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Caspase-9 activation is critical for intrinsic cell death. The activity of caspase-9 is increased dramatically upon association with the apoptosome and the apoptosome bound caspase-9 is the caspase-9 holoenzyme (C9Holo). In this study, we use quantitative enzymatic assays to fully characterize C9Holo and a leucine-zipper linked dimeric caspase-9 (LZ-C9). We surprisingly show that LZ-C9 is more active than C9Holo for the optimal caspase-9 peptide substrate LEHD-AFC, but is much less active than C9Holo for the physiological substrate pro-caspase-3. The measured Km values of C9Holo and LZ-C9 for LEHD-AFC are similar, demonstrating that dimerization is sufficient for catalytic activation of caspase-9. The lower activity of C9Holo against LEHD-AFC may be attributed to incomplete C9Holo assembly. However, the measured Km of C9Holo for pro-caspase-3 is much lower than that of LZ-C9. Therefore, in addition to dimerization, the apoptosome activates caspase-9 by enhancing its affinity for pro-caspase-3, which is important for pro-caspase-3 activation at the physiological concentration.
Caspase activation is a hallmark of apoptotic cell death (Riedl and Shi, 2004; Salvesen, 2002). According to their sequence of activation, caspases may be divided into two groups: initiator caspases such as caspase-8, 9 and 10, and effector caspases such as caspase-3 and 7 (Salvesen, 2002; Shi, 2002). In the classical paradigm of apoptosis, there are two general pathways for caspase activation and apoptosis induction (Danial and Korsmeyer, 2004). In the extrinsic apoptosis pathway, death ligands interact with and activate death receptors, leading to the assembly of the death inducing signaling complex (DISC) that activates caspase-8 and caspase-10. In the intrinsic apoptotic pathway, cellular insults lead to mitochondrial alteration and cytochrome c release. In the presence of cytochrome c and dATP or ATP, the adapter protein Apaf-1 assembles into an oligomeric structure known as the apoptosome, which recruits and activates caspase-9 (Li et al., 1997; Zou et al., 1997). Caspase-8, 9, and 10 then activate effector caspases such as caspase-3 and 7, which cleave a wide range of substrates to execute apoptosis.
Caspases are synthesized as single chain pro-caspases, which undergo intra-chain cleavage to generate the large and small subunits. The molecular requirements for caspases to be catalytically active have been revealed by previous elegant structural studies on active and inactive caspases (Chai et al., 2001; Riedl et al., 2001; Wei et al., 2000). First, a caspase needs to be dimeric to be active because regions from both its own chain and the neighboring chain form the caspase active sites. Specifically, these regions include the L1 and L2 loops of the large subunit and the L3 and L4 loops of the small subunit of one chain and the L2′ loop of the small subunit of the neighboring chain. Second, intra-chain cleavage facilitates formation of the active site because the L2 and L2′ loops are released upon cleavage to become part of the loop-bundle that supports the active site. This intra-chain cleavage may also stabilize dimerization of some caspases (Boatright et al., 2003; Donepudi et al., 2003). The requirement for cleavage differs among caspases in a manner that appears to depend on the length of the L2-L2′ linker region.
Because effector caspases are constitutive dimers, their activation is strictly a consequence of intra-chain cleavage by initiator caspases. In contrast to effector caspase activation, intra-chain cleavage does not appear to be the crucial factor for initiator caspase activation (Boatright et al., 2003; Donepudi et al., 2003; Srinivasula et al., 2001; Stennicke et al., 1999). For example, caspase-9 can be activated without proteolytic processing. Processed caspase-9 alone is not much more active than pro-caspase-9. A small population of dimerized uncleavable caspase-8 is as active as processed caspase-8.
An induced proximity model for initiator caspase activation was initially proposed from observations that initiator caspases fused to heterologous dimerizing partners underwent processing and induced cell death (MacCorkle et al., 1998; Muzio et al., 1998; Srinivasula et al., 1998; Yang et al., 1998a; Yang et al., 1998b). This model is consistent with the presence of prodomains in initiator caspases that participate in the assembly of oligomeric complexes in an apoptotic signal dependent fashion. In this model, the pro-form of an initiator caspase has low activity that is sufficient for autocatalytic processing when brought into close proximity in these complexes. More recent biochemical studies have led to a revised proximity-induced dimerization model for initiator caspase activation because caspase-8 and 9 are not constitutive dimers in solution and specific homo-dimerization appears to be crucial for their activation (Boatright et al., 2003; Donepudi et al., 2003; Renatus et al., 2001).
For caspase-9, its activity is increased dramatically (~103 fold) upon association with the apoptosome and the apoptosome bound caspase-9 is the caspase-9 holoenzyme (C9Holo) (Jiang and Wang, 2000; Rodriguez and Lazebnik, 1999; Srinivasula et al., 2001). Based on the proximity-induced dimerization model, the function of the apoptosome is to promote homo-dimerization of caspase-9 and therefore its activation. In the context of the cryo-EM structure of the heptameric apoptosome, this homo-dimerization could occur among caspase-9 chains bound on the apoptosome or the apoptosome-bound caspase-9 may dimerize with additional caspase-9 monomers from the solution (Acehan et al., 2002; Shi, 2004; Yu et al., 2005).
Although dimerization does enhance caspase-9 activity, it is not known whether dimerization is sufficient for caspase-9 activation. To address this question, a dimeric form of caspase-9 needs to be constructed and its activity needs to be quantitatively compared with C9Holo. In an effort to answer this question, a constitutive caspase-9 dimer was engineered by replacing the dimerization residues of caspase-9 with those from caspase-3 (Chao et al., 2005). This engineered caspase-9 dimer is more active than caspase-9, but much less active than C9Holo. Therefore, it was argued that dimerization of caspase-9 is not sufficient for its activation (Chao et al., 2005).
Because replacing residues at the dimerization interface may have compromised the catalytic activity of the engineered caspase-9 dimer, we took an alternative approach to address whether dimerization is sufficient for caspase-9 activation. We replaced the pro-domain of caspase-9 (caspase recruitment domain, CARD) with the leucine-zipper dimerization domain of GCN4 (Hu et al., 1990) to promote caspase-9 homo-dimerization through its intrinsic weak dimerization interface (LZ-C9). We surprisingly found that LZ-C9 is not less active, but more active than C9Holo in cleaving the tetrapeptide substrate LEHD-AFC. The measured Km values of LZ-C9 and C9Holo for LEHD-AFC are similar. The lower activity of C9Holo against LEHD-AFC may be attributed to incomplete C9Holo assembly. This establishes that dimerization is sufficient for catalytic activation of caspase-9. In contrast, LZ-C9 activity for pro-caspase-3, the physiological substrate of caspase-9, is much lower than C9Holo. This inverse activity of LZ-C9 and C9Holo for LEHD-AFC and pro-caspase-3 is entirely unexpected. It is not due to feedback cleavage of caspase-9 by caspase-3 or promotion of processed caspase-3 activity by C9Holo. Instead, Km determination reveals that the Km of C9Holo for pro-caspase-3 is much lower than that of LZ-C9, suggesting that apoptosome activates caspase-9 by enhancing its affinity to pro-caspase-3. This affinity enhancement is important for efficient pro-caspase-3 activation at the low physiological concentration and makes C9Holo a specific and optimal pro-caspase-3 processing machine. Collectively, these studies provide unforeseen insights into the molecular basis of caspase-9 activation by the apoptosome.
To determine whether caspase-9 dimerization is sufficient for its activation, we removed the CARD of pro-caspase-9 and replaced it with the leucine zipper dimerization domain of the transcription factor GCN4. A six residue linker was added in between the leucine zipper and the beginning of the catalytic domain of caspase-9 (LZ-C9, residues 151–416, Figure 1A). In the crystal structure of dimeric CARD-deleted caspase-9 (Renatus et al., 2001), residues L151 at the beginning of the two large subunits in the caspase-9 dimer reside at the same surface of the molecule with a Cα distance of 21 Å. In the crystal structure of the GCN4 leucine zipper, the last two residues are disordered and the distance between the last ordered residue and its symmetry mate is 13 Å (O'Shea et al., 1991). A minimal distance of 4 Å is required to bridge each end of the parallel leucine zipper to L151 of caspase-9. Because the two disordered leucine zipper residues and a linker of 6 residues could stretch to approximately 30Å, the LZ-C9 construct should be sufficient to allow intrinsic caspase-9 dimerization to occur without any constraints, as also shown in a molecular model of LZ-C9 we constructed (Figure 1B).
The calculated molecular weight of LZ-C9 is 34.7 kD. It elutes as a single peak at 14.6 ml in a Superdex 200 HR 10/300 gel filtration column, suggesting that it is a dimer (Figure 1C). Higher order oligomerization was not observed. In contrast, CARD deleted caspase-9 (C9ΔCARD) with a calculated molecular weight of 32.7 kD elutes at 15.5 ml in the same gel filtration column, which is consistent with the monomeric state of caspase-9. Since gel filtration column elution positions may be influenced by both mass and shape, we used static multiangle light scattering (MALS) in conjunction with refractive index measurements to determine the molecular mass of intact LZ-C9. Consistent with the gel filtration data, MALS measurements gave a molecular mass of 67.6 kD (0.7% fitting error) with a polydispersity of 1.000, confirming the dimeric state of LZ-C9 (Figure 1D). In contrast, MALS analysis showed that C9ΔCARD is a monomer with a measured molecular mass of 35.6 kD (0.7% fitting error) and a polydispersity of 1.000.
We used the fluorogenic peptide substrate LEHD-AFC, which is an optimal peptide substrate for caspase-9 (Thornberry et al., 1997), to compare the activity of LZ-C9 with C9Holo. Because intra-chain cleavage does not play a significant role in caspase-9 activation, both caspase-9 and LZ-C9 we used are the processed forms. Unexpectedly, at the same caspase-9 concentrations, LZ-C9 gave a significantly faster rate of substrate cleavage than C9Holo (Figure 2A).
Because the physiological substrate of caspase-9 is pro-caspase-3, we measured the activity of LZ-C9 and C9Holo for pro-caspase-3 activation. An indirect fluorogenic assay was used, in which cleavage of the caspase-3 substrate DEVD-AMC by caspase-9 activated caspase-3 was measured. In contrast to the higher activity of LZ-C9 for the LEHD-AFC peptide substrate, C9Holo is much more efficient in activating pro-caspase-3 than LZ-C9 (Figure 2B).
Since there are approximately 50 additional residues between the CARD and the beginning of the catalytic domain, we also constructed a longer LZ-C9 construct that includes this additional region (LZ-C9L, residues 98–416). MALS analysis showed that LZ-C9L, with a calculated molecular weight of 40.3 kD, is also a dimer with a measured molecular mass of 79.1 kD (2.0% fitting error) and a polydispersity of 1.000. We tested the activity of LZ-C9L against LEHD-AFC and pro-caspase-3. In comparison with C9Holo, LZ-C9L is more effective in cleaving LEHD-AFC, but is much less active against the physiological substrate pro-caspase-3 (Figure 2C, 2D). Therefore, LZ-C9 (or LZ-C9L) and C9Holo exhibit an inverse capability in cleaving the LEHD-AFC substrate and the physiological substrate pro-caspase-3.
This inverse activity of LZ-C9 and C9Holo for LEHD-AFC and pro-caspase-3 is intriguing. The human caspase-9 self-mediated intra-chain cleavage occurs at Asp 315 (sequence 312PEPD315) to generate the large (p35) and small (p12) caspase-9 subunits. However, it has also been shown that activated caspase-3 can further feedback cleave the N-terminus of the small subunit of caspase-9 at Asp330 (sequence 327DQLD330) and enhance its activity (Zou et al., 2003). Therefore, we wondered if this feedback cleavage could contribute to the observed difference between C9Holo and LZ-C9 towards the two different substrates.
We mixed the cell pellets of caspase-9 and LZ-C9 with those of non-tagged caspase-3 and purified the truncated caspase-9 forms (t-caspase-9 and t-LZ-C9) that have their p12 converted to p10 by the additional cleavage at Asp330. We then repeated the above experiments to determine whether our observations still hold true for these truncated caspase-9 forms (t-C9Holo and t-LZ-C9). Measurement of LEHD-AFC cleavage in the direct fluorogenic assay and pro-caspase-3 activation in the indirect fluorogenic assay showed that the inverse activity of t-LZ-C9 and t-C9Holo for these substrates is preserved (Figure 2E, 2F) Therefore, caspase-3 mediated cleavage of caspase-9 does not explain this discrepancy in capability between LZ-C9 and C9Holo for LEHD-AFC and pro-caspase-3.
We then examined whether the high efficiency of pro-caspase-3 activation by C9Holo is due to potential promotion of processed caspase-3 catalytic activity, making C9Holo processed caspase-3 a caspase-3 holoenzyme. To determine this, we first examined whether caspase-3 can stay associated with C9Holo after processing. We mixed Apaf-1, cytochrome c, dATP, full-length caspase-9 and excess pro-caspase-3 and subject the mixture to gel filtration chromatography. All pro-caspase-3 was cleaved. Indeed, some processed caspase-3 was associated with C9Holo in the early elution fractions (Figure 3A).
We then measured the DEVD-AMC cleavage activities of caspase-3 that was either co-eluted with C9Holo or eluted alone to determine whether C9Holo-associated caspase-3 exhibits higher activity. The experiment showed that similar protein levels of caspase-3 in either fraction gave similar catalytic activities (Figure 3B, compare fractions 4 and 9). Serial dilution of a high concentration caspase-3 fraction to match the caspase-3 level in the C9Holo fraction confirmed the same observation (Figure 3C, compare fraction 4 with 4-fold and 8-fold diluted fraction 12). Similar experiments replacing C9Holo with LZ-C9 showed the absence of caspase-3 protein or activity in the early elution fractions (Figure 3D). These experiments demonstrate that C9Holo-associated caspase-3 does not exhibit higher catalytic activity to cause the observed difference in LZ-C9 and C9Holo against LEHD-AFC and pro-caspase-3.
To further characterize the catalytic parameters of LZ-C9 and C9Holo, we measured the initial rates of substrate cleavage at different concentrations of LEHD-AFC, from which the Km values of LZ-C9 and C9Holo for the peptide substrate were determined. The Km values of LZ-C9 and C9Holo for LEHD-AFC are similar, 465.7 ± 118.4 μM and 686.2 ± 136.9 μM, respectively (Figure 4A–D, Table 1). The Km values for the feedback cleaved t-LZ-C9 and t-C9Holo are 260.0 ± 22.84 μM and 276.5 ± 65.48μM, respectively (Figure 4E–H, Table 1). They are high in comparison with the Km values of other caspases for their peptide substrates (Zhou et al., 1998).
