text:
We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase α, DNA polymerase δ and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell‐cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross‐linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G2/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.
DNA replication in eukaryotic cells takes place during a restricted period of the cell cycle: the S phase. The transition from G1 into S phase in mammalian cells is regulated by at least two cyclin‐dependent kinases (Cdks), cyclin E/Cdk2 and cyclin A/Cdk2. Cyclin E/Cdk2 activity peaks in late G1, while cyclin A/Cdk2 activity appears later, at the onset of DNA synthesis. Cyclin A is also required later for the S/G2 transition (Pagano et al., 1992). The initiation of chromosomal DNA replication in eukaryotes can be divided into two major events (Diffley, 1996; Dutta and Bell, 1997). The first event takes place during G1 phase, when a pre‐initiation complex is formed at the origin of replication by the ORC proteins. The second event occurs during G1/S transition, when Cdks promote the conversion of the pre‐initiation complex into an active replication form through the sequential recruitment of several proteins such as Cdc6, minichromosome maintenance proteins (MCMs), Cdc45, replication protein A (RP‐A) and DNA polymerase (pol) α. The Cdks appear also to exert a negative effect on DNA replication by catalysing a series of phosphorylation events that render these replication factors unable to re‐enter the pre‐initiation state, thus preventing re‐replication of the genome (Jallepalli and Kelly, 1997). Thus, a physical link between Cdk/cyclin complexes and essential replication factors at the origin of replication has been proposed (Fotedar and Roberts, 1991; Jaumot et al., 1994; Jallepalli and Kelly, 1997). DNA replication occurs in specific nuclear compartments, termed replication factories (Hozak et al., 1993, 1994; Montecucco et al., 1995), containing not only replication factors (Montecucco et al., 1998), but also other proteins that are not directly involved in DNA replication, such as cell cycle regulator factors (Cardoso et al., 1993; Sobczak‐Thepot et al., 1993) and DNA methyl transferase (Leonhardt et al., 1992). Each replication factory undergoes an assembly/disassembly cycle reflecting the recruitment of replicative factors to the replicons and the subsequent release of the same factors upon the completion of DNA synthesis (Dimitrova and Gilbert, 2000; Leonhardt et al., 2000; Montecucco et al., 2001).
The essential function of replicating DNA in eukaryotic cells is performed by a network of enzymes and proteins, which co‐operate together to rapidly and accurately duplicate the genetic information (Hübscher et al., 1996, 2002; Wang, 1996). Several reports have described the isolation of large macromolecular complexes of replication proteins from the extracts of eukaryotic cells (Jackson and Cook, 1986; Vishwanatha et al., 1986; Hickey et al., 1988). A fully functional 18–21S multiprotein complex called DNA synthesome was isolated from HeLa cells (Malkas et al., 1990; Applegren et al., 1995), HL60 cells (Lin et al., 1997b) and MDA MB‐468 human breast cancer cells (Coll et al., 1996, 1997). Using a combination of immunoblot analysis and functional assays, pol δ, DNA ligase I, DNA topoisomerase I (topo I) and II, replication factor C (RF‐C), RP‐A, proliferating cell nuclear antigen (PCNA), DNA helicase, pol α/primase and poly(ADP–ribose) polymerase (PARP) were found to be part of these complexes. The DNA synthesome was shown to be fully competent to support origin‐specific simian virus 40 (SV40) DNA replication in vitro. Studies on the organizational status of the DNA synthesome either in terminal differentiated or temporarily G1‐arrested HL60 cells (Lin et al., 1997a) showed that it remained inactive but intact in temporarily growth‐arrested cells, but disassembled in differentiated cells. Characterization of a replication‐competent (RC) multiprotein complex from calf thymus containing pol α, pol δ and RF‐C has been also described, and its biochemical and enzymological analysis suggested the existence of an asymmetric DNA polymerase complex in mammalian cells (Maga and Hübscher, 1996).
In an attempt to study the composition and regulation of the RC complex in human cells, we isolated from HeLa cells synchronized in G1/S, S and G2/M phases of the cell cycle a multiprotein complex that contains DNA replication proteins such as pol α, pol δ, DNA ligase I, RP‐A, RF‐C, topo I and PARP, together with cyclins and Cdks. In particular, we showed that cyclin A and Cdk2 associated with the complex in S phase, whereas cyclin B1 and Cdk1 were present only in G2 phase. This complex was capable of RF‐C dependent and independent DNA synthesis activity and also displayed histone H1 kinase activity. Cross‐linking of proteins to DNA in vivo in synchronized HeLa cells demonstrated the association of this complex to the chromatin and allowed the study of its composition along the cell cycle.
Isolation of an RC complex associated with cell cycle regulatory proteins from HeLa cells
NaCl nuclear extracts (0.2 M) from asynchronous HeLa cells were first loaded on a gel filtration column and the presence of the different proteins involved in DNA synthesis and cell cycle regulation (Cdk/cyclins) was systematically followed throughout the purification by immunoblot analysis. As shown in Figure 1A, DNA replication proteins (RF‐C, pol α) co‐eluted with cell cycle regulation factors such as cyclin A and Cdks with a retention volume of 9 ml, corresponding to a protein complex with an apparent mol. wt >669 kDa. The same proteins also eluted according to their respective weight: for example, RF‐C (p140) and pol α (p180) large sub units were found also in fraction 23, corresponding to a molecular weight range of 150–200 kDa (Figure 1A). When pol activity was assayed (see Materials and methods), two peaks of activity were detected in fractions 9 and 23, respectively, matching the elution profile of pol α (Figure 1A).
