that the promoter of the Myb-regulated adenosine receptor 2B gene co-localizes with a replication origin. Our work raises the possibility that a fraction of Myb target gene promoters is associated with DNA replication origins.
Cell culture
The BM2 and HD11 are lines of AMV-transformed chicken myeloblasts and MC29-transformed macrophages that were originally obtained from C. Moscovici and T. Graf, respectively. BM2 cells were grown in RPMI 1640 medium supplemented with 5 % fetal calf serum, 5 % chicken serum and 10 % tryptone phosphate broth (Gibco). HD11 cells were grown in basal Iscoves' medium supplemented with 8% fetal calf serum and 2% chicken serum. 10.4 is a derivative of the HD11 cell line expressing a v-Myb/ER fusion protein [25]. 10.4 cells were grown in the same medium as HD11 cells. To activate the v-Myb/ER fusion protein the growth medium was supplemented with 2 μM β-estradiol for 24 hours.
Northern blotting
Preparation of polyadenylated RNA and Northern blotting were performed as described [25]. Mcm4 and Prkdc mRNAs were detected using cDNA clones of chicken Mcm4 (riken1_3305r1) and Prkdc (riken1_9d19r2), kindly provided by H. Arakawa and J.M. Buerstedde. As an internal control a specific probe for the ribosomal S17 gene was used.
Electrophoretic mobility shift assay
The following pairs of single-stranded oligonucleotides were annealed and used for electrophoretic mobility shift assays:
MCM4-MBS-1_sense: 5'-CCGGGCAGCGTTGGGTCAGTCCGAGGGG-3',
MCM4-MBS-1_anti: 5'-GCCCCTCGGACTGACCCAACGCTGCCCGG-3',
MCM4-MBS-4_sense: 5'-CGCCGTGGGTCGAACGGCGCGTCCCCC-3',
MCM4-MBS-4_anti: 5'-GGGGGGACGCGCCGTTCGACCCACGGCG-3',
MCM4-MBS-6_sense: 5'-TAGCGCTGCCACAACGGTCCTACCG-3',
MCM4-MBS-6_anti: 5'-GCGGTAGGACCGTTGTGGCAGCGCTA-3'.
Mim-1A_sense: 5'-GCTCTAAAAAACCGTTATAATGTACAGATATCTT-3'
Mim-1A_anti: 5'-AAGATATCTGTACATTATAACGGTTTTTTAGAG-3'.
C/EBP_sense: 5'-CTGGCTCGGTTCTTTCACAACCACACATCC-3'
C/EBP_antisense: 5'- GGGATGTGTGGTTGTGAAAGAACCGAGCCAG-3'
After annealing, the oligonucleotides were radiolabeled by filling-in the ends using Klenow polymerase and α32P-dCTP. Preparation of bacterial full-length v-Myb protein and binding experiments were performed as described [47].
Chromatin immunoprecipitation
Approximately 108 BM2 cells were incubated in 1/10 cross-linking solution containing 0.1 M NaCl; 1 mM EDTA; 0.5 mM EGTA; 50 mM Tris-HCl, pH 8.0 and 11% formaldehyde for 10 min and quenched for 5 min in 125 mM glycine. After washing in ice-cold phosphate-buffered saline, cells were treated for 20 min each with washing solution A (0.25% Triton X100; 10 mM Tris-HCl, pH8.0, 10 mM EDTA; 0.5 mM EGTA) and B (200 mM NaCl; 10 mM Tris-HCl, pH8.0; 1 mM EDTA; 0.5 mM EGTA). Nuclei were sonicated on ice (4 times for 20 sec in 2 min intervals) in egg lysis buffer (ELB) (120 mM NaCl; 50 mM Tris-HCl, pH7.5; 20 mM NaF; 1 mM EDTA; 6 mM EGTA; 15 mM sodiumpyrophosphate; 1 mM phenylmethylsulfonyl fluoride and 0.1% Nonidet P-40). DNA-protein complexes were incubated with two different Myb-specific antibodies raised against the DNA-binding domain of v-Myb [48], normal rabbit serum or anti-Flag antibody (Sigma). After precipitation with protein-A-sepharose the samples were washed several times in ELB buffer. Following elution with 0.5% SDS the DNA was recovered by reverse-crosslinking for 6 h at 37°C and 65°C, respectively, in a buffer containing 0.5% SDS; 10 mM DTT and 100 μg proteinase K. The immunoprecipitated DNA was then purified by phenol-chloroform extraction and ethanol precipitation. PCR-amplification was performed by quantitative real-time PCR using primers specific for the chicken MCM4 promoter region (-0.55 kb) or primers derived from the -13 kb region of the gene.
Nascent strand abundance assay
Analysis of replication origin activity was performed according to Giacca et al. [26]. Total genomic DNA was isolated from approximately 108 exponentially growing HD11 or BM2 cells using the Nucleobond CB kit (Macherey & Nagel) following the manufacturer's instructions. DNA was resuspended in TNE buffer (10 mM Tris, pH 8.0; 100 mM NaCl; 1 mM EDTA) and heat-denatured for 7 min at 95°C followed by rapid cooling on ice. Denatured DNA was layered on a linear neutral sucrose gradient (5–30% in TNE) and size fractionated by centrifugation in a Beckman SW28 rotor at 26.000 rpm for 20 h at 20°C. Nascent strand DNA fractions with an average DNA size of 1–1.5 kb were pooled, precipitated with ethanol and and treated with T4-polynucleotide kinase and λ-exonuclease as described previously [49,50]. The purified nascent DNA strands were analyzed by quantitative real-time PCR using the following primer pairs:
A2B_-3.2kb_for 5'-TGTCCGCAGAGCAGTTCAG-3';
A2B_-3.2kb_rev 5'-GCAGAACTGGCACAGCTTTCT-3';
A2B_-0.9kb_for 5'-GGTGTTATTGGATAGGCGCTG-3';
A2B_-0.9kb_rev 5'-CACAAAAACATGAGGCAATCG-3';
A2B_+0.4kb_for 5'-CCCATCCCACCTCTCTCGT-3';
A2B_+0.4kb_rev 5'-CACCTTCACCATTGTCGCC-3';
A2B_+1.75kb_for 5'-CCTGGAGTGGGCTTTGACA-3';
A2B_+1.75kb_rev 5'-GCCCTGTTCCCATCCTTGTA-3';
A2B_+4.8kb_for 5'-AAGGTAATGCTGCGACGTCA-3';
A2B_+4.8kb_rev 5'-GTGAAAGCCACACGGAGCTA-3';
MCM4_-13kb_for 5'-CAGCCAGCAAACAGCACTATTTC-3';
MCM4_-13kb_rev 5'-GGCCTTGCTTTCACCTAAGTCA-3';
MCM4_-0.55kb_for 5'-TAGACGACTTTCCGGAGAGACAGA-3';
MCM4_-0.55kb_rev 5'-CGCTGGGCGAATCGTTT-3';
MCM4_+2.5kb_for 5'-CATGGCTGCCAATGAGATCT-3';
MCM4_+2.5kb_rev 5'-GGCCGTACTTGAATTTGATGTTC-3'.
Real-time PCR quantification
Q-PCR was carried out on a GeneAmp 5700 SDS (Applied Biosystems) with the qPCR Core kit for SYBR Green I (Eurogentec) following the instructions of the manufacturers. Primers were chosen with PrimerExpress 2.0 Software (Applied Biosystems). As DNA standard we used sonicated total genomic chicken DNA (average size 1 to 5 kb) serially diluted to 50.000, 10.000, 2.000, 400 and 80 genomic equivalents per reaction (1 genomic equivalent = 1.25 × 10-12 g). To produce a calibration curve, the CT value of each sample was plotted against the logarithm of its concentration. The number of genomic equivalents of each target sequence in the nascent DNA strand samples was then determined by extrapolation from the calibration curve. Several conditions were met by each assayed sample: The slope of the standard curve was around 3 to 3.5 (indicating a PCR efficiency close to 100 %). The sample was diluted such that its genomic equivalents fell within the central part of the calibration curve. The melting curve profile showed a single PCR product. The specificity of the PCR products was additionally confirmed for each primer pair by agarose gel electrophoresis.
Authors' contributions
HG carried out the in vitro DNA binding and nascent strand abundance assays. DB performed the chromatin immunoprecipitations and northern blot experiments. KHK conceived the study, participated in its design and prepared the manuscript. All authors read and approved the final manuscript.
We thank H. Arakawa and J.-M. Buerstedde for providing plasmids. This work was supported by a grant from the DFG (KL461/10-1).
Lipsick JS, Wang DM: Transformation by v-Myb.
Oncogene 1999, 18:3047-3055. PubMed Abstract | Publisher Full Text
Oh I-H, Reddy EP: The myb gene family in cell growth, differentiation and apoptosis.
Oncogene 1999, 18:3017-3033. PubMed Abstract | Publisher Full Text
Weston K: Myb proteins in life, death and differentiation.
Curr Opin Genet Dev 1998, 8:76-81. PubMed Abstract | Publisher Full Text
Biedenkapp H, Borgmeyer U, Sippel AE, Klempnauer K-H: Viral myb oncogene encodes a sequence-specific DNA-binding activity.
Nature 1988, 355:835-837. Publisher Full Text
Ness SA, Marknell A, Graf T: The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene.
Cell 1989, 59:1115-1125. PubMed Abstract | Publisher Full Text
Introna M, Golay J, Frampton J, Nakano T, Ness SA, Graf T: Mutations in v-myb alter the differentiation of myelomonocytic cells transformed by the oncogene.
Cell 1990, 63:1287-1297. Publisher Full Text
Frampton J, Ramqvist T, Graf T: v-Myb of E26 leukemia virus up-regulates bcl-2 and suppresses apoptosis in myeloid cells.
Genes Dev 1996, 10:2720-2731. PubMed Abstract | Publisher Full Text
Taylor D, Badiani P, Weston K: A dominant interfering Myb mutant causes apoptosis in T cells.
Genes Dev 1996, 10:2732-2744. PubMed Abstract | Publisher Full Text