Since caspase-9 and Apaf-1 need to be assembled into the large multimeric caspase-9 holoenzyme for caspase-9 activation, we asked whether the observed lower activity of C9Holo for LEHD-AFC is due to incomplete apoptosome and C9Holo assembly and hence lower effective concentration of C9Holo. Alternatively, a lower intrinsic turnover rate, kcat, of C9Holo, can also contribute to the observed lower activity. To distinguish these two possibilities, we titrated excess Apaf-1 into caspase-9 to facilitate assembly of C9Holo. This titration increased the rate of LEHD-AFC cleavage when caspase-9 concentration remained constant (Figure 4I). At the highest Apaf-1 concentration we could provide, the activity of C9Holo did not catch up with the activity of LZ-C9. These data suggest that holoenzyme assembly is a limiting factor contributing to the lower activity of C9Holo to the LEHD-AFC substrate. The data also suggest that the observed striking difference between C9Holo and LZ-C9 in activating pro-caspase-3 (Figure 2B) is still an underestimation because of incomplete holoenzyme assembly. On the other hand, it is likely that the catalytic activities of C9Holo and LZ-C9, exemplified in Km and kcat for LEHD-AFC, are equivalent, Thus dimerization appears to be sufficient for catalytic activation of caspase-9.
Then why does C9Holo activate caspase-3 much more efficiently than LZ-C9? Is it possible that C9Holo possesses a higher affinity for pro-caspase-3 than LZ-C9? To examine this, we measured the rates of substrate cleavage at different concentrations of pro-caspase-3 to determine the Km values of C9Holo and LZ-C9 for pro-caspase-3. We used the indirect fluorogenic assay, in which cleavage of the caspase-3 substrate DEVD-AMC was measured. The slope of the DEVD-AMC cleavage at a given time point is indicative of the active casapse-3 concentration at that time point and was used to derive Km values for the pro-caspase-3 cleavage reaction. Surprisingly, the Km of C9Holo for pro-caspase-3 is 139.3 ± 23.19 nM (Figure 5A–B, Table 1), about three orders of magnitude lower than that for LEHD-AFC. In contrast, the Km of LZ-C9 for pro-caspase-3 was estimated to be much higher; at the highest possible concentration of pro-caspase-3 we could supply, the reaction did not show any sign of saturation (Figure 5C-D). This difference in Km values between C9Holo and LZ-C9 is also exemplified in the much higher effect of increasing pro-caspase-3 concentration on the ability of LZ-C9 than on the ability of C9Holo to activate pro-caspase-3 (Figure 5A, 5C).
Similarly, we showed that the Km value of t-C9Holo for pro-caspase-3 is 79.8 ± 18.5 nM (Figure 5E–F, Table 1), similar to its non-truncated counterpart. These data demonstrate that both C9Holo and t-C9Holo have high affinity to pro-caspase-3. Therefore, we show unexpectedly that one important mechanism of caspase-9 activation by the apoptosome is by enhancing its affinity to the physiological substrate pro-caspase-3.
Caspase-9 is unusual in that it is only active when bound to the apoptosome. Such activation is so dramatic that the apoptosome-bound caspase-9 is known as the caspase-9 holoenzyme (Rodriguez and Lazebnik, 1999; Srinivasula et al., 2001). Other caspases, including caspase-8, an initiator caspase in the extrinsic apoptosis pathway, can directly activate their substrates without the requirement to be bound to an accelerator. Our data on the catalytic properties of LZ-C9 and C9Holo show that caspase-9 dimerized by a leucine zipper is not less active, but more active than the caspase-9 holoenzyme. This demonstrates that dimerization is sufficient for catalytic activation of caspase-9.
The measured Km values of caspase-9 holoenzyme and LZ-C9 for LEHD-AFC are similar. They are also similar to the previously reported Km values of caspase-9 for LEHD-AFC (Renatus et al., 2001; Zou et al., 2003). How about the intrinsic turnover rate of caspase-9 holoenzyme in comparison with LZ-C9? The similar Km but lower activity of the caspase-9 holoenzyme could indicate that it possesses a lower kcat than LZ-C9 for LEHD-AFC. However, because the apoptosome might also mediate caspase-9 dimerization, it is unlikely that its kcat is lower than that of leucine-zipper dimerized caspase-9. Since caspase-9 holoenzyme requires assembly, a more plausible explanation to the lower activity is that the effective concentration of C9Holo is lower than its apparent concentration. This is supported by the increase of activity upon titration of more Apaf-1. Therefore, we conclude that the catalytic activity of LZ-C9 is equivalent to that of caspase-9 holoenzyme for peptide substrate LEHD-AFC. Our data contradict the observation from an engineered dimeric caspase-9 made by replacing the weak dimerization residues of caspase-9 with the strong dimerization residues of caspase-3 (Chao et al., 2005). The difference may be that LZ-C9 likely promotes dimerization through its intrinsic dimerization interface, while the altered dimerization interface in the engineered dimer may have affected its catalytic ability, even against LEHD-AFC.
Although dimerization appears to be sufficient for establishing the catalytic competency of caspase-9, it is not sufficient for the high efficiency of caspase-9 holoenzyme to support pro-caspase-3 activation because LZ-C9 is much less efficient in pro-caspase-3 activation. Our data indicate that pro-caspase-3 is a much better substrate for the caspase-9 holoenzyme with dramatically increased affinity. Therefore, apoptosome activates caspase-9 by both dimerization and enhanced affinity to its physiological substrate, pro-caspase-3.
This affinity enhancement is important if the endogenous concentration of pro-caspase-3 in cells is low, for example, around the Km of C9Holo, but much below the Km of LZ-C9. Indeed, previous reports have shown this to be the case. Using quantitative Western analysis, the approximate concentration of pro-caspase-3 in 293 cells was found to be around 100 nM (Stennicke et al., 1998), which is similar to the Km of C9Holo for pro-caspase-3. Independently, caspase-3 concentration in Jurkat T cells was estimated to be below 100 nM (Pop et al., 2001; Saunders et al., 2000). At this pro-caspase-3 concentration, only C9Holo, but not dimerized caspase-9, can efficiently cleave pro-caspase-3 substrate. The small yet likely significant reduction in the Km of C9Holo for pro-caspase-3 by the feedback cleavage of caspase-9 (Table 1) may further enhance the ability of C9Holo to process pro-caspase-3 at the physiological concentration.
In the intrinsic apoptosis pathway, C9Holo appears to have a strict specificity for pro-caspase-3 and its relative pro-caspase-7. Other caspase-9 substrates have not been reported. This situation differs from the initiator caspase in the extrinsic apoptosis pathway, caspase-8, which cleaves pro-caspase-3 as well as BID, a BH3 only Bcl-2 family member (Li et al., 1998; Luo et al., 1998).
This strict specificity may be conferred by association of caspase-9 with the apoptosome in the form of C9Holo. In contrast, caspase-8 gets released upon activation from the death inducing signaling complex (Peter and Krammer, 2003). The intrinsic substrate specificity of caspase-9, as determined by its specificity for four-residue peptide sequences, is not stricter than caspase-8 or any other caspases (Thornberry et al., 1997). However, because the Km of caspase-9 or C9Holo for these peptides are unusually high, caspase-9 is essentially inactive against potential substrates. Only in the form of C9Holo, caspase-9 has an enhanced affinity specifically for pro-caspase-3, which makes C9Holo a specific processing machine for pro-caspase-3. Therefore, in addition to providing higher affinity for pro-caspase-3, association of caspase-9 with the apoptosome might also ensure its specificity for pro-caspase-3.
So what are the possible mechanisms for the enhanced affinity of caspase-9 holoenzyme for pro-caspase-3? First, the apoptosome may directly interact with pro-caspase-3 and therefore enhances the affinity of bound caspase-9 to pro-caspase-3 (Figure 6A). In this model, the apoptosome plays a direct contact role in pro-caspase-3 recruitment. Alternatively, the apoptosome may induce conformational changes in bound caspase-9 and therefore increases its affinity to pro-caspase-3 (Figure 6B). The conformational changes may possibly lie outside the immediate active site, as it does not affect the cleavage of LEHD-AFC.
It is also possible that this enhanced affinity may be due to avidity by a simultaneous recognition of two cleavage sites from two adjacent caspase-9 dimers (Figure 6C). The rationale is that in the platform of the oligomeric apoptosome, C9Holo may simultaneously interact with both cleavage sites on pro-caspase-3, leading to enhanced affinity via avidity. In contrast, LZ-C9, with its single productive active site (Chao et al., 2005; Renatus et al., 2001), cannot gain affinity through this avidity. We have tried to test this hypothesis by linking two active site mutant (C163A) of pro-caspase-3 sequences in tandem via a flexible covalent linker [1-277- (GS)7-1-277]. This single-chain pro-caspase-3-C163A construct harbors the entire heterotetrameric sequence of pro-caspase-3 and contains two caspase-9 cleavage sites. We then mutated the second caspase-9 cleavage site in this single chain pro-caspase-3 construct (D175A) to generate a pro-caspase-3 dimer with a single caspase-9 recognition sequence. However, the cleavage activities of C9Holo against the single chain wildtype or D175 mutant did not show dramatic differences (data not shown). It is possible that the lack of dramatic differences may also be due to the incomplete destruction of the second caspase-9 recognition site by the single D175A mutation. A more stringent test on this hypothesis is to generate monomeric pro-caspase-3. But previous (Bose and Clark, 2001) as well as our own experiments (data not shown) have indicated that monomeric pro-caspase-3 mutant cannot be generated without greatly altering the structure of pro-caspase-3. Therefore, it remains to be seen whether the potential concerted recognition of two cleavage sites by C9Holo contributes to this high affinity.
Regardless of the exact mechanism, it is clear that C9Holo has dramatically increased affinity to pro-caspase-3 and therefore exhibits dramatically increased activity and specificity for this physiological substrate. This characteristic places caspase-9 as a single unique caspase in the entire caspase superfamily in that it defies the otherwise universal activation mechanism of cleavage and/or dimerization. While dimerization of caspase-9 transforms it into an active enzyme when using its optimal peptide as the substrate, the Km of the enzyme for the substrate is high (~10−3 – 10−4 M) compared to those of other caspases (~10−4 – 10−5 M) (Zhou et al., 1998). Therefore, caspase-9 requires the additional step of activation, the association into C9Holo, to be fully activated for processing its physiological substrate at the low physiological concentration (~100 nM), ensuring a precise and versatile regulation. In summary, our quantitative characterization of C9Holo and LZ-C9 provided unexpected insights into the molecular mechanism of caspase-9 activation by the apoptosome and adds to our understandings of caspase activation in general.
His-tagged full length human caspase-9, leucine-zippered caspase-9 (LZ-C9 and LZ-C9L) and CARD-deleted caspase-9 (C9ΔCARD, residues 139–416) were expressed in E. coli. LZ-C9 contains the initiation Met, leucine zipper sequence from GCN4 (251–281), a linker (GSGSGS) and the C-terminally His-tagged catalytic domain of human caspase-9 (151–416). LZ-C9L is similar to LZ-C9 except that it includes the additional region between the CARD and the catalytic domain of caspase-9 (98–416). Both LZ-C9 and LZ-C9L were purified by Ni-NTA affinity chromatography (Qiagen), anion exchange at pH 8.0 with HiTrap Q (GE Healthcare) and gel filtration chromatography with Superdex 200 HR 10/300 (GE Healthcare). The buffer for gel filtration contains 20 mM Tris at pH 7.5, 150 mM NaCl and 5mM DTT. To generate caspase-3 feedback-cleaved full-length caspase-9 and LZ-C9, their respective cell pellets were mixed with those of non-tagged caspase-3 and purified similarly. His-tagged Apaf-1 and pro-caspase-3 were expressed in the baculovirus-mediated insect cell expression system. They were purified by Ni-NTA affinity chromatography followed by gel filtration. Caspase concentrations were determined by both the Bradford Assay and active site titration as described previously (Stennicke and Salvesen, 2000; Zou et al., 2003), which agree well with each other.
A molecular model of LZ-C9 was constructed using the GCN4 leucine zipper coordinates (PDB ID 2ZTA) and a caspase-9 coordinates (PDB ID 2AR9). The eight residue linker comprising the two disordered residues in GCN4 and the GSGSGS sequence was built from an eight residue loop in PDB ID 1Z3G containing a similar sequence. Model building was done in O (Jones et al., 1991) and the ribbon diagram was produced in PyMOL (DeLano Scientific).
C9Holo and LZ-C9 activities were monitored by turnover of fluorogenic tetrapeptide substrates in both direct and indirect assays. Fluorescence measurement upon substrate cleavage was performed on SpectraMax M2 plate reader (Molecular Devices) using 384-well plates (Corning). All measurements were performed in 20 μL reactions in Buffer A (20 mM Hepes pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA and 1 mM DTT) at 37 °C. Reaction mixtures also contain 10 μM dATP (Sigma) and 500 nM bovine heart cytochrome c (1 μM if Apaf-1 concentration is higher than 200 nM, Sigma) when measuring C9Holo activity. In direct assay, C9Holo or LZC9 of indicated concentration was incubated at 37 °C for 5 min. The reactions were initiated by adding the substrate Ac-LEHD-AFC (100 μM, Calbiochem). Release of the AFC moiety was monitored at emission wavelength of 505 nm with the excitation wavelength of 400 nm. In indirect assay, C9Holo or LZ-C9 was incubated together with Ac-DEVD-AMC (15 μM) at 37°C for 5 min before pro-caspase-3 was added to initiate the reactions. The reactions were then followed at emission wavelength of 465 nm with excitation wavelength of 360 nm. Reactions were monitored at 10-second intervals. V0 was drawn from the initial phase of the progress curve. Km values were determined using nonlinear regression method to fit Michaelis-Menton equation.
Molar mass of purified LZ-C9, LZ-C9L and C9ΔCARD was determined by static multiangle light scattering (MALS). Protein was injected onto a Superdex 200 HR 10/300 gel filtration column equilibrated in a buffer containing 20mM Tris at pH 7.5, 150mM NaCl and 0.2mM DTT. The chromatography system was coupled to a three angle light scattering detector (mini-DAWNEOS) and refractive index detector (Optilab DSP) (Wyatt Technology). Data were collected every 0.5 s at a flow rate of 0.2 ml/min. Data analysis was carried out using the program ASTRA, yielding the molar mass and mass distribution (polydispersity) of the sample.
Excess pro-caspase-3 (2μM) was incubated with 1 μM Apaf-1, 2 μM cytochrome c, 10 μM dATP and 1 μM caspase-9 at 30°C for 30 min in a total volume of 600 μL. Reaction mixture was then fractionated on a Superdex 200 column pre-equilibrated with Buffer A at 4 °C. The column was eluted with Buffer A and fractions of 1 ml were collected. Aliquots of 2 μL of each fraction were assayed for their cleavage activity against DEVD-AMC as described. Aliquots of 10 μL of each fraction were resolved by 15 % SDS-PAGE followed by western blot analysis (see below). Alternatively, excess pro-caspase-3 was incubated with 1 μM LZ-C9 for 30 min at 30 °C. The mixture underwent the same fractionation and analyses as described above.
Proteins were transferred from SDS-PAGE gel to PVDF membrane using Trans-Blot SD (Bio-rad). After transfer, membrane was probed with either mouse monoclonal anti-caspase-3 (Cell Signaling) (1:1000) or polyclonal anti-Apaf-1 (1:1000). Washed blot was then incubated with horseradish peroxidase-conjugated horse anti-mouse (caspase-3) or goat anti-rabbit (Apaf-1) IgG. Reactive bands were visualized using SuperSignal West Pico Kit (Pierce) as instructed.
The work was supported by the National Institute of Health (R01 AI-50872). S.C.L and Y.C.L are Cancer Research Institute postdoctoral fellows..
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text: I will be in Manhattan in May and want a really great burrito. Any suggestions on the best?? I love authentic but I also like the healthy version with great, fresh ingredients. A hole in the wall restaurant is not a problem -- actually it's a plus . .. .