Fractions containing to the peak of pol activity, were pooled and loaded onto a Mono S column. Proteins were eluted with a non‐linear gradient from 0.1 to 1 M NaCl and the fractions were analysed as described above. Immuno blot analysis showed that pol α, cyclin A, RF‐C and Cdks co‐eluted between 0.25 and 0.3 M NaCl (Figure 1B). Pol activity, assayed on poly(dA)/oligo(dT), peaked in fraction 10 (Figure 1B). Histone H1 kinase activity was found to co‐elute in the same fractions (data not shown). Fractions 8–12 were pooled and loaded on a heparin–Sepharose column. Proteins were eluted using a linear gradient of NaCl from 0.1 to 1 M. Pol α, RF‐C, cyclin A and Cdk2, revealed by immunoblot analysis, co‐eluted in fractions 28–33 (Figure 1C) and pol activity peaked in fraction 30 (Figure 1C). Histone H1 kinase activity was also found to co‐elute in the same fractions (data not shown).
Finally, the active fractions from heparin–Sepharose were pooled and loaded onto a Mono Q column. As shown in Figure 1D, pol α, RF‐C, cyclin A and Cdk2 co‐eluted in fractions 14–17. Pol activity peaked in fraction 15. Histone H1 kinase activity was also found to co‐elute in the same fractions (data not shown; see Figure 4B). The purification procedure is summarized in Table I. Thus, a multiprotein complex containing pol α, RF‐C, cyclin A and Cdk2 could be isolated from a HeLa cell nuclear extract. This complex was a relatively stable entity and survived successive purification steps.
DNA replication proteins and cell cycle regulatory factors co‐sedimented in a sucrose gradient as a high molecular weight complex
In order to confirm the presence of a multiprotein complex containing DNA replication and cell cycle regulatory factors, a 0.2 M NaCl nuclear extract from asynchronous HeLa cells was layered on a pre‐formed 10–40% sucrose gradient (see Materials and methods). After ultracentrifugation, the gradient was fractionated from the bottom of the tube and the protein distribution profile was monitored spectrophotometrically. As shown in Figure 2A, two peaks of polymerase activity were detected, one sedimenting faster than the thyroglobulin marker (19.2S) and the second one between 11.3S and 7.1S. The dot‐blot immunoassays shown in Figure 2B confirmed the presence of cyclin A, Cdk2 and pol α in the high molecular weight activity peak. In addition, pol α and Cdk2 were also sedimenting according to their native molecular weight.
The RC complex isolated from synchronized HeLa cells contains both DNA replication proteins and cell cycle regulatory factors
In order to determine the protein composition and to study the functional activities of the RC complex, we isolated the complex as described above from HeLa cells synchronized in S phase. The fractions of interest were analysed by SDS–PAGE and western blotting with different antibodies. As shown in Figure 3A, in the final purification step (Mono Q fraction) we detected the pol α p180, pol δ p125 and pol ϵ p210 large subunits, RF‐C (subunits p140 and p40), RP‐A (subunits p70, p32, p14), DNA ligase I, topo I, PARP together with cyclin A and Cdk2. No PCNA and cyclin E could be detected (data not shown). We confirmed the physical association of these proteins by two approaches. First, the Mono Q fraction was immunoprecipitated with antibodies against cyclin A and DNA ligase I. As shown in Figure 3B, anti‐cyclin A antibodies co‐immunoprecipitated the cognate antigen as well as other RC components, such as DNA ligase I, pol ϵ and pol δ. Control experiments shown in Figure 3B, bottom panel, demonstrated that antibodies against DNA ligase I also co‐immunoprecipitated cyclin A. Secondly, aliquots of fraction 9 of gel filtration were electrophoresed through a 5% native polyacrylamide gel followed by immunoblotting with antibodies directed against RF‐C p140, cyclin A and Cdk2 (Figure 3C). All the antibodies stained a band >669 kDa. Together, these results suggest that the proteins, purified through four different chromatographic steps, were part of high molecular weight multiprotein complexes.
The RC complex isolated from HeLa cells is active in DNA replication
Next, the pol activity within the RC complex was assayed on a singly primed (sp) M13 DNA template. In the presence of PCNA and Escherichia coli single‐stranded DNA binding protein (SSB), this template can be replicated either in an RF‐C‐independent (pol α) distributive way or in a RF‐C‐dependent (pol δ or pol ϵ) processive manner. Reaction products were separated on a 4% polyacrylamide gel containing 5 M urea. As shown in Figure 3D, lane 1, in the absence of PCNA and SSB, only short products could be detected. The synthesis of these products was inhibited by SJK 132‐20, a specific inhibitory antibody of pol α (compare lane 2 with lane 1), suggesting that they were due to the distributive activity of pol α. The addition of SSB and PCNA (lane 3) induced the synthesis of longer products and the concomitant decrease in the synthesis of short products. PCNA and SSB are known to inhibit pol α activity, whereas in the presence of ATP and RF‐C they allow processive synthesis by pol δ and/or pol ϵ (Maga and Hübscher, 1996). Addition of the pol α inhibitory antibody under these conditions did not abolish the synthesis of the larger products, showing that they were not due to pol α synthesis (compare lane 4 with lane 3). These results indicated that the RC complex was able to perform a switch from a distributive to a processive mode of DNA synthesis, which was dependent on RF‐C and PCNA, as has already shown with purified components (Maga et al., 2000).
Analysis of the cyclin/Cdk activity associated with the RC complex
Histone H1 phosphorylation activity was found in the final fraction of the RC complex (Figure 4A). To confirm that the phosphorylation was due to Cdk activity and not to other kinases present in the fractions, we treated the samples with olomoucin (500 μM; Vesely et al., 1994) or roscovitine (10 or 18 μM; De Azevedo et al., 1997), two potent inhibitors of Cdks that act as competitive inhibitors for ATP. The histone H1 phosphorylation activity in fractions obtained by gel filtration of S phase or G2/M phase HeLa cell extracts were inhibited by both drugs (Figure 4B). The most purified RC fraction (Mono Q) was also tested in a histone H1 phosphorylation assay in the absence or in the presence of the specific Cdk‐inhibiting protein p21cip. As shown in Figure 4B, bottom panel, already at the lowest concentration tested p21 completely abolished kinase activity. As shown in Figure 4A, a few other proteins contained in these fractions were also phosphorylated. In several experiments, we always observed the presence of phosphorylated bands with apparent molecular weights of 130, 70 and 18 kDa, respectively. The phosphorylation patterns detected by SDS–PAGE with the RC complex purified at different stages of the cell cycle were also analysed. The phosphoprotein with molecular weight of 18 kDa was present from G1/S borders to G2/M, whereas the phosphoproteins with molecular weights of 70 and 130 kDa were present only during S phase. In addition, a phosphoprotein with a higher molecular weight (170 kDa) was found at the beginning of S phase only (data not shown). Fraction 9 of the gel filtration was also tested in a kinase assay and when analysed on a native gel showed one phosphorylated band with a molecular weight >669 kDa (Figure 4C).