Burk O, Worpenberg S, Haenig B, Klempnauer K-H: tom-1, a novel v-Myb target gene expressed in AMV- and E26-transformed myelomonocytic cells.
EMBO J 1997, 16:1371-1380. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Hogg A, Schirm S, Nakagoshi H, Bartley P, Ishii S, Bishop JM, Gonda TJ: Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2.
Oncogene 1997, 15:2885-2898. PubMed Abstract | Publisher Full Text
Kowenz-Leutz E, Herr P, Niss K, Leutz A: The homeobox gene GBX2, a target of the myb oncogene, mediates autocrine growth and monocyte differentiation.
Cell 1997, 91:185-195. PubMed Abstract | Publisher Full Text
Schlichter U, Burk O, Worpenberg S, Klempnauer K-H: The chicken Pdcd4 gene is regulated by v-Myb.
Oncogene 2001, 20:231-239. PubMed Abstract | Publisher Full Text
Oelgeschläger M, Nuchprayoon I, Lüscher B, Friedman AD: C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter.
Mol Cell Biol 1996, 16:4717-4725. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Worpenberg S, Burk O, Klempnauer K-H: The chicken adenosine receptor 2B gene is regulated by v-myb.
Oncogene 1997, 15:213-221. PubMed Abstract | Publisher Full Text
Rushton JJ, Davis LM, Lei W, Mo X, Leutz A, Ness SA: Distinct changes in gene expression induced by A-Myb, B-Myb and c-Myb proteins.
Oncogene 2003, 22:308-313. PubMed Abstract | Publisher Full Text
Chayka O, Kintscher J, Braas D, Klempnauer K-H: v-Myb mediates cooperation of a cell-specific enhancer with the mim-1 promoter.
Mol Cell Biol 2005, 25:499-511. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kintscher J, Yamkamon V, Braas D, Klempnauer K-H: Identification of a Myb-responsive enhancer of the chicken C/EBPbeta gene.
Oncogene 2004, 23:5807-5814. PubMed Abstract | Publisher Full Text
Phi-van L, Sellke C, von Bodenhausen A, Stratling WH: An initiation zone of chromosomal DNA replication at the chicken lysozyme gene locus.
J Biol Chem 1998, 273:18300-18307. PubMed Abstract | Publisher Full Text
Phi-van L, Stratling WH: An origin of bidirectional DNA replication is located within a CpG island at the 3" end of the chicken lysozyme gene.
Nucleic Acids Res 1999, 27:3009-3017. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Braas D, Gundelach H, Klempnauer K-H: The glioma-amplified sequence 41 gene (GAS41) is a direct Myb target gene.
Nucleic Acids Res 2004, 32:4750-4757. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Beall EL, Manak JR, Zhou S, Bell M, Lipsick JS, Botchan MR: Role for Drosophila Myb-containing protein complex in site-specific DNA replication.
Nature 2002, 420:833-837. PubMed Abstract | Publisher Full Text
Ladenburger EM, Keller C, Knippers R: Identification of a binding region for human origin recognition complex proteins 1 and 2 that coincides with an origin of DNA replication.
Mol Cell Biol 2002, 22:1036-1048. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Connelly MA, Zhang H, Kieleczawa J, Anderson CW: The promoters for human DNA-PKcs (PRKDC) and MCM4: divergently transcribed genes located at chromosome 8 band q11.
Genomics 1998, 47:71-83. PubMed Abstract | Publisher Full Text
Fujimori A, Hashimoto H, Araki R, Daito T, Sato S, Kasama Y, Tsutsumi Y, Mori M, Fukumura R, Ohhata T, Tatsumi K, Abe M: Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene.
Radiat Res 2002, 157:298-305. PubMed Abstract | Publisher Full Text
Burk O, Klempnauer K-H: Estrogen-dependent alterations in differentiation state of myeloid cells caused by a v-myb/estrogen receptor fusion protein.
EMBO J 1991, 10:3713-3719. PubMed Abstract | PubMed Central Full Text
Giacca M, Pelizon C, Falaschi A: Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction.
Methods 1997, 13:3301-312. Publisher Full Text
Appl H, Klempnauer K-H: Targeted disruption of c-myb in the chicken preB-cell line DT40.
Oncogene 2002, 21:3076-3081. PubMed Abstract | Publisher Full Text
Ness SA, Kowenz-Leutz E, Casina T, Graf T, Leutz A: Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types.
Genes Dev 1993, 7:749-759. PubMed Abstract | Publisher Full Text
Burk O, Mink S, Ringwald M, Klempnauer K-H: Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP transcription factors.
EMBO J 1993, 12:2027-2038. PubMed Abstract | PubMed Central Full Text
Kattmann D, Klempnauer K-H: Identification and characterization of the Myb-inducible promoter of the chicken adenosine receptor 2B gene.
Oncogene 2002, 21:4663-4672. PubMed Abstract | Publisher Full Text
Jackson DA, Pombo A: Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells.
J Cell Biol 1998, 140:1285-1295. PubMed Abstract | Publisher Full Text
Berezney R, Dubey DD, Huberman JA: Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci.
Chromosoma 2000, 108:471-484. PubMed Abstract | Publisher Full Text
Davidson C, Tirouvanziam R, Herzenberg L, Lipsick J: Functional evolution of the vertebrate Myb gene family: B-Myb, but neither A-Myb nor c-Myb, complements Drosophila Myb in hemocytes.
Genetics 2004, 169:215-229. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Beug H, Blundell PA, Graf T: Reversibility of differentiation and proliferative capacity in avian myelomonocytic cells transformed by tsE26 leukemia virus.
Genes Dev 1987, 1:277-286. PubMed Abstract | Publisher Full Text
Prioleau MN, Gendron MC, Hyrien O: Replication of the chicken beta-globin locus: early-firing origins at the 5' HS4 insulator and the rho- and betaA-globin genes show opposite epigenetic modifications.
Mol Cell Biol 2003, 23:3536-3549. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Verbovaia L, Razin SV: Analysis of the replication direction through the domain of alpha-globin-encoding chicken genes.
Gene 1995, 166:255-259. PubMed Abstract | Publisher Full Text
Bell SP, Dutta A: DNA replication in eukaryotic cells.
Annu Rev Biochem 2002, 71:333-734. PubMed Abstract | Publisher Full Text
Stillman B: Origin recognition and the chromosome cycle.
FEBS letters 2005, 579:877-884. PubMed Abstract | Publisher Full Text
Takeda DY, Dutta A: DNA replication and progression through S phase.
Oncogene 2005, 24:2827-2843. PubMed Abstract | Publisher Full Text
Ishimi Y, Komamura-Kohno Y, Kwon HJ, Yamada K, Nakanishi M: Identification of MCM4 as a target of the DNA replication block checkpoint system.
J Biol Chem 2003, 278:24644-24650. PubMed Abstract | Publisher Full Text
Ishimi Y, Komamura-Kohno Y, Karasawa-Shimizu K, Yamada K: Levels of MCM4 phosphorylation and DNA synthesis in DNA replication block checkpoint control.
J Struct Biol 2004, 146:234-241. PubMed Abstract | Publisher Full Text
Hendrickson M, Madine M, Dalton S, Gautier J: Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex.
Proc Natl Acad Sci USA 1996, 93:12223-12228. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Zhu Y, Ishimi Y, Tanudji M, Lees E: Human CDK2 inhibition modifies the dynamics of chromatin-bound minichromosome maintenance complex and replication protein A.
Cell Cycle 2005, 4:1254-1263. PubMed Abstract | Publisher Full Text
Ishimi Y, Okayasu I, Kato C, Kwon HJ, Kimura H, Yamada K, Song SY: Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix.
Eur J Biochem 2003, 270:1089-1101. PubMed Abstract | Publisher Full Text
Huang XP, Rong TH, Wu QL, Fu JH, Yang H, Zhao JM, Fang Y: MCM4 expression in esophageal cancer from southern China and its clinical significance.
J Cancer Res Clin Oncol 2005, 131:677-682. PubMed Abstract | Publisher Full Text
Shima N, Alcaraz A, Liachko I, Buske TR, Andrews CA, Munroe RJ, Hartford SA, Tye BK, Schimenti JC: A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice.
Nat Genet 2007, 39:93-98. PubMed Abstract | Publisher Full Text
Oehler T, Arnold H, Biedenkapp H, Klempnauer K-H: Characterization of the v-myb DNA binding domain.
Nucleic Acids Res 1990, 18:1703-1710. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Klempnauer K-H, Bonifer C, Sippel AE: Identification and characterization of the protein encoded by the human c-myb proto-oncogene.
EMBO J 1986, 5:1903-1911. PubMed Abstract | PubMed Central Full Text
Gerbi S, Bielinsky A-K: Replication initiation point mapping.
Methods 1997, 13:271-280. PubMed Abstract | Publisher Full Text
Abdurashidova G, Deganuto M, Klima R, Riva S, Biamonti G, Giacca M, Falaschi A: Start sites of bidirectional DNA synthesis at the human lamin B2 origin.
Science 2000, 287:2023-2026. PubMed Abstract | Publisher Full Text