subset: commoncrawl


text: Here awhile back I found a new source for bellies and other gastronomical delicacies. When I thawed out my bellies they were all freezer burnt. I was slightly miffed (being polite here). Beside they were the worst bellies I ever saw. A friend here talked me out of just chopping 'em up for cracklins (and I wasn't even that sure about that). Since my cured smoked hocks were depleted also, I thought a nice happy curing bucket might be nice. I made two gallons of Pop's brine, thought I would need help to get the bucket up on the old plastic shelf in the beer fridge, but I did and it didn't break the plastic. They just don't make good plastic like that any more. LOL Honestly I should not admitt it, I have not been ton the beer reefer in a long time, and I just forgot they were in there. Been out there submerged for 19 days now. I tasted the brine (I always do) and it was soooo mellow. I am guessing maybe so much pork, it had reached equalization? I took it out, rinsed the meat, and spread it out in the project reefer. Will continue the story tomorrow maybe. If I allow it to dehydrate for 2 days in the reefer it should require less of a dewater cycle. BTW as to the freezer burnt belly. It appears that the curing cycle makes that a mote point. Maybe it will rear its ugly head again after smoking? I hope not, got another belly in the freezer from the same source. Chuckles, the neighbors Mother came by to what I was smoking for supper, Tenderloin. Guess I can show yas. We got to talking about food prices. She's like me and likes all the less traveled trails. We talked about oxtails, hocks, bellies, gizzards, chicken feet, etc etc etc...... She finially laughed and said since delux tripe and ribeyes cost about the same her family was eatting better than ever before. They can't understand all the steak and ribs but no gumbo's or jambalayas. I thought it hilarious, true but funny. OK, be back tomorrow. I did not post any on the tenderloins, I have done them too many times for them to be interesting, but if you have a question or want step by step, I really don't mind helping. Demain