Analysis of the RC complex composition at different stages of the cell cycle
Next we studied the dynamics and structure of the multiprotein complex during the cell cycle. HeLa cells were synchronized using a double thymidine block. Aliquots were withdrawn at different times after block release and cell cycle distribution was determined by flow cytometry (Figure 5A). The RC complexes from 0.2 M NaCl nuclear extracts of HeLa cells synchronized in G1/S, early S, late S and G2/M phases were purified and analysed as described before.
The cell cycle regulatory proteins showed a dynamic association with the complex (Figure 5B): cyclin A was detected within the complex from G1/S to G2 phase, while Cdk2 was associated with the complex along the S phase but disappeared in G2. Interestingly, Cdk1 and cyclin B1 associated with the complex only at G2/M. Pol α, DNA ligase I and RF‐C were detected in the complex throughout the S phase. Interestingly, DNA ligase I was not found associated with the RC complex in G2/M. Total pol activity was measured in the samples obtained from synchronized HeLa cells and it steadily increased along the S phase and decreased in G2/M (Figure 5C).
Thus, the protein composition of the RC complex varied during the cell cycle, indicating that this higher order complex is a dynamic entity.
Cross‐linking of the RC complex to chromatin in vivo
To investigate whether the multiprotein complex was associated with chromatin, we in vivo cross‐linked proteins to DNA with formaldehyde in synchronized HeLa cells. We used a modified cross‐linking procedure (Göhring and Fackelmayer, 1997) that minimizes the formation of non‐specific cross‐linking and largely excludes contamination with non‐cross‐linked material. DNA was labelled in vivo with tritiated thymidine and HeLa cells were synchronized in S phase or G2/M phase as described above, before treatment with formaldehyde, which prevents artefactual redistribution of proteins along the DNA by freezing the native conformation of chromatin. Then, DNA–protein complexes were isolated by centrifugation in CsCl gradients. Density of individual fractions was determined by refractometry and relative DNA concentrations as well as the position of DNA–protein complexes were measured by liquid scintillation counting. As shown in Figure 6A, DNA–protein complexes were found at a density of 1.4 g/ml corresponding to a mass ratio DNA–protein of 1:1, characteristic of native chromatin (Göhring and Fackelmayer, 1997). Purified complexes were de‐cross‐linked by boiling in SDS–PAGE sample buffer and analysed for their protein components by gel electrophoresis. Based on the intensities of the Coomassie Blue staining, we were able to verify that the amount of proteins loaded on the gel and subsequently analysed by western blot was comparable for all the cell cycle points. Immunoblot analysis was performed to determine whether the proteins of the RC complex were cross‐linked to DNA in HeLa cells synchronized in S and G2 phase. RF‐C, RP‐A, PCNA, DNA ligase I and topo I were found in DNA–protein complexes as the cell cycle regulatory protein cyclin A. Interestingly Cdk1, Cdk2 and cyclin B1 were not found in the DNA–protein complexes and were detected in the fraction of free proteins (Figure 6B). The presence of the RC complex in the free protein fraction was confirmed by native gel electrophoresis (Figure 6C). The complex isolated from 0.2 M NaCl nuclear extracts (Figure 1), probably represents a fraction that is not stably associated with the chromatin. Thus, by comparing the proteins present in the chromatin‐bound and in the free protein fractions of the CsCl gradient, with the proteins purified as a complex by chromatography, it appeared that the composition of the multiprotein complex seem to change when it was either unbound or associated with the chromatin. PCNA, on the other hand, was found within the complex bound to the chromatin (Figure 6B), whereas it cannot be detected in the unbound complex (Figure 5B). Cyclin A was always present (Figures 4B and 5B), whereas Cdk2 and Cdk1/cyclin B1 were found associated with this complex in the unbound form both in S phase and G2 phase (Figures 4B and 5B).
It is known that upon hypotonic lysis of eukaryotic cells, the bulk of pol activity is in the cytosolic fraction, and this approach has been used, for example, to purify all the replicative pols from calf thymus (Weiser et al., 1991). Other DNA replication enzymes, for example RF‐C, are retained in the nucleus and standard purification protocols include a high salt (0.35 M NaCl) nuclear extraction (Tsurimoto and Stillman, 1989). When we analysed HeLa cell cytosolic extracts by gel filtration, very little pol activity eluted with an apparent high molecular weight, and the three major replicative pols (α, δ and ϵ) eluted at a position corresponding to their native molecular weights, as confirmed by western blot analysis (data not shown). Gel filtration analysis of 0.35 M NaCl nuclear extracts showed a similar elution profile, with very little pol activity associated with high molecular weight material. On the other hand, we found that mild nuclear extraction conditions (0.2 M NaCl), as well as buffer optimization (for example with the inclusion of ATP), allowed the preservation of a high molecular weight complex with associated pol activity. These observations might explain why classical purification protocols for DNA replication enzymes failed to detect such large complexes. Thus, in this paper we present the purification of a RC complex containing DNA replication proteins like pol α, pol δ, RF‐C, RP‐A, DNA ligase I and topo I, and cell cycle regulatory factors like cyclin A, cyclin B1, Cdk2 and Cdk1 through different chromatographic columns (gel filtration, Mono S, heparin–Sepharose, Mono Q) from 0.2 M NaCl nuclear extracts of HeLa cells synchronized at different points of the cell cycle. Gel filtration and native PAGE showed that these proteins were associated in multiprotein complex with a molecular weight >600 kDa, which exists from the G1/S border throughout the entire S phase. This multiprotein complex displayed both an RF‐C‐independent and an RF‐C‐dependent polymerase activity, with a switch from a distributive to a processive mode of DNA synthesis that was dependent on PCNA. Immunoblot analysis showed that, while cyclin A was associated with the complex from G1/S to mitosis, Cdk2 was present in the complex only during S phase and disappeared in G2. It is very probable that in G2/M, Cdk2 is replaced by Cdk1, which was found to be one component of the complex, together with the cognate cyclin B1 only in G2/M phase. The complex contains a kinase activity able to phosphorylate exogenous histone H1 and different proteins within the complex itself. Altogether, our data suggest the existence of a multiprotein complex competent for DNA synthesis, the composition and activity of which could be regulated by specific cyclin/Cdk complexes during the cell cycle.