text: http://forum.xda-developers.com – Encounter somebody? Don't know where to meet again? Falling for somebody deeply? Don't know when and how to tell? Maybe somebody likes you too. Post your thinking at the place you meet her/him. Maybe she/he'll find by her/himself. Vote to help make more touching posts. Subscribe places nearby to find interesting things. Will add "tag search" for "encounter scenario" if you like it. It depends enough more users. Pls search "Spark LBS" in Google Play to make a try (HowTos)

subset: commoncrawl


text:
"We have a committed program," he says of Massachusetts General Hospital. " We have a guilty-'til-proven-innocent mentality regarding diversity issues. It's not about blame, but knowing there are gaps to fill."
The Massachusetts General Hospital established a disparities committee in 2002 after the IOM issued its report documenting the disparity problem nationally. According to Betancourt, the MGH committee continues to identify and address disparity issues "wherever they may exist" at the hospital. The committee receives information from subcommittees that target "experience, education and awareness" involving racial and ethnic issues, he says. On a regular basis, the committee's planning and findings are presented to the MGH board, executive council and other hospital leadership officials. Their reports range from non-adherence to policies, educational programs and data collection issues.
The hospital established cross-cultural training as well as interactive learning programs to allow them to "provide quality to patients of diverse cultural backgrounds," Betancourt says.
As a result, the hospital program resulted in significant awareness improvements among physicians after training 3,000 front line staff.
"If a certain group of diabetic patients are doing less well, maybe you can find some explanation, maybe it's a [literacy] issue, maybe it's a cultural issue, and then you can target interventions to meet those patient needs," Betancourt says.
Coaching programs and patient navigators who are important tools to overcome racial and disparity issues, he says. While a doctor visit may be about 15 minutes, the coach may then spend an hour with a patient, Betancourt says.


text:
- Home
- News
- Sports
- Opinion
- Obituaries
- Features
- Special Sections
- Classifieds
- Public Notices
Forgot Password? | Not a User? Sign up
Group gets tax status
The group Friends of Pewee Valley officially received their 501(c)(3) status from the Internal Revenue Service. This status makes them an official tax exempt non-profit organization. Mayor Bob Rogers announced at January's city council meeting that the Friends applied for the status in February of 2011 and just recently received notification of acceptance.
Comprehensive plan passed
If you currently subscribe or have subscribed in the past to the Oldham Era, then simply find your account number on your mailing label and enter it below.
Click the question mark below to see where your account ID appears on your mailing label.
|ZIP Code:


text:
- 800.878.7323
This item may be
Check for Availability
This title in other editions
Heart and Soulby Maeve Binchy
Synopses & Reviews
Publisher Comments:
A modern day women's writer in the Jane Austen sense. -Lauren Daley, The Standard-Times
"Sweet indulgence...Heart and Soul is a delicious delight." -Lindsey Losnedahl, Las Vegas Review-Journal
"Heart and Soul is a pleasant escape into an entertaining fantasy world across the ocean... Binchy] once again paints a delightful picture of Ireland that elevates the everyday joys and tragedies of her characters to ones of pure romance." -Karin Greenberg, Woodbury Magazine
A new Maeve Binchy novel is always welcome...Binchy has a true gift of creating characters we either know or wish we knew...Heart and Soul creates a perfect escape. -Michele Ross, Cleveland Plain Dealer
At the end of a long week, a long winter, a long economic downturn, Maeve Binchy's Heart and Soul is] exactly what we need. -Laurie Hertzel, Minneapolis Star Tribune
"Good-hearted and] entertaining... Heart and Soul] reflects a pervasive generosity of spirit and] offers many honest pleasures." -Bill Sheehan, The Washington Post
"Another delightful Binchyesque amalgamation of intersecting lives...the collective, charming effect of these story lines suggests that individuals are more connected than they might think." -Publishers Weekly
"Only a curmudgeon could resist this master of cheerful, read-by-the-fire comfort." -Kirkus Reviews
"Interweaving the domestic narratives of a dissimilar collection of individuals is beloved Binchy's stock-in-trade, and once again, she does so with sublime ease, inventively engaging readers through a reassuring and persuasive combination of gracious warmth, gentle humor, and genuine affection." -Carol Haggas, Booklist
Ambitious and intelligently conceived...A heart clinic is really the perfect metaphor for how this book feels. It's a warm and comfy world and reading Heart and Soul is] not unlike getting a hug from your mother...Binchy's millions-strong readership...will not be disappointed. -William Kowalski, The Globe and Mail (Toronto)
"Oh, the bliss...Maeve's back, on top form...The heart is the theme, literally and metaphorically, and this is heartwarming stuff-sweet but never cloying." -The Times
" Maeve Binchy] knows how to fashion a minor drama into a crisis, and the book rattles along from one gripping story to another, leaving the reader with a satisfying glow...It does exactly what it says on the tin: gives heart and soul." -Daily Mail
" Heart and Soul] brings together the secret hopes and dreams of a disparate group of characters...with Binchy's] trademark warmth and empathy." -Irish Sunday Independent
"Maeve Binchy's latest novel is packed as usual with wonderful characters...Full of warmth, caring and commonsense." -CHOICE
From the Hardcover edition.
Given the difficult task of building an underfunded clinic in an Irish community caught between the past and present, Dr. Clara Casey finds her task complicated by two difficult adult daughters, the unwanted attentions of her ex-husband, her colorful and diverse staff, and the demanding, often difficult patients they serve. 350,000 first printing.
With the insight, humor, and compassion we have come to expect from her, Maeve Binchy tells a story of family, friends, patients, and staff who are part of a heart clinic in a community caught between the old and the new Ireland.
Dr. Clara Casey has been offered the thankless job of establishing the underfunded clinic and agrees to take it on for a year. She has plenty on her plate already—two difficult adult daughters and the unwanted attentions of her ex-husband—but she assembles a wonderfully diverse staff devoted to helping their demanding, often difficult patients.
Before long the clinic is established as an essential part of the community, and Clara must decide whether or not to leave a place where lives are saved, courage is rewarded, and humor and optimism triumph over greed and self-pity.
Heart and Soul is Maeve Binchy at her storytelling best.
About the Author
Maeve Binchy is the author of numerous best-selling books, including her most recent novel, Whitethorn Woods, in addition to Night of Rain and Stars, Quentins, Scarlet Feather, Circle of Friends, and Tara Road, which was an Oprah's Book Club selection. She has written for Gourmet; O, The Oprah Magazine; Modern Maturity; and Good Housekeeping, among other publications. She and her husband, Gordon Snell, live in Dalkey, Ireland, and London.
What Our Readers Are Saying
Be the first to add a comment for a chance to win!
Product Details
Related Subjects
Fiction and Poetry » Literature » A to Z


text:
Let Ohio Business College be your first choice for healthcare education, business degrees, information technology programs and truck driving training. We've designed our advanced career training specifically to prepare students for the competitive job market. So if you're ready to further your career with high-quality, convenient career education and hands-on training, you don't need to look any further. Take advantage of our flexible class schedules, career-specific coursework, relevant training and accessible campus locations to get started on the way to your professional future.
Practical career training and support
In the career training programs here at Ohio Business College, you can learn job functions you'll apply directly in the workplace after you graduate. This will allow you to start your new career confidently. Check out our complete list of programs to see which one is for you. Regardless of your choice of career training programs, we can help you with everything you need to hit the ground running once you graduate.
Our faculty understands your goals and works closely with you to make them happen. In addition to personal attention from our faculty members, students can receive free tutoring. Graduates from our associate degree and diploma career education programs benefit from career placement assistance and free refresher courses.
Explore our associate, diploma, and certificate, and workshop programs below to find your fit.


text:
Haleakala Crater
Tours & Tickets
Viator Exclusive: Private Maui Helicopter Tour Including West Maui, Molokai and Sunset Landing
Soak in unbeatable views of West Maui on this thrilling 55-minute helicopter tour! Flying over famous West Maui and Molokai, you'll be treated to unobstructed ... Read more
Maui, Hawaii
Duration: 90 minutes (approx.)
Spectacular Haleakala Maui Sunrise Tour
Witness the dawn of a new day on a Haleakala Sunrise Tour. Standing at the top of majestic Mt. Haleakala, renowned for its stunning sunrises and beauty, ... Read more
Maui, Hawaii
Duration: 7 - 8 hours
Small-Group Luxury Day Trip to Haleakala National Park and Hana Coast Rainforest
Experience Maui's diverse landscapes – from the countryside to the coast – on this small-group luxury day trip. Aboard a luxury limo-van, soak in sweeping ... Read more
Maui, Hawaii
Duration: 9-10 hours
Haleakala, Iao Valley and Central Maui Day Tour
See the best central Maui on this scenic and informative guided day trip, with hotel pickup and drop-off. Your knowledgeable local guide will take you to the ... Read more
Maui, Hawaii
Duration: 8 - 9 hours
Small-Group Tour: Luxury Haleakala Sunrise Experience
Start your day on Maui at the top of the world! This small-group guided tour, for just up to eight guests, gives you an exclusive view of the famed Haleakala ... Read more
Maui, Hawaii
Duration: 8 hours (approx.)
Best Small-Group Maui Tour: Haleakala National Park, Lavender Farm and Wine Tasting
Awaken your senses on this small-group day trip highlighting Maui's best sights, scents and tastes! With your guide, travel to the top of Maui's highest peak ... Read more
Maui, Hawaii
Duration: 9 hours (approx.)
Haleakala Crater Hike
Explore the vastness of one of Maui's most iconic landmarks with a Haleakala Crater Hike. Experience the ethereal energy of the national monument as you ... Read more
Maui, Hawaii
Duration: 8 hours (approx.)


text: Hazel Nelson | IBDB: The official source for Broadway Information IBDB: The official source for Broadway Information Official Broadway League sites: - sites - broadway.org broadwayleague.com broadwayfanclub.com kidsnightonbroadway.com stagespecs.org tonyawards.com Quick Search For: In This Category: All Show People/Org. Theatre Season Hazel Nelson Performer Female Productions ProductionsDate of ProductionsThe Dollar Princess[Musical, Comedy, Operetta, Original] Performer: Hazel Nelson [Nellie, Typewriter Girl, Tennis Girl]Aug 06, 1909 - May 14, 1910 Find out where Hazel Nelson and are credited together © 2001-2015, The Broadway League, All Rights Reserved. (Copyright and Disclaimer)


text:
Sometimes going to the pub can almost feel like work. Last Thursday at Against the Grain was one of those. Dungarvan's Black Rock was on cask and in fine form. I could have happily drank it all evening. Except Clanconnel's excellent McGrath's Black was on special offer so I had to put one of those away too. And then there was the new Beer of the Month: Krombacher Dark.
I'm always pleased to see a new dark lager on the market in Ireland. I think it's a healthy sign of diversity in our beer culture. That this is being brought in by one of the big mainstream importers is a positive indicator. So what's it like?
Well, sadly, it's no Zeitgeist, nor even a Herold Dark. It does hit most of the dark lager targets of roasted grain and burnt caramel, no mean feat in a very sessionable 4.3% ABV bundle. There's even a faint creaminess, enhancing the body and rounding it off quite well. But through it all there's one niggling little bug that would make me think twice about buying more: that plasticky buzz found in some German lagers which may or may not be down to their use of hop extract (this certainly does). Add roasted malt to plastic and you get burnt plastic, which is not nice at all.
The beer isn't ruined by any means, but it's not all it should be either. I should also mention that it wasn't a great match for the chicken tajine I ate alongside it: the flavours, good and bad, just weren't robust enough to stand up to even a moderate level of spice.
Overall a slightly flawed but generally decent easy-drinking lager and well worth a punt. Maybe you won't notice the plastic.
19 May 2011
Dark out
8 comments:
I know the hop extract tang you're talking about but didn't get any plastic from the Dark. I rather enjoyed it.ReplyDelete
Beer for Autons.ReplyDelete
That certainly explains why the guy at the next table's laser-gun-hand kept flipping open. But I didn't like to stare.ReplyDelete
- Bobsleigh10:06 p.m.
I'm relatively new to the Beer scene, would you recommend it to a lover of the Dark Budvar?ReplyDelete
Yes, it's along similar lines, though I don't think it's as good.ReplyDelete
I'm glad you also associate that plasticky flavour with hop extract. I haven't ranted about that in a long time, it seems. Shit, does that mean I'm getting used to it?ReplyDelete
I think I got the association entirely from your tasting notes.ReplyDelete
Bugger. There goes the affirmation.ReplyDelete
Subscribe to: Post Comments (Atom)