text:
Making a strategic use of A-to-B
Brand strategy and collateral
Arts University Bournemouth
Arts University Bournemouth (AUB) needed a campaign that tied together all of the different touch-points across the whole recruitment cycle. They wanted to reflect the diversity of opportunity within the creative industries and position themselves as a pathway to them, rather than an end destination for students.
The campaign had to be flexible enough to work across online, print and physical spaces, while remaining distinctive enough to achieve cut-through in a competitive market. It had to remain relevant throughout the various key stages in the recruitment cycle; from discovery through to conversion and, finally, enrolment of new students.
Working with AUB's abbreviation, we developed the concept of 'A to B'. Emphasising the route from A to B as the start of a creative education and the end result of a creative career. We applied various treatments to the letters A and B, creating a set of different letterforms for each, showing the diversity of both students and their careers as graduates.
To further emphasise the individual journey that students take at AUB, we produced an animation with a ball motif that travels along a landscape of abstract A and B shapes. By moving fast, slow, tentatively and confidently across the landscape, it provided a visual cue to the varied journey that students at AUB take.
We produced a full range of collateral for the recruitment period, covering everything from AUB's House of Cards prospectus, to the hoardings surrounding building works on campus, and a double-decker bus with full livery used on the routes to and from the university.
Working with AUB, we helped to reflect the visual identity of the campaign in the copy that accompanied it. By focussing on the journey that students take, rather than the institution, we created a unique voice that helped AUB stand out in the competitive HE marketplace.
The campaign has been taken even further with Aberdeen to Bournemouth, a project that sees an AUB alumna travel through the United Kingdom documenting the work of other alumni.


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5/129 Argyle Street St Kilda 3182 VIC
Enter the market with complete confidence
Peacefully positioned at the rear of a boutique block, this spacious entry-level ground floor apartment and high-flying portfolio performer delivers on all measures. Refreshed and rejuvenated, discover a newly painted apartment with great living and dining spaces flowing through to a large modern kitchen with meals area and stainless steel appliances. Enjoy the light and bright double bedrooms with built-in-robes and a sparkling bathroom with separate shower, bath, laundry facilities and separate toilet. The secure entrance offers privacy with the added convenience of off-street parking. Moments from Chapel Street, St Kilda's iconic amenities and transport. This is classic St Kilda apartment living, with all the lifestyle benefits.
* 2 large bedrooms with BIRs
* Spacious, light-filled open-plan living and dining
* Modern kitchen with meals area
* Sparkling bathroom with shower, separate bath and laundry facilities
* Separate WC
* Off street car parking
* Newly painted throughout
* Secure intercom entry
* Moments from transport options, shopping, and the hub of St Kilda
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text:
Presenting a designer push bra that integrates style with comfort at the best level. Special soft and stretchable polyamide with spandex fabric has been used to craft this exquisite piece. This underwired bra has padded cups for better support and lift. Smooth straps provide the much-needed supportive feel covering your upper bosom properly.
Key Features
Ravishing red colour
Contrast lace on the cups
Demi cups
Supportive underwiresClovia Says A little push in the right direction.
Polyamide with spandex and lace
Colour: Red
A little oomph is better for your stylish look.


text:
Well Established Business
Price: ~ ~ SOLD ~ ~
Property Description: Exceptional opportunity to become the new owner of one of the most well known businesses in Dillon, MT! This business has been owned and operated by the same family for 60 years, has developed a very loyal, consistent clientele of colorful local personalities! Well maintained and improved facilities are in very good functional condition. Diversified income generated from Bar operations, Electronic Gaming Machines, furnished Apartment and 8 Hotel Room rentals (90%+ occupancy rate). Offering includes Real Estate, Equipment, Furnishings and All Beverage Liquor License. A great opportunity to continue a local tradition and be your own boss in downtown Dillon, Montana!
Business Type: Bar / Tavern / Hotel
Property Address: 24 East Center Street


text: Is there a charge for the coffee, milk, and/or juice that comes with the room service continental breakfast? I'm just wondering if I should plan on getting up early in the morning & grabbing DH a cup of coffee from the Deck 9 drink station, or if we'll order it from room service every day and have it brought right to us (almost too luxurious to imagine, really). Thanks for any help.


text:
The product has been added to yourWISHLIST
Take a screenshot, call/Whatsapp us at +92 321 1999902 or email at email@example.com
Mint green net dress with an organza neck cuff. Resham floral embroidery with sequins and beads embellishment on dress bodice and sleeves. Bead tassel detailing on sleeve cuff. Back bodice has button detailing and embroidered motifs. Pleats on bodice hem. Lower net dress has embroidered motifs.
Each of our products are designed with high quality standards and careful attention to detail.


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3D Artist | Motion Graphics Designer working on various creative projects and always looking for the next challenge.
I am open for commissions, the areas I could help you with are:
Conceptual art (characters & environments), 2D Illustrations, Portraits, 3D modeling, Lighting and Rendering & Compositing. If you have any questions, just send me a message please.
Watch my latest showreel here!
You can also follow me on:


text:
You can download iOS 6.0 and 6.0.1 now for all the devices listed below. These download links are official Apple download links coming directly from the Apple site. The fruit companies 6th generation operating system has been available for a while now, almost going for a few months. It released around about the same era as the iPhone 5. The IOS 6.0.1 is a new firmware which brings along many bug fixes with it.
So what does this bring with it if I should choose to download and install it on my device? It's well-known to have not been a massive update of what we know as Apple's software. It was more the timing of it that had its name everywhere, and everybody wanted to use it. With that said, there is a handful of neat new features that are well worth a mention.
Sometimes it's the simple things that satisfy us the most, and when it comes to the device, there is a good reason for that. There is often a simple task that you might do a lot of because it's so easy. So when you add up all those times, it equals a lot of time spent on it. If we were able to tweak this a bit so that it becomes more efficient for us, or at least more useful, then suddenly we have something well worth talking about here. That is probably the best way, to sum up, what the Cupertino company have managed to do with the new call reminder add-on. Have someone call your iPhone and instead of answering it, do a swipe gesture upwards from the bottom of the screen to test this out. It will now bring in two new options available for you to use. One of them is to set a reminder and even gives the option of choosing how long to wait before it issues an alert. For example, if I swipe I can select two hours, and now my iPhone will inform me in two hours time about that call that I elected not to take. It's a bit cheeky, but if you're someone who doesn't like or talk on a mobile a lot, or you are busy and genuinely don't have time to answer a lot of the calls that are coming to you, then this will a fantastic inclusion for you.
The choice of whether to update or not is purely up to you. If you wish to update to the 6.0 version, you can do so by following this guide on how to install iOS 6. If you already have iOS 6.0 and are looking to update to iOS 6.0.1, it's very simple. All you need to do is go to the Settings, General > Software update and from there it will update for you.
Direct Links for all the firmware in question has been given out underneath:
iOs 6.0.1 for iPhones
For iPhone 3GS, 4 GSM (ATT), 4 CDMA (Verizon/Sprint), 4S, 5 GSM (ATT) and the 5 CDMA (Verizon/Sprint)
For iPad's
For iPad 2 Wi-Fi (Original iPad 2), 2,4 (Current iPad 2), 2 GSM (ATT), 2 CDMA (Verizon), iPad 3 Wi-Fi, 3 CDMA, 3 GSMl, iPad 4 Wi-Fi and the iPad mini.
For iPod Touch
For iPod Touch 4th Gen and the 5th
Direct Links to download iOS 6.0:
For the iPhone:
Download iOS 6 ipsw firmware for iPhone 5 (GSM), 5 (CDMA), 4S, (Global), (AT&T), (Verizon – CDMA) and iPhone 3GS can all be used from the same links given in the earlier models.
For iPad:
Download 6 ipsw firmware for iPad 3 – CDMA, Wifi+3G(GSM), Wifi, CDMA, Wifi+3G (GSM) and the iPad 2 – Wifi
For iPod Touch:
iOS 6 ipsw firmware for iPod Touch 5G and the 4G can also be taken from the same place.