As already described for the RC complex isolated from calf thymus (Maga and Hübscher, 1996), we did not find PCNA associated with the complex. However, PCNA is known to interact with multiple protein partners (Jónsson and Hübscher, 1997; Loor et al., 1997; Warbrick, 2000), including the replication proteins pol δ (Zhang et al., 1999) and pol ϵ (Maga et al., 1999), RF‐C (Mossi et al., 1997), Fen1 (Li et al., 1995), DNA ligase I (Levin et al., 1997; Montecucco et al., 1998), topo I and the cell‐cycle regulation factors cyclin/Cdks (Xiong et al., 1992; Koundrioukoff et al., 2000), which are part of this complex. The complex isolated after 0.2 M NaCl nuclear extraction probably represents a soluble or loosely DNA‐associated form. One possibility to account for the lack of PCNA is to hypothesize that PCNA remains bound to the chromatin, which is consistent with its proposed role to tether the RC complex to DNA. Previous experiments, have shown that the association of DNA ligase I with PCNA was cell cycle dependent (Rossi et al., 1999) and DNA ligase I could not be co‐immunoprecipitated with PCNA in G2/M. Accordingly, DNA ligase I was not found in the RC complex in G2/M, suggesting a possible common regulatory mechanism for the association of DNA ligase I with replication proteins. Cross‐linking of proteins to DNA in vivo in synchronized HeLa cells supported this hypothesis. These experiments revealed the presence of PCNA, along with the DNA replication proteins RF‐C, DNA ligase I and topo I in the cross‐linked chromatin. Interestingly, a cell cycle regulatory factor, cyclin A, was also found within the chromatin‐associated complex. Cyclin A has been already found associated with replicating DNA (Fotedar and Roberts, 1991) and co‐localized with replication foci in S‐phase nuclei (Cardoso et al., 1993; Sobczak‐Thepot et al., 1993). In addition, Kim and Kaelin (2001) have hypothesized that cyclin A and cyclin E might be concentrated at certain regions of genome in a temporally controlled manner by virtue of their ability to form stable complexes with specific proteins that directly or indirectly bind to DNA.
We did not find Cdk2 associated with the chromatin‐bound complex in S phase, but only in the free complex. Similarly, Cdk1/cyclin B1 were associated exclusively with unbound complexes in G2/M. In a previous study carried out in asynchronous HeLa cells (Jaumot et al., 1994), it was shown that cyclin A and Cdk2 co‐migrated with the replicase complex on sucrose gradient, whereas only small amounts of Cdk1/cyclin B1 were found in the same fractions. By enriching cells in, respectively, S and G2/M phases, we were able to distinguish which complex (either free or chromatin bound) contained Cdk1/cyclin B1 or Cdk2/cyclin A. Cdk2/cyclin A has been shown to co‐localize at the nuclear foci (Cardoso et al., 1993) and to interact directly with PCNA. This PCNA–Cdk2/cyclin A complex was active in phosphorylating the PCNA binding region of RF‐C p145 as well as DNA ligase I (Koundrioukoff et al., 2000). Thus, our results suggest the presence of two complexes: (i) one bound to the chromatin that contains replication proteins, cyclin A and no Cdks; and (ii) a soluble complex in the nucleus containing the same replicative proteins as the chromatin bound complexes, except PCNA, which is absent. This DNA‐unbound complex is associated with Cdk2/cyclin A in S phase and Cdk1/cyclin A and B1 in G2 phase. This complex displays a kinase activity that is due to Cdks as confirmed by the inhibition by roscovitine, olomoucine and the Cdk inhibitor p21. Cdk/cyclins are known to phosphorylate several DNA replication proteins, such as SV40 T antigen (McVey et al., 1993), RP‐A (Dutta and Stillman, 1992), pol α (Nasheuer et al., 1991), pol δ (Zeng et al., 1994) and PCNA (Prosperi et al., 1994). Accordingly, we have detected Cdk‐dependent phosphorylation of different proteins within our complex. Cdk‐dependent phosphorylation of DNA replication proteins appears to have a regulatory role. For example, cyclin A/Cdk2 has been shown to inhibit the replication activity of human pol α primase in an SV40 initiation assay, whereas the activities of pol α and the tightly associated primase were not impaired in simpler in vitro assays (Voitenleitner et al., 1997, 1999). In addition to the role in modulating the activity of DNA replication enzymes, our results seem to suggest a role for cyclin/Cdk complexes in regulating the association of replication complexes to chromatin during the cell cycle. It could be that a stable association of cyclin A to replication complexes during S phase has the role of recruiting Cdk2, which in turn can regulate the dynamic association of the replication proteins to the chromatin. This might represent an example of intra‐phase regulation, perhaps correlated to a different timing of origin firing. Cyclin A could have an 'informational' role, independent of its association to a Cdk, as suggested by Kim and Kaelin (2001) for its role in the repression of transcription during the S phase. The appearance of Cdk1/cyclin B1 associated with replication complexes in G2/M, concomitantly with the disappearance of Cdk2, could reflect an inter‐phase regulatory mechanism, which prevents re‐binding of replication complexes to chromatin during G2/M phase. This hypothesis fits well with the so‐called Cdk‐driven 'replication switch' model (Kelly and Brown, 2000), which predicts that Cdk activity serves both to activate initiation complexes and to inhibit further initiation complex assembly.