text:
Health & Pregnancy
Vaginal Birth After Cesarean (VBAC) - References
American College of Obstetricians and Gynecologists (2010). Vaginal birth after previous cesarean delivery. ACOG Practice Bulletin No. 115. Obstetrics and Gynecology, 116(2): 450-463.
Miller DA (2010). Vaginal birth after cesarean. In Management of Common Problems in Obstetrics and Gynecology, 5th ed., pp. 52-56. Chichester: Wiley-Blackwell.
Cunningham FG, et al. (2010). Prior cesarean delivery. In Williams Obstetrics, 23rd ed., pp. 565-576. New York: McGraw-Hill.
Other Works Consulted
Institute for Clinical Systems Improvement (2011). Health care guideline: Management of labor, 4th edition. Available online: https://www.icsi.org/_asset/br063k/Labor-Interactive0511.pdf.
National Institutes of Health consensus development conference statement: Vaginal birth after cesarean: New insights March 8-10, 2010. Obstetrics and Gynecology, 115(6): 1279-1295.
In this article
This information is produced and provided by the National Cancer Institute (NCI). The information in this topic may have changed since it was written. For the most current information, contact the National Cancer Institute via the Internet web site at http:// cancer .gov or call 1-800-4-CANCER.
WebMD Medical Reference from Healthwise


text:
Useful Notes: Djibouti
Djibouti (Arabic: جيبوتي Jībūtī, French: Djibouti, Somali: Jabuuti, Afar: Gabuuti), officially known as the Republic of Djibouti (Arabic: جمهورية جيبوتي Jumhūriyyat Jībūtī, French: République de Djibouti, Afar: Gabuutih Ummuuno, Somali: Jamhuuriyadda Jabuuti), is a little country near the Horn of Africa, right next to Somalia, Eritrea and Ethiopia. Actually, thats the best way to describe its culture, because Djibouti is ethnically composed by Somalis and Afars, ethnic groups from the aforementioned countries. But most of all, the inhabitants of the country tend to consider themselves as brothers with Somalia (except for the European descendants, obviously). The land was originally known as Obock, where Afars and Somalis shared the land. When the French came in the mid-1800s, the sultans in the area began to sign treaties with them, giving them each time more control until Frances influence was big enough to firmly establish a colony, the French Somaliland. In 1958, when the independence of Somalia was imminent, a referendum was held to decided if the people wanted to go independent (and presumably join later with Somalia) or keep being a French protectorate. Since most of the European residents and the Afars were in favor of the French, they rigged the vote by expelling thousands of Somalis (who obviously wanted to join Somalia proper). In 1967, another referendum was due, and they did the same thing again and just to piss off the losers they renamed the colony French Territory of the Afars and the Issas (a take that against the Somalis, because the Issas were just a sub-clan of them). In 1977 came a third referendum, but the Somali population was big enough to avoid the rigging and ended gaining independence. They didnt join with Somalia, though. The president elected turned the country into a one party state, eventually triggering a civil war in 1991 that ended in 2001. Considering what happens with Somalia, maybe they should be glad of being an independent country; on the other hand, the country has had enough problems on its own. Its highly unlikely that you ever see a reference to Djibouti in the media; usually its treated just as a part of Somalia or as a non-entity. There was a mention of Djibouti in According to Jim, but it was mainly just so they could say ye-booty. On the other hand, it is the subject of an incredibly lame pickup line/meme in Model United Nations circles, namely, "I want to invade Djibouti with the aid of Greece", or "Swiggity Swooty I'm coming for Djibouti." Incidentally, this area was one of the first places where Islam came to Africa. Accordingly, the country is highly Islamic (94%). The remaining is Christian. The Djiboutian flag
The white isosceles triangle symbolizes peace; inside the triangle is a star, colored red for unity, and whose five points symbolize five areas inhabited by Somalis: British and Italian Somaliland (modern-day Somalia), French Somaliland (Djibouti), Ogaden (eastern Ethiopia) and North Eastern Province, Kenya; the blue upper half symbolizes both the sea and sky as well as the Issa clan of the Somali people; and the green lower half symbolizes both the earth and the Afar people.


text: > View Product Details Please select the size of the product. Please enter valid quantity. James Jeans warm grey stretch denim 'Twiggy' side zip skinny jeans overview details Retail: $178.00 Reduced: $34.97 $34.97 color : We're sorry. This style is sold out. Warm Grey Cured to perfection and fit to flatter, James Jeans are what all the celebrities and fashionistas alike are wearing. Color: Warm Grey Soft, lightweight stretch cotton blend denim Zip fly with button closure at belt looped waistband Classic four-pocket construction with signature pintuck detail at back pockets Skinny leg with 11'' zipper detailed opening Fitted throughout 8�'' rise 28'' inseam Approximate measurements taken from a size 28, and may vary by size 52% Cotton / 26% Rayon / 21% Polyester / 1% Spandex; Machine Wash; U.S.A; style#317501301


text:
Programs offer ways to track senior citizens if they go missing
The senior citizen who suffered dementia and sat in her car in the middle of Vero Beach ranchland for several days wasn't the only person Treasure Coast authorities were looking for last weekend.
Indian River County sheriff's spokesman Deputy Jeff Luther said his agency searched for three other missing persons as well as Vivian Vander Zyl, who was discovered safe by hog hunters Sunday.
"Luckily, we found all of them safe," Luther said.
Law enforcement agencies across the Treasure Coast said they have tools to track down patients of dementia or Alzheimer's disease when they wander off. In Indian River and Martin counties, families can register their loved ones through Project Lifesaver, a program which provides a tracking bracelet for users. The bracelet emits a unique frequency for its wearer. If the person drives or wanders away from home, law enforcement can use equipment to track the frequency, officials said.
"We had 20 people enrolled in Indian River," Luther said. "We should have a lot more. It's never taken more than 17 minutes to track a person if they had the bracelet."
The St. Lucie County Sheriff's Office started a similar program two years ago using the Care Trak transmitter bracelet, said Sgt. Anthony Cavallaro of the agency's K9 Unit. Cavallaro said the loved ones of a missing senior citizen shouldn't hesitate to call 911 if they go missing.
"It's everyone's natural instinct to do some searching themselves before they call," he said. "We lose time if people wait, and it disrupts the scent trail for our dogs. It's better to be safe than sorry by calling us."
Agencies in all three counties have used fundraising efforts and grant money to provide the bracelets at no cost to their users, officials said. In Indian River, officials ask for a $50 deposit for the bracelet.
In Martin County, the Sheriff's Office also offers the Return Safe program, in which families can provide deputies with pertinent information about their loved ones to keep on file in case they go missing, spokeswoman Rhonda Irons said.
"The deputies will immediately have a picture of the person if they are registered," Irons said. "If the person has wandered off before, we'll have a list of places where they have gone before."
Subscribe to TCPalm.com and our Treasure Coast Newspapers
Need Help? Call us at 1-877-710-6180.
Monday-Friday: 6am-3pm / Saturday: 6:30am-10:30am / Sunday: 7am-11am


text:
Montacute House
2015 Programme
Find out about the activities we can offer on a learning visit to Montacute House. This resource includes a description of the tours we offer, price, curriculum links and other essential information
Planning your visit
A resource of essential information to help plan your visit. This includes information on how to book, arriving, the location of facilities such as toilets and the all important where to eat lunch.
Risk assessment
Hazard information to help with writing your own risk assessments for visiting Montacute House.
Tudor inspired packed lunch
Some ideas for creating your own Tudor themed packed lunch to bring along on your visit.
Helpful information on the house
Information for accompanying teachers and helpers on your visit around the house.
Explore the garden and beyond
A fun trail for children to explore beyond the house to find out more about the gardens.