text:
The Siemens Murray 100 Amp Ringless Horn-Bypass Overhead/Underground Meter Socket features a corrosion-resistant construction with a galvanized carbon steel shell and an electrostatic polyester paint powder coating. The design of this meter socket offers a 4-jaw, 2-position, horn-bypass assembly. For safety, this meter socket is UL listed and ANSI certified.
- Type-G90 hot-dipped galvanized carbon steel construction for corrosion protection
- Single-phase, 3-wire, 4-jaw, 2-position, horn-bypass design
- Electrostatic powder coating of polyester paint enhances corrosion resistance and helps provide a finish free of drips and runs
- Designed for overhead and underground entrance feeds
- UL listed and ANSI certified


text:
RedZee Offers Very Real Benefits For Virtual Businesses
Headquartered in Tampa, Florida, RedZee, Inc. is among the Top 100 web-based search engines. The site was originally designed with an orientation toward family-friendly online searching in mind. Since initial inception, its primary focus has shifted to cutting-edge technological integrations intended to drastically alter the landscape of virtual querying.
RedZee pay per click ads are especially valuable for web-based marketers wanting to find high favor with younger members of both genders. According to RedZee officials, the secret of success for this internet advertising service runs far deeper than the initial draw of its famous red zebra imagery.
In a recent news report, RedZee Search President John Stewart stated that his firm's emphases on high-quality visual displays are especially attractive to the younger generation. This is particularly true of those who form intimate acquaintances that lead to undying love of graphics applications (like games), iPods, and Smart Phones.
RedZee PPC Engine Stats
Quantcast is an internationally-renowned industry analyst firm. Quantcast is also widely regarded as the world's only entirely objective internet advertising service evaluator. Its new media measurement open source tool permits online marketers to compile accurate audience analyses for millions of websites and online services.
A 2008 Quantcast research study revealed the following demographic characteristic prevalence among RedZee-based web viewers:
- An even split between males and females
- 37 percent were 18 to 24 years of age
- 20 percent were 25 to 34 years of age
As numbers never lie, the lesson contained within the above figures is clearly evident: More than half of all RedZee end consumes consisted of those under the age of 35.
RedZee facilitates ethnically-diverse marketing efforts
Additional Quantcast findings have shown that a whopping 68 percent of RedZee site visitors are of Hispanic ethnicity. On average, African Americans comprise another 13 percent, with whites making up only 11 percent of total viewer populations.
Pictures worth thousands of words
This pervasive visual orientation is probably the best explanation for RedZee's distinguishing characteristic. Other search engines display results lists generated in response to user inquiries in a textual format. By contrast, visual content carries the day and rules the roost for RedZee search results.
What's the big deal?
Per official sources of the RedZee website, this revolutionary high-tech imagery innovation enables a tenfold return of relevant site listings. Rather than a maximum return of the "Top Ten" sites offered by Yahoo, Google and other text-based search engines, RedZee Search proprietary software reveals up to 100 relevant "hits" for users' viewing pleasure.
The RedZee Search™ software makes it all possible with its display of search results within a "fanned" format of individual web page images. Users may browse search results by merely mouse-hovering over any displayed image. This permits an in-depth preview to determine which sites are worthy of further exploration.
Get in on ground floor of fantastic search innovations
At present, RedZee is touted as Cyberspace's sole metasearch engine that features the highest-quality metsearch results, state-of-the-art features, and an interface that is ultra user-friendly. For more details about the definite advantages of this virtual marketing tool, visit www.RedZee.com or call (888-399-5534.


text:
A modern surgery center, opened in 2010, specializing in elective surgeries. Medical procedures carriedout at the center include general surgery, urology, pediatrics, orthopedics, oncology, plastic and cosmetic surgery, ophthalmology, pain management.
Laser Lithotripsy
upon request
Trust based hospital that treats around 7,000 international patients a year. India's first JCI and comprehensive NABH accredited hospital.
This information is intended for general information only and should not be considered as medical advice on the part of Health-Tourism.com. Any decision on medical treatments, after-care or recovery should be done solely upon proper consultation and advice of a qualified physician.
What is laser lithotripsy?
This is a medical procedure used to remove stones, from the ureter, kidney, bladder, or urethra (urinary tract). It is a minimally invasive outpatient procedure that is considered safe and effective. It is recommended by your doctor when you suffer a large stone that cannot be passed through urine and especially after medications.
While the procedure is carried out you will be under local or general anesthesia. Laser lithotripsy is done by an urologist by inserting a scope into the urinary tract- through your urethra, bladder and up the ureter- to locate the stone. A laser light is directly emitted to the stone through an optical fiber which is inserted through the working channel of the scope. The laser fragments the kidney stones and any remaining pieces are washed out through the urinary tract. To ensure the kidney drains urine well after procedure, a ureteral stent may be left in place.
You should call your doctor if:
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text:
Upcoming Events
The Office of the Dean of Students and Community Development invites all students to attend the programs taking place at Seton Hall University. The calendar below lists the programs planned for the spring semester. Please make sure to check back as the calendar is updated daily with programs sponsored by the Student Activities Board, Student Clubs and Organizations, Greek Life, and Commuter Council.


text: The very high unemployment rate and the very high political problems in the country lead to this work. The main thematic of this master thesis is "The prerequisites and needs for the automotive supplier industry to become established in Bosnia and Herzegovina". This work contains information about the actual situation in Bosnia and Herzegovina and the actual automotive supplier industry in Bosnia and Herzegovina. Also a historical overview of the automotive industry in Bosnia and Herzegovina is provided. The work shows the actual problems and gives ideas and plans to overcome these problems. The objective of this work is to define preconditions and proposals, together with solutions,for Bosnia and Herzegovina to be able to establish a automotive supplier industry in the country and to gain more investors in that area. There will be defined a set of criterions which are relevant for investors for there decisions. It will be shown how these criterions must look like and how should they be fulfilled by the country of Bosnia and Herzegovina. We will see that the political situation and the corruption are the most problematic things which block the country to have more prosperity in the country


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Results 1 to 9 of 9
February 7th, 2005, 10:18 PM #1Anyone who's purchased through EveryTicket, StubHub, etc.
Or even if you just have knowledge of them...that'd be fine too.
They all seem to be the same, so this question should be universal. Their price listing confuses me a bit. For instance, 2 tickets to a certain section of the stands at Talladega Superspeedway (just choosing something I know what the prices are) are listed as:
Section --- Seat #(s) Qty. --- Sale Method --- Sale Price (USD)
TALLADEGA --- 2 tickets together --- Fixed Price --- $143.00 each
(made a nice table, apparently I can't post HTML in this forum)
Now is that $143.00 per 2 tickets, or is that $143.00 per ticket? If that's per ticket, these sites sure do rip people off. If that's per pair, then that's a fair price.
I ask because I've been contacted by several of these sites to sell tickets on my Talladega fan site, but there's no way I'd do that to my visitors if that a per ticket price. As it is I just point them to the offical site for free.
February 7th, 2005, 10:51 PM #2
I'm pretty sure it's for one ticket, I think these companies sell hard to get/ sold-out tickets.
February 7th, 2005, 10:58 PM #3
I agree with Electropulse. No cheap lunch out there.
February 7th, 2005, 11:05 PM #4
Tickets to the races (especially months ahead) are not hard to get, at all. Maybe for the Daytona 500. I've bought good Talladega tickets a week or two before the race. For that particular section I quoted above, I've sat in it before, and the tickets are $70.00 flat (which would match the roughly $140 they have listed for 2).
EveryTicket for example charges depending on where in the section you sit! There's a flat price for every section, regardless of where you sit. The most expensive Talladega ticket (very top center stands) is like $160.00. EveryTicket has them listed for over $350.00!
I too believe that's a per ticket listing. I had a warning up on my site about these types of sites, but before I stepped too far, I thought I'd get 100% clarification. I emailed EveryTicket but have yet to hear back from them.
February 8th, 2005, 09:11 AM #5
It is a per ticket listing. Supposedly they sell the "best" tickets for whatever event is out there. I went thru this conversation on another board - I was looking to buy tickets to go see Chicago The Musical which will be here in Rochester in April.
The asking price of the tickets thru EveryTicket was like $250. The price for tickets via Ticketmaster - the main source of tickets was $50.50 - While its true that I wasn't able to get in the first 3 rows via Ticketmaster which I could have done via EveryTicket -- I was able to get tickets for Row 23 which is still excellent seating in my book and considering I could get 5 tickets for the price of 1 at Everyticket I was quite happy with their location.
Had I not waited another month past the time I first started looking for tickets, I could have actually had better seats but I kept forgetting to ask my Mom if she wanted to go see it with us Girls.Heidi
February 8th, 2005, 10:30 AM #6
- Join Date
- January 18th, 2005
- Location
- St Clair Shores MI.
- Posts
- 17,328
Ticket scalping is a profitable business with less then savory practitioners.Webmaster's... Mike and Charlie
"What have you done today to put real value into a referral click...from a shoppers viewpoint!"
February 8th, 2005, 05:19 PM #7
Thanks everyone, looks like I'll keep my warning up.
February 8th, 2005, 08:25 PM #8
I affiliate with some of these companies but figure most people know how to shop for tickets and anyone who MUST have something and is willing to pay for it is very welcome to pay the price.
February 13th, 2005, 12:03 AM #9
- Join Date
- January 18th, 2005
- Location
- Neyland Stadium West
- Posts
- 56
TicketMaster is essentially the box office for a lot of events. They charge face value plus a handling fee. But they get sold out of the best tickets on the good events.
Places like everyticket cater to people who are either 1) more interested in the very best seats as opposed to the very best price or 2) people who have more money than time to invest in getting a better deal.
If you've got time, poke around forums or classified ad sites. You'll almost always get better prices. For the past few years, we've just always bought tickets to our fav football team at the game. That works for us as you can almost always find plenty of people with extra tickets.
Similar Threads
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text:
Celebrity News For Feb. 20, 2013 | Late Edition
Anne Hathaway Will Perform at the Oscars
- Anne Hathaway will sing with her Les Mis cast at the Oscars — Lainey Gossip
- Matthew McConaughey has no beef with his exes — HuffPost Celebrity
- Jessica Biel attends Beverley Mitchell's baby shower! — Us Weekly
- Michael Bublé reveals his wife's strange pregnancy cravings — People
- Which celebs have come out as bisexual? — ET Online
- Stars who have gotten virtually hacked — The Daily Beast
- See Rob Kardashian's reported 40-pound weight gain — BuzzFeed
- Did Julianne Moore do Elizabeth Taylor's jewelry justice? — Dlisted
- Kim Kardashian is making plans to depart her reality show — JustJared
- See Kendra Wilkinson make a splash in a swimsuit — Hollywood Tuna
- Watch the Downton Abbey cast sing a One Direction hit — Pink Is the New Blog
- Details on the ring John Mayer gave Katy Perry — The Superficial
- Which star was best dressed at the Paris Spring Breakers premiere? — The Blemish
Image Source: Getty


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Serving more than 600,000 patients each year, this Atlanta not-for-profit healthcare system was experiencing multiple issues with their current site which included poor user experience, a botched implementation of an antiquated CMS, and an reliance on IT for Marketing to publish time-sensitive information.
Thanks to a technology overhaul which included website redesign and re-platforming on SharePoint, the healthcare system has seen a reduction in patient complaints, an increase in site traffic, and improvement in visitor engagement. The website redesign has allowed visitors to find a treatment, doctor, or nearby location with just one click from the home page.
- Industries: Healthcare
- Strategies: CMS, Portals