Materials and methods
[3H]dTTP (25 Ci/mmol), [3H]dTTP (30 Ci/mmol), [3H]dATP (73 Ci/mmol), [γ‐32P]ATP (3000 Ci/mmol) and [α‐32P]dTTP (3000 Ci/mmol) were from Amersham Pharmacia Biotech. GF/C fibre glass filters were purchased from Schleicher & Schuell. Nitrocellulose membrane (Hybond ECL) was from Amersham Pharmacia Biotech. Olomoucin and protease inhibitor cocktail were purchased from Sigma and roscovitine was a gift from E.Prosperi (CSI‐CNR, Pavia, Italy). All other reagents were of analytical grade and purchased from Sigma, Fluka and BDH.
Buffer A: 1 mM KH2PO4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA pH 8.0, 10% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF). Buffer B: buffer A containing 0.35% (v/v) Triton X‐100. Buffer C: phosphate‐buffered saline (PBS) containing 5 mM MgCl2, 1 mM EDTA pH 8.0, 10% (v/v) glycerol, 0.5 mM PMSF. Buffer D: 50 mM Tris–HCl pH 7.5, 0.35 M NaCl, 1 mM EDTA pH 8.0, 1 mM dithiothreitol (DTT), 10% (v/v) glycerol, 1 mM PMSF, 1/1000 protease inhibitor cocktail. Buffer E: buffer D containing 0.01% (v/v) Nonidet P‐40 (NP‐40). Buffer F: 25 mM bis‐Tris pH 6.6, 50 mM NaCl, 0.5 mM ATP, 1 mM DTT, 5% (v/v) glycerol, 1 mM PMSF. Buffer TDB: 50 mM Tris–HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.2 mg/ml bovine serum albumin (BSA).
Cell culture
HeLa cells S3 were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM; Sigma–Aldrich) supplemented with 10% fetal bovine serum (Sigma), 50 μg/ml gentamicin and 2 mM l‐glutamine. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. Synchronization of Hela S3 cells at the G1/S border was obtained by growing cells in 2 mM thymidine as described by Stein (1989). After thymidine removal, cells were washed twice with DMEM, harvested immediately (G1/S border) or further cultured in complete medium for 1.5 h (early S phase), 3 h (late S phase) or 6 h (G2/M phase). Cells were then harvested and washed three times with PBS. The cells were pelleted by low‐speed centrifugation (1500 r.p.m., 5 min, 4°C), and the cell pellets stored at −80°C until use.
Isolation of the RC complex
Isolation of HeLa cell nuclei. Cells (1 × 108) were resuspended in 1 ml of buffer A and lysed for 1 h on ice in 9 ml of buffer B. The suspension was centrifuged at 900 g for 10 min at 4°C in a Sorvall RC‐5B centrifuge, and the pellet containing the nuclei washed with 1.5 ml of buffer C and centrifuged at 900 g for 10 min at 4°C.
Preparation of nuclear extracts. Nuclei were resuspended in 1 ml of buffer D containing 0.2 M NaCl, stirred for 30 min at 4°C and centrifuged at 12 000 g for 30 min at 4°C. The supernatant was kept as the 0.2 M NaCl nuclear extract. The pellet was resuspended in 1 ml of buffer E containing 0.35 M NaCl, stirred for 1 h at 4°C and centrifuged at 10 000 g for 20 min at 4°C. The supernatant was kept as the 0.35 M NaCl nuclear extract.
Size exclusion chromatography. Half a millilitre of 0.2 M NaCl nuclear extract from HeLa cells was loaded onto a Superdex 200 HR 10/30 gel filtration column (Pharmacia) equilibrated with buffer F. The column was previously calibrated with blue dextran (2000 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), ovalbumin (43 kDa) and myoglobulin (17.5 kDa) as molecular weight standards. Each calibration was performed twice.
Mono S column chromatography. Gel filtration fractions that contained the proteins of interest were loaded on a Mono S column (Pharmacia) equilibrated with buffer F. Proteins were eluted with a non‐linear gradient from 0.1 to 1 M NaCl.
Heparin–Sepharose chromatography. Active fractions from Mono S column were pooled and loaded onto a HiTrap heparin–Sepharose column (Pharmacia) equilibrated with buffer F. The column was eluted with a linear gradient from 0.1 to 1 M NaCl.
Mono Q chromatography. Active fractions from heparin–Sepharose column were pooled and loaded onto a Mono Q column (Pharmacia) equilibrated with buffer F. The column was eluted with a linear gradient from 0.1 to 1 M NaCl.
Sucrose gradient analysis. Nine hundred micrograms of 0.2 M NaCl HeLa nuclear extract were layered on top of a 4.5 ml 10–40% sucrose gradient formed in buffer F (without glycerol) in a 13 × 51 mm Beckman polyallomer ultracentrifuge tube. The gradient was centrifuged in a Beckman SW‐50 rotor using a Beckman L8‐50 M/E ultracentrifuge at 40 000 r.p.m. for 16 h at 4°C. In a parallel run, molecular weight markers were loaded onto an identical gradient. Both gradients were fractionated from the bottom of the tube and the protein distribution monitored spectrophotometrically.
Immunoblot analysis of column fractions (spot test)
For spot test analysis, 4 μl of each fraction were spotted on a nitrocellulose membrane without electrophoretic separation and the membrane was treated as in standard immunoblot protocols.
For SDS–PAGE and western blot analysis, aliquots of the fractions that contained proteins of interest were electrophoresed in a 7.5 or 10% SDS–polyacrylamide gel and transferred by standard procedures to a nitrocellulose filter. The membrane was blocked for 1 h with PBS containing 10% newborn calf serum and 0.2% Tween (PTN) and then incubated for 3 h with the different primary antibodies. After washing with PBS containing 0.2% Tween‐20, the membrane was incubated with either anti‐mouse (Amersham) or anti‐rabbit (Pierce) IgG conjugated to horseradish peroxidase. Immunodetection was carried out using a light‐enhanced chemiluminescence (ECL) detection system according to manufacturer's instructions (Amersham, Arlington Heights, IL).