text:
BigOven Pro required (try it free)
Notice a categorization error? Let us know by clicking [alert editor] on the recipe page, in the ingredients box.
Tiramisu Cupcakes
Tiramisu Cheesecake
Brazil Nut Mousse
Tiramisu - eggless
Twinkie Tiramisu
Discover new recipes, add your own, create grocery lists and plan your menus.
Glad you've found something to try! Let BigOven share your recipe discoveries on Facebook. Click "Okay" on the Facebook prompt.
Got it


text:
The .exe extension on a filename indicates an executable file. Executable files may, in some cases, harm your computer. Therefore, please read below to decide for yourself whether the rundll.exe on your computer is a Trojan that you should remove, or whether it is a file belonging to the Windows operating system or to a trusted application.
The process known as Generic Host Process for Win32 Services or Run a DLL as an App or bitcoin-miner or Windows Security System or padFecVgm
belongs to software Microsoft® Windows (version 2000 Operating System) or Windows Update or Generic Host Process for Win32 Services or UnJapan or rundll32 or WSS or Microsoft Windows Operating System or PTTmMBmbvAhSe
by Microsoft (www.microsoft.com) or bit or Microsoft Co (www.microsoft.com) or GoHacking.INC or Ufasoft (www.ufasoft.com).
Description: Rundll.exe is not essential for Windows and will often cause problems. Rundll.exe is located in the C:\Windows\System32 folder.
Known file sizes on Windows 8/7/XP are 1,245,190 bytes (20% of all occurrences), 40,960 bytes and 12 more variants.
The rundll.exe file is not a Windows core file. The program is not visible. There is no information about the author of the file. The rundll.exe file is an unknown file in the Windows folder. The software starts upon Windows startup (see Registry key: Run, MACHINE\Run, Winlogon\Shell, MACHINE\RunServices, User Shell Folders). The software uses ports to connect to or from a LAN or the Internet. Rundll.exe is able to monitor applications, record keyboard and mouse inputs and hide itself. Therefore the technical security rating is 77% dangerous, however you should also read the user reviews.
Recommended: Identify rundll.exe related errors
External information from Paul Collins:
There are different files with the same name:
Important: Some malware also uses the file name rundll.exe, for example not-a-virus:Monitor.Win32.007SpySoft.g or Worm.Win32.VB.fi (detected by Kaspersky), and Win32:Haxpa (detected by Avast). Therefore, you should check the rundll.exe process on your PC to see if it is a threat. We recommend Security Task Manager for verifying your computer's security. This was one of the Top Download Picks of The Washington Post and PC World.
Summary: Average user rating of rundll.exe: based on 17 votes with 8 user comments. 8 users think rundll.exe is essential for Windows or an installed application. One user thinks it's probably harmless. One user suspects danger. 7 users think rundll.exe is dangerous and recommend removing it. 2 users don't grade rundll.exe ("not sure about it").
A clean and tidy computer is the key requirement for avoiding problems with rundll. This means running a scan for malware, cleaning your hard drive using cleanmgr and sfc /scannow, uninstalling programs that you no longer need, checking for Autostart programs (using msconfig) and enabling Windows' Automatic Update. Always remember to perform periodic backups, or at least to set restore points.
Should you experience an actual problem, try to recall the last thing you did, or the last thing you installed before the problem appeared for the first time. Use the resmon command to identify the processes that are causing your problem. Even for serious problems, rather than reinstalling Windows, you are better off doing a repair of your installation, or in the case of Windows 8, executing the DISM.exe /Online /Cleanup-image /Restorehealth command. This allows you to repair the operating system without losing data.
To help you analyze the rundll.exe process on your computer, the following programs have proven to be helpful: Security Task Manager displays all running Windows tasks, including embedded hidden processes, such as keyboard and browser monitoring or Autostart entries. A unique security risk rating indicates the likelihood of the process being potential spyware, malware or a Trojan. Malwarebytes Anti-Malware detects and removes sleeping spyware, adware, Trojans, keyloggers, malware and trackers from your hard drive.
hkwnd.exe a2srchas.dll stk02nm.exe rundll.exe lmworker.exe stdrt.exe adirss.exe smarterdownloader.dll googlequicksearchbox.exe soundmax.exe sansadispatch.exe [all]


text:
Continuing Resolution
Congress Is Back. Is Another Government Shutdown Coming?
September 08, 2014
Here's what Congress has to do before the midterms
Senate Republicans Don't Get It: The Democrats Aren't Bluffing
October 13, 2013
Mitch McConnell, leader of the Senate Republicans, approached Democrats with a new offer over the weekend: He and his colleagues would vote to open the government and increase its borrowing authority, as long as Democrats would agree to accept the de
Boehner: Surrender on Obamacare, Or It's Shutdown City
September 18, 2013
House Republican leaders finally have a plan.
The House GOP Is About to Crack Up: Three Theories Why
September 12, 2013
Lots of people think John Boehner has lost control of the House Republican caucus. Apparently John Boehner does, too.


text:
For the past several years, Spilman Thomas & Battle has been an active participant in the West Virginia Business Court Division ("BCD").
After two years of cases, the BCD's leaders presented improvements to the West...more
- Business Court Division Proposed Amendments
On November 12, 2013, the West Virginia Supreme Court of Appeals released its decision in Hersh v. E-T Enterprises, Ltd., et al., No. 12-0106. This landmark decision overturns more than 100 years of West Virginia precedent by...more
- Foreseeability Negligence Premises Liability Trespass
On September 25, 2012, Spilman Thomas & Battle released an article discussing the key features of the new West Virginia Business Court Division ("BCD"). Since the date of that release, there have been multiple...more
- Business Court Division Judicial Appointments
Business Court Division
Judicial Appointments
Premises Liability
Proposed Amendments
Your first step to building a free, personalized, morning email brief covering pertinent authors and topics on JD Supra:
Sign up to create your digest using LinkedIn*
Back to Top
Copyright ©


text:
Saheed O Olagunju
Chicago, IL
Get contact information on Saheed O Olagunju, Chicago IL. Find out Saheed O Olagunju's age, gender, possible aliases, associates, professional, and education info.
Also Known As
Saheed O Olagunju
Saheed Ola
Saheed O Olagunju is in his 40's
Saheed Has Lived In
Chicago, IL
Riverdale, IL
Calumet City, IL
Saheed's Relatives
Saheed O Olagunju
Zodiac SignAries
Additional Infomation on Saheed O Olagunju
Saheed O Olagunju's Contact Information
Known Cities Lived In
Find out where Saheed O Olagunju has lived as well as Saheed O Olagunju's phone numbers and email addresses.
Saheed O Olagunju Has Lived in 2 States
Illinois Address for Saheed O Olagunju
7301 N Rxxxx Bxxx
Chicago IL
Chicago IL
Has Lived InGet Full Address Report
Chicago, IL (7 Addresses)
Riverdale, IL
Phone Numbers Associated with Saheed O Olagunju
Get Full Phone Report
(773) ***-**** - Chicago, IL
(773) ***-**** - Chicago, IL
(773) ***-**** - Chicago, IL
Email Address Associated with Saheed O Olagunju
Get Email Report
All trademarks, product names, company names or logos on this page are the property of their respective owners.
Related People Searches