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The La Crosse Public Library offers free computer classes for those who need to brush up on their skills or have had minimal experience with technology. Whether you're a job seeker looking to brush up on entry-level computer skills or a smart shopper looking to find great bargains online, our classes offer something for all skill levels and areas of interest. Unless noted, all classes meet in our lower level training room at the Main Street library.
Due to popular demand, these classes now require registration. Please call (608) 789-7122 or stop by the Reference Desk (2nd floor at Main) to register or to get more information about all of our upcoming classes. View the full schedule of our May 2017 classes below. Participants must have some familiarity with using a keyboard and mouse.
May 2017
Click here to access class handouts and to find more resources


text:
Thursday: February 5, 2009. Review yesterday's quiz! PowerPoint on Trait Perspective Personality Test Ch. 15 Reading Quiz 2 Learning Objective : Understand the many components of the Trait Perspective of Personality. The Trait Theory of Personality.
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Trait Dimension Description
Neuroticism Calm versus anxious
Secure versus insecure
Self-satisfied versus self-pitying
Extraversion Sociable versus retiring
Fun-loving versus sober
Affectionate versus reserved
Openness Imaginative versus practical
Preference for variety versus
preference for routine
Independent versus conforming
Agreeableness Soft-hearted versus ruthless
Trusting versus suspicious
Helpful versus uncooperative
Conscientiousness Organized versus disorganized
Careful versus careless
Disciplined versus impulsiveThe Trait Perspective


text:
We all have stresses in our lives. Your boss is a jerk. Your kids are jerks. Everyone's a jerk, really. Daily stresses are "harshing" your "mellow", as the kids these days are saying (and we totally confirmed with some kids that we paid 5 dollars that they are indeed saying that).
What better way to release your inner turmoil than going to a safe, judgement-free place where you can just be ridiculous for a little while? Just ask one of our many class attendees... they keep coming back time and time again, so there must be something to it.
See? Told you.
All classes and performances are held precariously close to the expensive artwork at Derek Gores' art gallery in the Eau Gallie Arts District.
587 W Eau Gallie Blvd, Melbourne, FL 32935
Check the calendar for upcoming events, then do yourself a favor and order some tickets.
Buttons not working / out of date? Try using the calendar instead. If neither of those work, email us or check out our facebook page. Sorry. Our web guy only works on saltines and Gatorade, which are in limited supply.


text:
Brenda from Schooling A Monkey [and one of our very own writers] grew up in the country, and moved to the city when her husband got a job there. What struck me the most after talking with her was her adaptability, interest in trying new things, and her determination to try again if she didn't succeed the first time. Perseverance is a good trait to have if you are trying to live a homestead / preparedness lifestyle in city.
Listen to hear how they find fresh, farm-grown food to eat; how she cooks in advance and freezes a lot of foods; what plants they can grow indoors; her struggle with only having a shady patio outside; tips for finding and shopping at farmer's markets; how they keep their emergency things in their vehicle, how she makes fermented tea to drink, and more.
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Mentioned in this podcast:
- Community Supported Agriculture [CSA]
- All About Kombucha Tea
The Complete Idiot's Guide to Small-Space Gardening
- Secrets of Successful Shade Gardeners
- The Best Plants To Grow In The Shade
We have a new podcast every Wed. Make sure you are subscribed to MomPrepares so you don't miss any! You can subscribe to our podcast on iTunes , Stitcher, or Beyond Pod. If you would like to be a guest on our podcast or sponsor the podcast – email firstname.lastname@example.com with ATTN: podcast in the subject line.
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MomPrepares loves to join link-ups and meet other bloggers. Here is the list of our favorite link-ups so you can participate too!


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By Bob Thompson
Washington Post Staff Writer
Monday, March 2, 2009
When David Foster Wallace killed himself last September, his death shocked and saddened the literary world -- and provoked immediate speculation about what posthumous work might emerge.
This week's New Yorker offers at least a partial answer to that question. In a pile on Wallace's Claremont, Calif., desk when he died were nearly 200 pages from an unfinished novel called "The Pale King," on which the author of "Infinite Jest" had worked for years. Much more material related to the novel turned up in Wallace's files.
The magazine, due on newsstands today, is publishing a short excerpt from the novel as well as a long article on Wallace by D.T. Max that tells the story of the unfinished work.
Michael Pietsch, Wallace's editor at Little, Brown, said in an interview that he had a tentative agreement with Wallace's agent to publish "The Pale King" in the spring of 2010.
The unfinished novel, Max writes, was in part Wallace's attempt to move beyond the "self-consciously maximalist style" of "Infinite Jest."
"I think he didn't want to do the old tricks people expected of him," Max quotes Wallace's wife, Karen Green, as saying.
"It was different from what he had written before," said New Yorker fiction editor Deborah Treisman, who has seen portions of Wallace's manuscript -- though it's also clear, from Max's reading of part of the new work, that some of it recalls the expansive, trick-filled David Foster Wallace many readers came to love.
The characters in "The Pale King" are Internal Revenue Service agents working at an IRS facility in the Midwest. The intense tediousness of their jobs and their attempts to transcend boredom reflect Wallace's preoccupation with the concept of "mindfulness" -- the idea, as he put it in a 2005 commencement speech, that you should be "conscious and aware enough to choose what you pay attention to and to choose how you construct meaning from experience."
The unfinished novel did not surface for more than two months after Wallace's death, said his agent, Bonnie Nadell.
Nadell said that she and Green had cleaned out Wallace's office at Pomona College, where he taught, on the second weekend after he died because "it was not particularly secure." They didn't find much there, however, because Wallace mostly worked at home, in a garage crammed with old couches, chairs and a multitude of the lamps the writer collected.
"It took us about two months before we could face going into this room," Nadell said. "It was like he was there."
Sometime after Thanksgiving, they finally started in. There was a great deal of old material to sort through, including "many, many drafts of 'Infinite Jest.' " But when they got to the pile of "Pale King" pages on Wallace's desk, Nadell said, "Karen clearly felt that it was there for us to find."
Nadell read just enough to realize she was dealing with a book, and that she should call Pietsch. "I wasn't sure that this would be a coherent book," she said, "but I knew it was enough to say, 'Get on a plane.' "
Pietsch had been working with Wallace ever since he acquired "Infinite Jest," which Little, Brown published in 1996. He flew to California in January to examine what he called the "rather huge collection of manuscripts" Nadell and Green had assembled "in various bags and boxes and bins." There were handwritten drafts, marked-up typescripts and notebooks filled with thoughts on the novel-in-progress. He started in on the "several hundred pages that read continuously."
It's still not easy for him to talk about this.
"I had the uncanny experience of feeling joy where I expected to find grief," he said, because he could feel Wallace's presence in his "astounding, levitating, daring" work. Yet it was agony "to realize that he was not here, and not here to finish it."
A couple of days later, he flew home. In the overhead bin was one of Wallace's duffel bags, crammed with material.
In the meantime, New Yorker editor David Remnick had assigned Max to write about Wallace and told him to take the time he needed. "There are different ways to be competitive," Remnick said. Being first with a story is one of them; "better and deeper" is another.
The reporter had never met his subject, though he'd once seen Wallace across the room at the kind of publishing event the writer hated, wearing his trademark bandanna and looking "horrified to be there." Max was also one of many would-be readers of "Infinite Jest" who hadn't made it through all 1,079 pages, though he recalls being "stunned" by Wallace's first novel, "The Broom of the System."
"There was a guy who was basically my age," he said, and at a time when minimalist fiction was the vogue, "he just exploded it."
Given time, Max persuaded a number of Wallace's friends and associates, including novelist Jonathan Franzen, to show him letters and e-mails from Wallace. With Nadell's help, he persuaded Green to talk with a reporter for the first time since her husband's death.
And in a Wallace letter to novelist Don DeLillo -- which he'd obtained earlier for another article -- he noticed a line he hadn't paid attention to before:
"I believe I want adult sanity," Wallace had written, "which seems to me the only unalloyed form of heroism available today."
"This was going to be the theme of his novel," Max said.
Sanity is also a theme of Max's piece, as it must be of anything written about Wallace. After his diagnosis of depression as an undergraduate, he had been on medication ever since. He decided to wean himself from the antidepressant Nardil -- a decision that may have had fatal consequences, though nothing about suicide is ever certain -- in part, Max writes, because he thought the drug "might be getting in the way of 'The Pale King.' "
Whether that was true or not, Wallace had a bigger, non-clinical problem with the novel: He was trying to evoke a way of being in the world that he himself had not achieved.
" 'The Pale King' had many ambitions," Max writes. "It would show people a way to insulate themselves from the toxic hyperactivity of American life. It had to be emotionally engaged and morally sound, and to narrate boredom while obeying the physics of reading. And it had to put over the point that the kind of personality that conferred grace was exactly the kind that Wallace did not have."
View all comments that have been posted about this article.


text:
Commemorated on July 3
SAINT OR FEAST POSTED THIS DATE 2014(with 2013's link here also and further:, 2012, 2011, 2010, 2009 and even 2008!):
Taking Orthodoxy As It Is - Fr. Marc Dunaway, who was one of the leaders of the Antiochian Evangelical Orthodox Mission, which 30 years ago were received into the Orthodox Church by...
46 minutes ago


text:
Small yet bold numerals stand out in modern style on this executive alarm clock.More Details >
The sleek design of this executive alarm clock takes into account style and utility. It is ergonomically angled at 22 1/2 degrees for easy reading when placed on a desk or table top. A white pvc dial matches the matte white finish barrel and is highlighted by polished chrome. An orange alarm indicator stands out among chrome plated hands.


text:
[Ed. – Poor babies.]
Funny how when the shoe is on the other foot it doesn't fit quite so well, isn't it? As part of its War on Trump, the establishment media consistently conflates two only superficially related, but contextually quite different or actually unrelated things — "Russian hacking" and "changed the vote for Trump" is a good example. But conservatives are wise to their game, and so they gave "fake news" right back to them with both barrels. Of course, it's an easy case to make when you have a former purveyor of fabulism like Dan Rather now teaching journalism ethics.
So, naturally, the [Washington] Post writer blames the current non-utility of the "fake news" slam on… the Right:
"The speed with which the term became polarized and in fact a rhetorical weapon illustrates how efficient the conservative media machine has become," said George Washington University professor Nikki Usher.
As Jeremy Peters wrote in the New York Times: "Conservative cable and radio personalities, top Republicans and even Mr. Trump himself . . . have appropriated the term and turned it against any news they see as hostile to their agenda."
So, here's a modest proposal for the truth-based community. Let's get out the hook and pull that baby off stage. Yes: Simply stop using it. Instead, call a lie a lie. Call a hoax a hoax. Call a conspiracy theory by its rightful name. After all, "fake news" is an imprecise expression to begin with.