Native PAGE
Gel filtration fractions (30 μg) were resolved using a 5% native polyacrylamide gel with the exclusion of SDS in the polyacrylamide gel, running, sample buffer as described in Tom et al. (1996). 2‐mercaptoethanol was excluded from the sample buffer. All samples were electrophoresed at 4°C. Proteins were electrophoretically transferred to nitrocellulose at 12 V for 12–16 h at 4°C. Immunoblotting was carried out as described previously. Thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and BSA (66 kDa) were used as molecular weight standards (Amersham Pharmacia).
Mono Q fraction (5 μg) was ajusted to 10 mM Tris–HCl pH 7.4, 50 mM NaCl, 2.5 mM MgCl2 and protease inhibitor cocktail (IP buffer) and then were incubated for 1 h at 4°C with a suspension of protein A–Sepharose beads (Bio‐Rad) coupled with polyclonal L‐β antibodies against DNA ligase I (Rossi et al., 1999) or cyclin A. After centrifugation, the pellet was washed three times with IP buffer. The supernatant and the immunoprecipitated material were analysed by western blot analysis.
Antibodies against Cdc2 p34 sc‐54, Cdk2 sc‐748, Cdk2 sc‐6248, cyclin B1 sc‐245 and PCNA (clone PC10) were purchased from Santa Cruz Biotechnology. The antibody against cyclin A (clone CY‐A1) was purchased from Sigma. The polyclonal antibody against the 40 kDa subunit of human RF‐C was a gift from J.Hurwitz (Memorial Sloan‐Kettering Cancer Center, NY). Antibody against calf thymus pol α catalytic subunit was a gift from A.M.Holmes (Holmes et al., 1986). Antibody against the 140 kDa subunit of human RF‐C was a gift from B.Stillman (Cold Spring Harbor Laboratory, NY). Monoclonal antibody against the large subunit pol δ was a gift from H.P.Nasheuer (Abteilung Biochemie, Jena, Germany). Monoclonal antibody against human DNA ligase I (2B1) was produced by Rossi et al. (1999). Antiboby against RP‐A was as described by Georgaki and Hübscher (1993). Monoclonal antibody against PARP was a gift from G.Poirier (Université Laval, Québec, Canada). mAb 6B5 against DNA topo I was a gift from A.I.Scovassi (IGBE‐CNR, Pavia, Italy). The polyclonal antibody against the cyclin A was a gift from J.Sobczak‐Thépot (Université Paris 6, Paris, France).
Enzymatic assays
Pol α/pol ϵ. A final volume of 25 μl contained the following components: buffer TDB, 8 μM [3H]dNTP (1.5 Ci/mmol), 10 mM MgCl2 and 0.5 μg of poly(dA)/poly(dT).
Pol δ. A final volume of 25 μl contained the following components: buffer TDB, 8 μM [3H]dNTP (1.5 Ci/mmol), 10 mM MgCl2, 0.5 μg of poly(dA)/poly(dT) (10:1 base ratio), in the presence of PCNA (100 ng). All reactions were incubated for 30 min at 37°C and precipitated with 10% trichloroacetic acid. Insoluble radioactivity was counted in a Beckman liquid scintillation counter and all values were corrected by subtracting the blank values.
RF‐C‐dependent polymerase assay. A final volume of 10 μl contained the following components: 50 mM Tris–HCl, pH 7.5, 1 mM DTT, 0.2 mg/ml BSA, 10 mM MgCl2, 130 ng of sp DNA, 1 mM ATP, 10 mM MgCl2, dATP, dCTP, dGTP each at 100 μM and 0.1 μM [α‐32P]dTTP (3000 Ci/mmol). One hundred nanograms of PCNA and 500 ng of SSB, either alone or in combination, were added to the mixture. All reactions were incubated for 45 min at 37°C. The reaction was stopped by adding 1% SDS and 20 mM EDTA pH 8.0. Two microlitres of GBL 5X (10% w/v sucrose, 0.2% w/v Bromophenol Blue, 0.2% w/v xylene cyanol and 95% formamide) were added and samples were heated for 3 min at 95°C. Samples were loaded on a 4% polyacrylamide gel for 25 min at 250 V. The gel was fixed with 12% (v/v) methanol and 10% (v/v) acetic acid. DNA synthesis was visualized by PhosphorImager (Molecular Dynamics).
Kinase assay. This was carried out in a final volume of 10 μl containing histone‐kinase buffer [50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 0.5 μM [γ‐32P]ATP (3000 mCi/mmol), 0.4 mg/ml histone H1]. Reactions were carried out for 20 min at 37°C. Proteins were then separated by 10% SDS–PAGE and radioactive bands were visualized by PhosphorImager (Molecular Dynamics). For kinase inhibition assay, 0.5 μg of protein from gel filtration or 0.15 μg of Mono Q fraction were incubated with olomoucin (500 μM), roscovitine (10 and 18 μM) or p21cip (1, 2 and 10 μM) for 5 min at room temperature, and then kinase assay was performed as described above. One unit of kinase activity was defined as the amount of kinase incorporating 1 nmol of ATP into histone H1 in 60 min at 37°C under the conditions described above. For determination of the specific activity of the nuclear extract and of the active fractions throughout the purification, a recombinant Cdk2/cyclin A complex (0.4 mg/ml, 48 000 U/ml) was used as the standard reference.
Experiments were performed in duplicate. Three independent experiments were carried out.
In vivo cross‐linking experiments
HeLa S3 cells were grown on 145 mm plastic dishes in DMEM with 5% fetal calf serum (Sigma). For DNA labelling, the medium of semi‐confluent cultures was replaced by 20 ml of medium containing 15 μCi of [methyl‐3H]thymidine (25 Ci/mmol), and cells were incubated for 24 h under normal culture conditions. Then cells were synchronized using a double thymidine block.