text:
Police investigate area burglariesMarch 27,2013Police investigate
burglaries in area
Police are investigating several burglaries around Rutland County.
Vermont State Police responded Monday to the home of Lee Hadley on Cold River Road in Rutland Town. Police said someone threw a brick through the window of Hadleys home and stole some jewelry.
Around 10 a.m. Monday, state police responded to the home of Donna Smith on Lincoln Hill Road in Shrewbury to investigate the theft of antique sterling silverware.
A Danby homeowner returned home Friday to learn she had been the victim of a burglary, police said. Sometime between Feb. 14 and Friday, police said,someone broke into the residence of Jadalyn V. Smietana on Danby-Pawlet Road and stole 200 feet of copper piping.
Anyone with information about these crimes can call Vermont State Police at 773-9101. Tips can also be submitted anonymously online at www.vtips.info.MORE IN This Just InKABUL, Afghanistan Confusion reigned in the wake of the deadly bombing Saturday of a hospital... Full StorySPRINGFIELD A fast-moving fire destroyed a freestanding two-bay garage on 432 South St. Full StoryIt will be a busy week in gubernatorial politics with two candidates holding formal campaign... Full Story
- Most Popular
- Most Emailed


text:
Skip to: Main Content / Navigation
You are here:
New Search
Web: http://www.fieldwork.com
Company Address:
2030 Main St.
Suite 300
Irvine, CA 92614
View Map
Phone: 949-252-8180
Fax: 949-252-1661
Send E-mail: E-mail this company
Kami Celano, President
Extensively remodeled in 2013, Fieldwork OC is the hottest place to be in Southern Cal. Come see our beautiful new creative spaces, including rooms with floor-to-ceiling mirrors for an unmatched viewing experience. Suites of various sizes can accommodate a range of people and methodologies. Only five minutes from John Wayne/Orange County airport, Fieldwork OC has quick access to fine restaurants, shopping, the beach and Disneyland. E-mail us your research requirements and take a vacation while you're here. Capabilities include: digital audio recording, DVD recording, digital video upload, video streaming, usability lab, high-speed wireless Internet, transcription services.
Fieldwork Los Angeles is located in sunny Orange County, only five minutes from John Wayne/Orange County airport. Completely remodeled in 2013, the facility has creative spaces and features such as floor-to-ceiling mirrors to make the most of your research experience. The five large conference rooms have viewing rooms that comfortably accommodate up to 50 clients, plus separate client lounges. All with top-ranked recruiting, hosting and project management. E-mail us your research requirements today. And take a vacation while you're here! Capabilities: digital audiorecording (complimentary), DVD recording, digital video upload, videostreaming, usability lab, high-speed wireless Internet, transcription services.
1 miles
5 minutes
Office building
Include this firm in my Request For Proposal (bid request).
These companies, organizations and individuals have opted to be listed in this Directory. Quirk's does not endorse these companies, organizations and individuals and makes no representations, warranties or guarantees as to, and assumes no responsibility for, the products or services provided by them. Quirk's expressly disclaims all liability for damages of any kind arising out of the use or performance of the products or services provided by those listed.
New Search
4662 Slater Road, Eagan, MN 55122 651-379-6200
Copyright 1986-2015 Quirk Enterprises, Inc.


text: Emma Grattan | IBDB: The official source for Broadway Information IBDB: The official source for Broadway Information Official Broadway League sites: - sites - broadway.org broadwayleague.com broadwayfanclub.com kidsnightonbroadway.com stagespecs.org tonyawards.com Quick Search For: In This Category: All Show People/Org. Theatre Season Emma Grattan Performer Female Productions ProductionsDate of ProductionsChow Chow[Musical, Burlesque, Extravaganza, Original] Starring: Emma Grattan Sep 09, 1872 - Oct 05, 1872The Forty Thieves[Musical, Burlesque, Extravaganza, Spectacle, Original] Starring: Emma Grattan Feb 01, 1869 - May 28, 1869 Find out where Emma Grattan and are credited together © 2001-2015, The Broadway League, All Rights Reserved. (Copyright and Disclaimer)


text:
We're sorry. The information you have requested is temporarily unavailable.
Banks AutoMind Programmer - 66104
Part Discontinued
This part has been discontinued by Banks . We apologize for the inconvenience.View all options for this product
Have a Question?
Have a Question? Ask a product expert! (800) 874-8888 Live Chat
Popular Searches
- banks diesel
- banks iq review
- banks six gun diesel tuner and banks iq
- automind 66105
- 2012 mercedes benz chip
- best ford diesel tuner
- jeep performance tuners
- performance programmers for ford f-150
- 98 dodge dakota gas mileage
- 04 ford f250 sct livewire buy
- suspension parts
- suv vehicle mats
- tent for back of truck


text:
Your cart is empty!
Chinese Red Ginseng generally recommended for yin situations, such as weakness due to illness or injury, lack of strength or stamina, coolness, poor circulation, vegetarian diet, sluggishness and for improved athletic performance
Please read all label information carefully on delivery and prior to use.
I routinely use this product, these are very high quality. been using over the counter drugs with uncomfortable side effects. I definitely recommend this product!
Centrum Silver Ultra Mens Tablets - 200 Ea
Our Price:$19.99
Cerave Moisturizing Skin Cream - 16 Oz
Our Price:$13.07
Mason Natural Korean Ginseng 518 Mg White Panax Ginseng Root Capsules - 100 Ea
Our Price:$5.10
Prima Care Medical Aneroid Self-Taking Kit Manual Blood Pressure Monitor For Adults - 1 Kit
Our Price:$13.81
White Cloverine Salve Tins - 1 Oz, 3 Pack
Our Price:$10.07


text:
106 8th Avenue South
Clinton, IA 52732
Sunday closed (show)
Monday 8:00am - 5:00pm
Tuesday 8:00am - 5:00pm
Wednesday 8:00am - 5:00pm
Thursday 8:00am - 5:00pm
Friday 8:00am - 5:00pm
Saturday 8:00am - 1:00pm
Sunday closed (hide)
Billboard - The latest coupons and news on this business!
Call Today for our Latest Specials!
We Pay Top Prices And Will Beat Our Competitions Offers!
|MON 8:00am - 5:00pm
|TUE 8:00am - 5:00pm
|WED 8:00am - 5:00pm
|THURS 8:00am - 5:00pm
|FRI 8:00am - 5:00pm
|SAT 8:00am - 1:00pm
|SUN closed
Category: Salvage and Surplus|Sanitation|Recycling
Service Area: Serving: Sterling IL (Metro), Scott County IA, Davenport IA, Bettendorf IA, Clinton IA, Cedar Rapids IA (Metro), Clinton IA (Metro), Whiteside County IL
Service: Recycling Collection, Recycling Centers
Material: Appliance Recycling, Battery Recycling, Scrap Metal, Aluminum, auto batteries, brass, copper, scrap iron, stainless steel aluminum siding
Type: Commercial Recycling, Industrial Recycling
Service: Trash Cleanup, Trash Hauling, Dumpsters
Type: Home Sanitation, Commercial Sanitation, Industrial Sanitation
Specialty: Junk and Scrap, Industrial Salvage, Appliance Salvage