text:
Fedora 15 Is Golden
In a mailing list post today, Fedora Program Manager Robyn Bergeron announced that Fedora 15 is ready for release on May 24, 2011.
"At the Fedora 15 Final Go/No-Go meeting today, the Fedora 15 Final Release was declared GOLD and ready for release on May 24, 2011," Bergeron writes.
Before Fedora 15 can be released, it must meet specified criteria. The beta release rolled out in mid-April with the Gnome Shell and Gnome 3 desktop, LibreOffice productivity suite, BoxGrinder, a dynamic firewall, and language updates.
The Fedora 15 Release Wide Readiness meeting will take place this Thursday in IRC.
Issue 205/2017
Buy this issue as a PDF
Linux Kernel 4.14 Released
Torvalds lashes out at a Canonical developer who introduced a regression.
Fedora 27 Is Released
Developers can now access Red Hat Enterprise Linux Developer subscriptions on Fedora 27 at no cost.
Samsung to Bring Linux to the Galaxy Phone
Samsung is testing Linux desktop for its DeX experience.
System76 Releases Pop!_OS
An Ubuntu-based operating system designed for professionals.
News and views on the GPU revolution in HPC and Big Data:
HIP: CUDA Integration with ROCm
Exploring AMD's Ambitious ROCm Initiative
Crypto-Currency Mining Drives a Surge in GPU Sales
New Platform for GPU-Enabled HPC and Ultra-Scale Computing
Radeon Instinct: Evolving, Adapting & Learning Evolving, Adapting & Learning
Linux Comes to Windows
Run multiple Linux distributions in Windows 10.
Docker Embraces Kubernetes
Docker is now offering Kubernetes as an orchestration platform.
Kubernetes 1.8 Announced
The new release comes with two new features for better security.
Final Ubuntu Desktop 17.10 Beta Arrives
Ubuntu completes the cycle of life and goes back to letter A and Gnome.
Linus Torvalds Invites Attackers to Join the Kernel Community
He wants attackers to join the community instead of attacking the code.


text:
- Please select a date to see the proper prices!
Description of the accommodation:
Rooms, services Balmoral Hotel San Francisco is offering 122 tastefully furnished rooms to those seeking accommodation in San Francisco. The exact scope of services offered by Balmoral Hotel San Francisco (including those for an extra fee) is available on the information sheet below.
Payment Depeding on the selected room, date and price, you may be asked to provide credit card guarantee or to pay by credit card in advance.
Arrival, opening hours The earliest time to arrive is 09:00, the latest time to leave the rooms is 11:00. English is spoken by the personnel.
Balmoral Hotel San Francisco rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Hairdryer, Fridge, Microwave oven


text:
Mondo Homo Trailer (2009)
"Inquiry into 70'S gay French porn." 01 January 2009 History, Factual 110 mins
Between 1975 and 1983 a new kind of film could be seen in French cinema; home-grown gay pornography. They were essentially the work of three production companies: Les films de La Troika (Norbert Terry), AMT Productions (Anne-Marie Tensi) and Les Films du Vertbois (principally Jacques Scandelari). The genre met an untimely end with the advent of video, the last being made in 1983 'Mon ami, mon amour (My friend, my lover)'.
Watch the official Mondo Homo 2009 trailer in HD below or find more Mondo Homo videos on Vidimovie.


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Okay, I don't get Brad Maddox. At all.
And I say this because I've seen a ton of people online write that they think he's hilarious. I dunno. Maybe I'm not "with it." He came out do be the third person on commentary on Smackdown and he was terrrrrrible. And then last night on RAW, he couldn't even get through a simple bit. The problem is that he's treating everything like a goof. It's one thing to have a gimmick where you're a goofball, but we get absolutely nothing serious out of him. Remember when he was trying to threaten The Shield and he just came off like Bane? He's not a character. He's, like, the way I used to write e-fed announcers. He won't sell any story because he's either high as s*** or he doesn't give a f***. He changes his delivery style, cadance, inflections and beats with every single sentence. He comes out to act overly-important on Smackdown, after becoming Vicki's RAW assistant, and says "I'm now the most important man in the WWE. You're all my employees." See, that's actually too much.
There's a line between coming off like an arrogant, comedic heel and coming off brain-dead. It's hard to explain. Hyperbole can be funny. It's the current currency of the entire internet, in fact. But going overboard within wrestling just makes it sound like you don't give a damn. Like you don't have a grasp on your character or what your character would say. Maddox is like the worst improv partner ever. Every other line out of his mouth sounds like a flub. Like a director should be angrily yelling "CUT!" right after it. Maybe his gimmicks is that of "guy so untalented it's revolutionary."
He's bad. And you should feel bad.
Speaking of things I'm already tired of, Fandango. And, reportedly, WWE officials are already sick of the gimmick too. Fandango's dancing partner can stay though. She has potential. *points at crotch* *notices salsa stain* *sheepish grin* *cries in shower*
Oh, and don't think I forgot about AJ suffering the pinnacle of shame and humiliation by getting drenched in water on Smackdown.
How funny would it be if someone no-sold water by the way. Like someone got a bucket of water thrown on them and instead of acting like it's the worst thing to happen in the course of human events, they just shrugged it off and walked around in wet clothes. That would put Undertaker to shame. That would be more powerful than a "Hulk Up."
"The water... it had no effect! It didn't phase him! WHO IS THIS MAN???"
After AJ got doused, we were shown a segment with her and Bryan where Bryan beat her to the 18 second premature ejaculation joke. Yes, the same joke that was used on him when he was a heel. He just transferred it over to Ziggler. Because villains climax before their sexual partners are satisfied.
Okay, before I get into TNA's Lockdown, I wanted to talk about the Gutcheck segment from last week's Impact. It featured Ivelisse and Lei'd Tapa, but only one of them could go to the ring to face the vote. And Ivelisse, one of the most talented gals working today, got cut first, meaning she didn't even make it to Gutcheck. So... what the f*** is Gutcheck these days? Is it real? Was this a real decision? When it first started the contestants swore up and down that it was a shoot. That the votes were real. It was legit. Then they brought Joey Ryan back, using his Gutcheck loss as a story. Now Gutcheck is totally used for storyline purposes, like with the D-Lo/Wes Brisco thing. So is it sometimes used for story and sometimes not? Is it bulls*** all the time? Regardless, I'll say this. When TNA, under the guise of a real vote, cuts Ivelisse, I get mad at TNA. If Ivelisse's cut was real, then they're horrible. So why have a segment that could possibly turn fans against the company? And, since Gutcheck has been fake, for the sake of letting Aces members "infiltrate" TNA, do the people who've been legitimately denied a contract have any sort of legal ground to stand on? Can you have a promoted bit where sometimes people will be given, or denied, a real paying job but then, you know, sometimes it's all a farce?
Yes, I was hopping mad about the Ivelisse thing. And I never hop. Ask anyone. Also, D-Lo was revealed as an Aces member, just like we knew here and here. Yes, more non-star power to not care about. Of course, Angle "knew" this secret for a week but didn't tell anyone. Even though he claimed "Nobody's safe in this company!"
So at Lockdown, as predicted here, Bully Ray turned on Jeff and revealed himself as the leader of Aces & Eights. But - my god - was it sloppy. First off, in the middle of the match, Garett and Wes came into the ring to attack. But Bully didn't choose to turn then. He fought them both off, siding with Hardy. And he didn't escape the cage when he demanded to door open so they could throw the Aces members out. Why didn't he turn then? From a "this is real" standpoint, he could have lost the match to Jeff after that point and then the plan would have been wasted. No, he waited until Aces returned a second time, after a few "miraculous" kickouts from both men, to turn right at a certain moment when Devon showed up.
My question is: Why did Wes and Garett come out mid-match? Why not just have the swerve at the end? Why have him act like a hero and not take an easy opportunity to win right then and then during the first Aces run-in? I don't mind the twist itself. Whatever. Turn Bully heel again. But it was poorly executed. Then some fans who were planted to throw trash into the cage at the end threw some trash into the ring at the end.
And poor Taz. If there's a harder job out there than having to, sincerely, put over Aces & Eights, I can't think of it. When complaining about the lack of Aces in the Lockdown Main Event, he said, in all seriousness, "How much better would this be if D-Lo were competing against Bully?" That's, like, something Maddox would say.
Now, that being that, it was nice seeing Lockdown in front of a larger, non-Impact Zone crowd. Jeff suffered a concussion thanks to Bully's top rope Bully Bomb so he'll probably be down and out for a while. Also, the triple Tag Team match, with Chavo/Hernandez vs. Aries/Roode vs. Daniels/Kaz, was excellent. But will this mark, finally finally finally, the moment that TNA takes off and soars to new heights? On Impact, Hogan literally said, of his time in TNA, that "this small company became... a good little company!" So apparently, after years of saying "Now's the time!," now is the time.
Speaking of finally, after years of litigation, that I'm positive TNA would have stretched out for years and years to come, Daffney got a settlement deal out of them for her Workers Comp claim.
From her Twitter.
"I'd like to take this opportunity to sincerely thank all the love and support over the past two years. I appreciate so much that you recognize the HUGE injustice that is being done to Professional Wrestlers throughout the industry. I hope my case has opened up a few blind eyes and maybe now we, the wrestlers who put our bodies on the line every time we step in the ring, are one step closer to being provided with medical coverage and all the benefits that go along with being a true employee and not an independent contractor."
Again, this all comes down to that loophole that wrestling promotions use to deny their workers compensation and benefits by categorizing them as "contractors" and not "employees." There's nothing about this practice that isn't universally s***ty. The more we can all chip away at this boulder of garbage the better.
Let's touch d***s online. Sword fight, social media-style! I can show you the world. Don't you dare close your eyes. Also, if you do "like" thing to my Facebook page, you get the Wrap Up cyber-delivered to your news feed.
Hobnobbin' Kneelift!
Watch Wrestling!


text: An enormous portion of his net worth comes from his participation into film and music business. Every one of these occupations of his net add lots of sales to the total number of Juicy J net worth. His net is also called an Academy Award winner which he received due …Read More »


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SPECIAL UPDATE FOR SUNDAY, Aug. 25th.
Exceptionally, we will be jamming on Oderberger Str., near the entrance of Mauerpark, at around noon. There is a festival on the baskett-ball field, so we moved to an alternative location a few steps away. https://www.facebook.com/events/1405719052981773/?ref=22
On Sunday afternoons, from about noon to 14h, we will be jamming, improvising and dancing at Mauerpark, in Berlin. Find us on the baskett-ball field in front of the amphitheatre.
We'll do it on those Sundays: Aug.18, Aug. 25, Sept.1, Sept.15
Follow us on Facebook to make sure we are there.
Is there an ice rink in Mauerpark? Not yet. But we have roller-skates and developed a way to dance on those, as a summer side-project.
We are currently living in Prenzlauer Berg and are happy to make this little gift to our temporary local community.
What we do on those improvised dance sessions on wheels is only spontaneous and far from the more sophisticated artistry of our creations for the ice. But it's still quite fun!
If you like dancing or rolling yourself, why not join in?
We hope this will convince Berliners to give us a chance to initiate them to contemporary ice skating, at our proper performances at Erika-Hess-Eisstadion, not far from Mauerpark.
We'll be on that black square, the baskett-ball field.
A few videos of us jamming.