Formaldehyde cross‐linking in vivo and DNA–protein complexes preparation were performed as described in Göhring and Fackelmayer (1997), with some modifications. Briefly, five dishes (145 mm) of HeLa cells, one of which contained labelled cells, were washed once with 37°C warm PBS and incubated for 4 min at room temperature in 20 ml of DMEM medium (without serum) containing 1% formaldehyde (freshly added from a 37% stock solution; Merck). The reaction was stopped with 2.2 ml/dish of a 7.5% solution of glycin in PBS. Cross‐linked cells were rinsed twice with cold PBS, scraped off the Petri dishes in 10 ml PBS with a rubber policeman, and centrifuged for 5 min at 800 g in a swinging bucket rotor. After resuspension in 10 ml of RSB buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl and 3 mM MgCl2 pH 8.0), cells were homogenized on ice by 15 strokes in a chilled Dounce homogenizer and nuclei were collected by centrifugation (8 min, 800 g). After nuclei were washed twice with RSB, and proteins were extracted in 10 ml of buffer E (10 mM Tris–HCl pH 8.0, 10 mM Na2S2O5, 1 M NaCl, 0.1% NP‐40, 1 mM EDTA, protease inhibitor cocktail). After extraction, nuclei were pelleted as above, resuspended in 2.7 ml of buffer E with 0.1 M NaCl, and lysed by addition of 0.3 ml of 20% sodium sarkosyl solution. The sample was then layered over a preformed CsCl step gradient consisting of 3 ml of 1.75 g/ml CsCl solution, 3.5 ml of 1.5 g/ml CsCl solution and 3 ml of 1.3 g/ml CsCl solution in a SW40 polyallomer ultracentrifugation tube; all CsCl solutions were prepared in 20 mM Tris–HCl pH 8.0, 1 mM EDTA and 0.5% sodium sarkosyl. The gradient was centrifuged for 24 h at 34 000 r.p.m. in an SW40 rotor at 20°C and then fractionated from the top in 18 aliquots of 700 μl each. DNA–protein complexes sedimented at a density of ∼1.4 g/ml and could usually be seen as a slightly turbid band by visual inspection of the gradient. Aliquots of the fraction were sheared by brief sonication, and the density of individual fractions was determined by refractometry. Relative DNA concentrations were measured by liquid scintillation counting. DNA‐containing fractions were briefly sonicated and dialysed against a buffer containing 20 mM Tris–HCl pH 8.0, 50 mM NaCl and 2 mM EDTA overnight. SDS sample buffer was then added and cross‐links were cleaved by incubation in a boiling water bath for 40 min. Samples were then analysed by SDS–PAGE and western blot as described above.
The authors thank E.Prosperi (Centro di Studio per l'Istochimica del CNR, Pavia, Italy) for the cell cycle distribution analysis by flow cytometry and Dr J.Breton (Pharmacia & Upjohn, Nerviano, Italy) for the recombinant Cdk 2/cyclin A complex. This work was supported by the EU‐TMR Grant (ERBMRXCT 970125) to S.S. and U.H., by the CNR‐Target Project on Biotechnology to S.S., by a grant from Associazione Italiana per la Ricerca sul Cancro to A.M., and from Ministero dell'Università e della Ricerca Scientifica e Tecnologica‐Consiglio Nazionale delle Ricerche 'Biomolecole per la salute umana' L. 95/95 to G.B. I.F. was supported by a fellowship from the EU‐TMR Network (grant ERBMRXCT 970125).
- Copyright © 2002 European Molecular Biology Organization


text:
Let's bring your vision to life.
At Left Coast Catering, we love helping our clients create memorable experiences built around exquisite food. Tell us about your vision. We'll bring it to life.
Custom Events
I want to create a memorable event.415-934-0600 (Ask for Kathryn)
Workplace Delivery
I need to order great food for my office.415-934-0600 (Ask for Jordan)
To create a delicious event
Ask for Kathryn
To order workplace delivery
Ask for Jordan
Employment inquiries:
Head Chef: Laurine Wickett
Photography and videos by Henry Dombey / Club Soda Productions.
Logo by Kevin Wansley / Zanaman Design
Site by Rena B Design
Logo by Kevin Wansley / Zanaman Design
Site by Rena B Design

subset: commoncrawl


text:
Imelda Light
Imelda Light
The Imelda Wall Light is handmade from wheel-thrown porcelain, each strand starts life as a circle on the potter's wheel and is then cut, folded and assembled to create a lattice.
All Imelda Lights are made to order as a simple lampshade for an existing light.
dia. 29 x 15cm
3-4 week delivery time
Delivery Costs:
Uk Next Day - Not available
Europe £14.50
Global £20.00
Delivery costs refer to a single transaction and will be the same no matter how many items are bought in one go.


text:
Please use this identifier to cite or link to this item:
|Evaluating the quality of research into a single prognostic biomarker: A systematic review and metaanalysis of 83 studies of C-reactive protein in stable coronary artery disease
|Hemingway, H.
Philipson, P.
Chen, R.
Fitzpatrick, N. K.
Damant, J.
Shipley, M.
McAllister, K. S. L.
Hingorani, A. D.
Abrams, Keith R.
Moreno, Santiago
Palmer, S.
Kaski, J. C.