text:
The LCA-MA 2008 Report Card on Lung Cancer is widely distributed among public policy leaders, medical professionals and health care associations working to improve outcomes for lung cancer.
For more information on the services provided by Lung Cancer Alliance or to view the complete LCA-MA 2008 Report Card on Lung Cancer visit www.lungcanceralliance.org
Lung Cancer Alliance, www.lungcanceralliance.org, is the only national non profit dedicated exclusively to patient support and advocacy for those living with or at risk for lung cancer. LCA is committed to reversing decades of stigma and neglect by empowering those with or at risk for lung cancer, elevating awareness, and changing health policy.
Media Contact: Joanne O'Connor email@example.com 202-463-2080
|SOURCE Lung Cancer Alliance
Copyright©2008 PR Newswire.
All rights reserved


text:
2016 memberships are now OPEN. Sign up below!
View 2016 membership fees and ad rates
Sign up right here:Sign-up for a new account here!
Search for an Associate Partner:
2016 memberships are now OPEN. Sign up below!
View 2016 membership fees and ad rates
Sign up right here:Sign-up for a new account here!
Search for an Associate Partner:
Receive a listing in the Indiana Festival Guide and on IndianaFestivals.org, the website for travelers. Network and receive valuable info at the annual workshops and convention. Apply for an ISFA grant and much more!
Sign up at the top of this page and become a member today!
ISFA Board meetings are open to guests and members to attend.
Sunday, Nov 15 - 2pm at Marriott East Indianapolis (7202 E 21st St, Indpls)
Just Published! A manual to help you plan every aspect of your event is a new resource presented by the Indiana Office of Tourism Development and the Indiana State Festivals Association.
The Indiana State Festivals Association is the state's largest membership organization for festivals and events. ISFA publishes the Indiana Festival Guide each year and maintains this website featuring searchable festival listings.
Over 550 festivals and events receive promotion, leadership and education opportunities as members of ISFA.
Visit ISFA's website for travelers, Indiana's best online resource for festival info! Over 550 searchable festival and event listings.
Now's the time to purchase your 2016 ISFA membership and secure your spot in the ALL-COLOR 2016 Bicentennial edition of the Indiana Festival Guide! Login or create a new account above to get started. Also, there are opportunities to add-on a graphic to your Festival Guide listing or place a display ad. There are more options to promote your event than ever before!
Young women who represent their local ISFA member festival can compete in the ISFA scholarship pageant in January. Scholarships of $3,000 will be awarded!
Click below for info and application.
Deadline to apply for the pageant is in the fall each year. Check back, the 2015 application will be available soon.
Join the conversation! The ISFA Discussion Board is now open. It's the place to share your questions, comments and expertise.
The Ann Retseck Memorial Grant makes attendance to the annual ISFA Fall Convention possible for ISFA members who need assistance in paying their representatives' costs.
This grant is dedicated to Ann Gettinger Retseck for her donation of time and effort into her community's festival, the Merom Bluff Chautauqua.
The Charlene "Shorty" Harrell Memorial Grant makes attendance to the annual ISFA Fall Convention possible for ISFA members whose festival budget is less than $10,000.
This grant is dedicated to Charlene "Shorty" Harrell of the Lagro Good Ole Days Festival, who died on November 9, 2012 while attending the annual ISFA Fall Convention.
To request that the ISFA queen make an appearance at your festival, contact Chris Benson or call at 260-569-9391
Signing up will be completed in just a few easy steps! At the end of the form, you can select which listing type you would like to purchase. The annual cost ranges from $225-$525. All listing types need to complete this form.


text:
Doud Drexler and startup
View Single Post
October 4 2012, 03:33 PM
Location: Staten Island, NY
Doud Drexler and startup
Does anyone know how to reach him. I have a sci-fi franchise pilot he might be interested in. I see why he wasn't interested in the design aesthetics of Space Command. I don't like it either and I believe that along with the music is very important to the visual experience. Is it going to make money? If getting a few big people involved can net a quarter million and sidestep network interference, it's worth it for the integrity of the show and those involved else we get ringleaders like JJ Adams, er, Abrams and his sadistic predatorial lot or more Bermans. There's plenty of them. It's either a suit or a son of a suit. Take your pick.
View Public Profile
Send a private message to xortex
Find More Posts by xortex


text:
a little portrait session for shits and gigs next to my place. it's so important to experiment and try new things
I had the opportunity to photograph these lovely humans for quick shoot around this gorgeous 12th century city in the
I love when a bride & groom are willing to get back into their wedding attire for some photos! There is no stress
What do you get when 6 photographers head out to the PNW on an adventure, a kick ass shoot filled with gorgeous
This was one of my favorite weddings of the year! It was surrounded by love, family, laugher & more love. It was
Congratulations Tyler & Christina!! Tina, it's such an honor to know you & to call you my best friend
This couple deserves an award! We met up in Big Bear on a stormy afternoon in November. It was freezing out
This wedding was an adventure! First of all, just to get to the wedding location it took a plane, train &
∆ available worldwide ∆
e. firstname.lastname@example.com
p. 949 | 374 3124
© 2014 Nicole Leever Photography|ProPhoto Blogsite | La Lune Creative


text:
Ohio Farm Bureau Statement on approval of Free Trade Agreements
Ohio Farm Bureau Federation thanks members of Ohio's congressional delegation who voted to approve Free Trade Agreements with South Korea, Columbia and Panama.
Reporters, please visit our news room located in the Media and Publications section of this site.
Ohio Farm Bureau Federation thanks members of Ohio's congressional delegation who voted to approve Free Trade Agreements with South Korea, Columbia and Panama.
Multiple $1,000 to $3,000 grants support porgrams and projects focusing on agricultural education, environmental and/or economic development in Ohio communities. Forms must be received by Nov. 15.
Ohio Farm Bureau Foundation, Animals for Life Foundation, Ohio Center on Agricultural Law hosting
online auction during the month of November, with a live bidding finale during Ohio Farm Bureau Federations Annual Meeting in Columbus Nov. 30.
Asked about his philosophy on the role of government during a recent taping of Ohio Farm Bureaus radio show 'Town Hall Ohio,' Ohio Gov. John Kasich said government should do what people cant do for themselves.
American Farm Bureau is asking Congress to spread farm bill cuts across key program areas. The organizations proposal represents a balance of multiple commodity and regional interests.
About 30 young agricultural professionals, Ohio Farm Bureau staff and board members went on the three-day trip in September to learn how the political process works in D.C.
Ohio Farm Bureau is pleased with a recent ruling from the Ohio Supreme Court that upholds the private property rights of landowners along Lake Erie.
When it comes to transition planning, farmers face some unique challenges and need to take proactive steps to help ensure that their legacy is protected.
Ohios top conservation farm families for 2011 were honored for their long-standing dedication to natural resource conservation on the land they farm during ceremonies at the Farm Science Review.
A new Ohio Farm Bureau staff team is helping give members the leadership tools they need to help be better leaders and manage their busy lives more effectively.


text: A CENTURY OF NCIS: On Tuesday, September 4, 2007, the cast of NCIS gathered to celebrate the start of production on the hit series' 100th episode. From left: David McCallum, Lauren Holly, Pauley Perrette, Sean Murray, Cote de Pablo, Brian Dietzen, Mark Harmon and Michael Weatherly. CLIFF LIPSON / CBS


text: Andy Lyons/Getty Images Sport Minnesota Gives Richard Pitino $400K Annual Raise, 2-Year Extension By Associated Press August 26, 2015 | ESPN 99.1 Minnesota has finalized a two-year contract extension for coach Richard Pitino through the 2020-21 season, including a $400,000 raise. Read More|Comment Category: Homepage, Sports Tags: Minnesota Golden Gophers, NCAA, Regional Sports Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!