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The Preston Passion – A contemporary and dramatic re-telling of the Passion story, featuring an exciting combination of live performance and spectacle, coupled with innovative pre-recorded drama, presented by Fern Cotton.
The ambitious Preston Passion live spectacle aims to involve 3,500 local residents as performers and spectators. In addition to local choirs singing traditional Easter music, large-scale choreographed crowd movements across the city will re-create 'human crosses'. The stunning live spectacle will also include contemporary dance pieces and traditional local brass band music. And, to close Preston Passion's live programme of performance, M People's Heather Small will sing Candy Staton's gospel classic, You Got The Love.
The three 13-minute pre-recorded dramas are the culmination of detailed research into the history of Preston and its community undertaken by the writers Colin Heber-Percy and Lyall Watson (Casualty 1909).
Channel: BBC1
Producer: BBC North / Walk the Plank
TX: 6th April 2012
Source: BBC press release


text:
Britain's economy grew by 1.1 percent in the second quarter of 2010 from output in the previous three months, far higher than market expectations, official data showed on Friday.
"Gross domestic product (GDP) increased 1.1 percent in the second quarter of 2010," the Office for National Statistics (ONS) said in a statement. Analysts' consensus forecast had been for 0.6-percent expansion in the April-June period.
"The increase in output was due mainly to increases in business services and finance and construction," the ONS added.


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Acer Mobile Service Center in Davangere
Acer Mobile Service Center in Davangere, Karnataka – Address, Contact Number are listed below. If you have any problem with your Acer mobile, kindly contact the service centers officials. There is 1 authorized mobile service centers in Davangere
List of Acer Mobile Service Centers in Davangere:
Acer mobile service center in Davangere
Name: Microline Technologies Pvt Ltd
Address: #2931/1,Vidyanagar,(E) Davangere 577005
City: Davangere
State: Karnataka
Pincode: 577005
Contact Number 1: 08192-260306
Contact Number 2: *
Contact Number 3: *
Email: email@example.com
Working Hours: 10:00 AM to 6:00 PM
Working Days: Monday to Saturday [Sunday Holiday]
Categories: All Product Support


text:
Ted Crawford
Listed by Prestige Real Estate Of Killington
Shuttle to the mountain and ski home to your own backyard. Beautiful contemporary condominium with cathedral ceilings, wood fireplace, master bedroom suite and open floor plan. Lots of windows, efficient gas heat and Energy Star appliances. Unit is right next to ammenities of indoor pool with jacuzzi tub. Extremely well managed association and beautifully maintained grounds.
Date Showings Begin:
Delayed Showing: No


text:
OLYMPIA – The Department of Corrections today announced initial operational changes following the death of a correctional officer at Monroe Correctional Complex.
The agency will pursue the following changes immediately:
Prisons will begin counting staff members whenever an offender cannot be accounted for.
Response and Movement Officers will begin monitoring single-officer posts on a periodic basis.
Prisons will conduct drills on the use of silent alarms that are on all hand-held radios.
The agency will no longer have one-day modified lockdowns at all eight major prisons. The agency began the pre-planned lockdowns in November so that additional staff members would have temporary layoffs.
"We will take every action we can to make prisons safer for our staff," Secretary Eldon Vail said. "These are only the initial actions that we will pursue immediately. We will make additional changes once we conduct a thorough review of all our policies and procedures."
At 1:35 p.m. Sunday all 13 prisons will have a moment of silence for Correctional Officer Jayme Biendl. Sunday would have been her 35th birthday.


text: Good evening my Black hat beauties! I'll get straight to the point my uncle has been trying to sell his holiday home for some time but going about it in an extremely half arsed way. Either way after a conversation about it the estate agent he was using planned to take 20% I'm sure you've guessed it by now but I can take the same cut if I can get a buyer for the house. (Asking price is about £600k) The question I have is; Would/could I get in any sort of trouble for a mass listing on Craig's list? I've never really used it as all my IM'ing experience is in niche sites and auto blogs Other suggestions would be greatly appreciated Thanks


text:
The Chinese media is reporting this story in the mainland, while a deepening sense of impasse grips Hong Kong after four weeks of pro-democracy protests. Government officials see limited options to end this crisis and demonstrators are now willing to confront police.
Dozens were injured after clashes in the Chinese-controlled city, including 22 police officers. So far a few have been arrested on Sunday for assault. Protesters remain on the streets.
Hopes of easing the worst political crisis in Hong Kong since Britain handed the free-wheeling city back to China in 1997 rest on talks between the government and student protest leaders.
But few are expecting any resolution given the two sides are poles apart on how the city will elect its next leader in 2017.
It is interesting to see the Chinese government exercise unusual restraint and allow limited news coverage of this developing news story.
City Island Images is proud to offer "Front Pages" from newspapers all over the globe. They are today's chosen front pages from many cities and countries. For copyright protection, watermarks are occasionally placed on front pages that cover news events of historic significance.
Through a special agreement with more than 800 newspapers worldwide, the Newseum displays these front pages each day on its website. The front pages are in their original, unedited form, and some may contain material that is deemed objectionable to some visitors. Discretion is advised.
Anyone seeking permission to use a front page must credit the Newseum and contact the newspaper directly for permission. U.S. copyright laws apply.


text:
|01 Jul 2011
Windows 7 Home Premium 64bit
Onboard graphix won't let go
Im having problems updating my video drivers. I have a Gateway DX4600 with Windows 7 home premium 64 bit. I go to AMD and get the correct drivers and it goes through its install process but always hits the same wall. It tells me I have the incorrect drivers and when I look at the log it has my onboard video listed and not the HD4650 that my monitor is plugged into. I have gone into the device manager and it has both cards listed. I have disabled the onboard one but it keeps re-enabling itself whenever the system if restarted. Please help me get this issue fixed.
|My System Specs
|01 Jul 2011
Windows 7 Ultimate SP1 (64-bit)
Hello there, Moolro. Welcome to SevenForums.
Have you disabled the Onboard GFX Card in the BIOS instead of disabling it via device manager? that could be the problem.
Try to do this:
1) uninstall the video driver (if you have one installed)
2) boot up and go into the BIOS, disable the onboard graphics (can't tell you exactly where is that option)
3) keep the monitor connected to the HD4650, boot into windows
4) install updated drivers and directx
5) chill back and enjoy some nice coffee.
Here's what you need:
AMD Graphics Drivers & Software
Download Details - Microsoft Download Center - DirectX End-User Runtime
|My System Specs
|Thread Tools
|Similar help and support threads
Onboard Graphics Gives Out
I'm not quite sure how to describe the issue(hence the title) but through several diagnosis I think the Graphics on the motherboard is failing. There is no add-on graphics, the system is a HP Pavilion P6310Y, AMD Athlon 2, 6GB of RAM, 1 TB of HDD Space. The onboard Graphics is NVidia GeForce...
|Graphic Cards
graphix problems
Recently we had a power failure in our area, and it seems it altered the graphix on my computer, everything now looks a lot bigger and different than before the resolution is set at the same size (1440x900) Ive tried: reinstalling the ati drivers for the card, no use Ive tried resetting all...
|Graphic Cards
Onboard vs Win 7 RAID 1
Hi Been looking around and decided just to ask directly. What is the better option in terms of performance and future proofing (reinstalling windows or upgrading hardware) for a RAID 1 Array; Windows or an onboard MB controller? I have 2 WD 1.5TB drives i am going to be using for data only...
|Hardware & Devices
A little help with onboard graphics!!
Where is the Onboard Graphics here? I entered Integrated Prepherals menu when i pressed Del on the restart screen and i see these: Onboard IDE Controller: Both OnBoard PCI S-ATA Controller: IDE USB Controller: Enabled Legacy USB Support: Enabled OnBoard AUDIO Function: Enabled Onboard LAN...
|Graphic Cards
Onboard LAN drivers
Is there a universal Microsoft driver for on board Ethernet?? I have a Gigabyte GA-M57SLI-S4 (rev. 2.0) Onboard Lan Lan Chipset Marvell 88E1116 Max Lan Speed 10/100/1000Mbps I don't want to buy a new Ethernet card but looks like I can never find a solution. Whenever doing gaming on...
Onboard Sound
I have seen a couple threads about having RealTec onboard sound and when Win 7 is installed there is a problem. Is this a common thing? If I get a new sound card, would it automatically disable my onboard sound?
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text:
Quick Details
- Standard or Nonstandard: Standard
- Structure: Safety
- Pressure: Other
- Power: Manual
- Material: Casting
- Temperature of Media: Medium Temperature
- Media: Oil
- Port Size: Standard
- Place of Origin: Zhejiang, China (Mainland)
- Model Number: CHQIE-DN40(1-1/2")
- Brand Name: Chunhui
- Application: Fuel dispenser parts
- Keyword: cut off valve
Packaging & Delivery
|Packaging Details
|cut off valve
carton box, wooden package
|Delivery Time
|Shipped in 15 days after payment
home>>product categories>>hot product
Alibaba hot sell Emergency cut off valve/ shut off solenoid valve
|Inlet connection thread(female thread)
|Outlet connection thread(female thread)
|Cut off torque
|Other description
|Product features
|High quality, cost-effective
|Market share
|70% in China market
|Cooperation case
|Hengshan, Gilbarco, Censtar,Furen,Dresser, Jia Li Jia
|domestic explosion-proof certification
|Shortlisted in the suppliers of sinopec or not
|stablize existing main customers, develop international market, try to be the international leading brand in the industry
|Brief introductio
|the cut off valve is the reliable protection device of the submerged pump fuel dispenser, it uses metable material(melting point 76℃) and cut off ring, when there has fire or the fuel dispenser is bumped and damaged , the valve core can seal off automatically, and the oil flow of the pipeline will be cut off, so that the danger is prevented
Transaction History of the Supplier
Below is the information about the supplier's transactions conducted via Alibaba.com. If you require further details regarding the transaction data, please contact the supplier directly.
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