Timmis, A. D.
|First Published:
|Public Library of Science
|PLoS Medicine, 2010, 7 (6)
|Background Systematic evaluations of the quality of research on a single prognostic biomarker are rare. We sought to evaluate the quality of prognostic research evidence for the association of C-reactive protein (CRP) with fatal and nonfatal events among patients with stable coronary disease. Methods and Findings We searched MEDLINE (1966 to 2009) and EMBASE (1980 to 2009) and selected prospective studies of patients with stable coronary disease, reporting a relative risk for the association of CRP with death and nonfatal cardiovascular events. We included 83 studies, reporting 61,684 patients and 6,485 outcome events. No study reported a prespecified statistical analysis protocol; only two studies reported the time elapsed (in months or years) between initial presentation of symptomatic coronary disease and inclusion in the study. Studies reported a median of seven items (of 17) from the REMARK reporting guidelines, with no evidence of change over time. The pooled relative risk for the top versus bottom third of CRP distribution was 1.97 (95% confidence interval [CI] 1.78–2.17), with substantial heterogeneity (I2 = 79.5). Only 13 studies adjusted for conventional risk factors (age, sex, smoking, obesity, diabetes, and low-density lipoprotein [LDL] cholesterol) and these had a relative risk of 1.65 (95% CI 1.39–1.96), I2 = 33.7. Studies reported ten different ways of comparing CRP values, with weaker relative risks for those based on continuous measures. Adjusting for publication bias (for which there was strong evidence, Egger's p<0.001) using a validated method reduced the relative risk to 1.19 (95% CI 1.13–1.25). Only two studies reported a measure of discrimination (c-statistic). In 20 studies the detection rate for subsequent events could be calculated and was 31% for a 10% false positive rate, and the calculated pooled c-statistic was 0.61 (0.57–0.66). Conclusion Multiple types of reporting bias, and publication bias, make the magnitude of any independent association between CRP and prognosis among patients with stable coronary disease sufficiently uncertain that no clinical practice recommendations can be made. Publication of prespecified statistical analytic protocols and prospective registration of studies, among other measures, might help improve the quality of prognostic biomarker research.
|DOI Link:
|Journal Article
|Copyright: © 2010 Hemingway et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
|Appears in Collections:
|Published Articles, Dept. of Health Sciences
Files in This Item:
|Publisher version
|722.03 kB
|Adobe PDF
Items in LRA are protected by copyright, with all rights reserved, unless otherwise indicated.


text: In message <001c01bee380$08d5c920$1232ba8c@merkur> "Andrew M. Steere" writes: > Personally, I find that a pair of Cibie Oscar+ pencil beams do a great job > of spotting moose from several thousand feet away... This is of course the ultimate approach. I forgot to mention that one of my design criteria was stealth - but it remains true that good accessory lighting will beat any contrivance using the stock fittings. -- Phil Payne UK Audi quattro Owners Club Phone: 0385 302803 Fax: 0870 0883933


text:
Results 1 to 2 of 2
Thread: Epic Mahabharat on the floors!
Epic Mahabharat on the floors!Follow us on Social Media
Follow @iamdesirulez
Tweet #iamdesirulez
With the popular mythological series like Devon Ke Dev. Mahadev, Ramayan, Durga, etc. already doing well on-screens, another epic mythological series Mahabharat is all set to make its come back...
Siddharth Kumar Tewary, who is known for producing shows like Agle Janam Mohe Bitiya Hin Kijo, Phulwa and Navya is now coming back with the epic show Mahabharat. There had been a lot of speculations about the making and scrapping of the show. Now, the wait is over and our sources have informed us that the shoot has finally begun.
The war sequence is being shot in the outskirts of Jaisalmer. Buzz is going around that the show will be aired daily and not weekly but is yet to be finalized. Earlier, the show was being expected to take the time slot of Satyamev Jayate but it couldn't make it. The making of the show has not been easy for the makers, as it involves three years of re-search and pre-production.
The star cast of this show is going to have a mix of many newcomers and few known faces. All India auditions have been done but the final casting is still on as it will be having lots of characters. According to our reliable sources, Shaheer Sheikh has been approached to play Arjun and Deblina Chatterjee to play Draupadi. Anita Raj has been approached to play the character of Satyavati but due to some reasons it didn't got finalized.
The show is expected to go on-air after about 3-4 months.


text:
LHF Outlaw REG Fonts
<link href="//db.onlinewebfonts.com/c/83be85e883af5385ef801ebaaf80cffc?family=LHF+Outlaw+REG" rel="stylesheet" type="text/css"/>#2 Using @import CSS directive, put the following line in add to your css file.(http | https)
@import url(//db.onlinewebfonts.com/c/83be85e883af5385ef801ebaaf80cffc?family=LHF+Outlaw+REG);#3 Use font-face declaration Fonts.(http | https)
@font-face {font-family: "LHF Outlaw REG"; src: url("//db.onlinewebfonts.com/t/83be85e883af5385ef801ebaaf80cffc.eot"); src: url("//db.onlinewebfonts.com/t/83be85e883af5385ef801ebaaf80cffc.eot?#iefix") format("embedded-opentype"), url("//db.onlinewebfonts.com/t/83be85e883af5385ef801ebaaf80cffc.woff2") format("woff2"), url("//db.onlinewebfonts.com/t/83be85e883af5385ef801ebaaf80cffc.woff") format("woff"), url("//db.onlinewebfonts.com/t/83be85e883af5385ef801ebaaf80cffc.ttf") format("truetype"), url("//db.onlinewebfonts.com/t/83be85e883af5385ef801ebaaf80cffc.svg#LHF Outlaw REG") format("svg"); }
Brisko Sans W01 Bold Italic
Dusan Jelesijevic & Tour de Force Font FoundryAuthor
Dusan JelesijevicTrademark
Brisko Sans Bold Italic is a trademark of Dusan Jelesijevic & Tour de Force Font Foundry.Copyright
Copyright (c) 2013 by Dusan Jelesijevic & Tour de Force Font Foundry. All rights reserved.Description
Coneria Script Demo
Coneria Script DemoStyle
Mns GrebckAuthor
Mns GrebckTrademark
Coneria Script is a trademark of Mns Grebck.Copyright
Copyright (c) 2012 by Mns Grebck. All rights reserved. Personal use only. Please visit www.mawns.com for a commercial license.Description
Copyright (c) 2012 by Mns Grebck. All rights reserved.License
Copyright (c) 2012 by Mns Grebck. All rights reserved. Personal use only. Please visit www.mawns.com for a commercial license.
Waukegan LDO Oblique
Style : ObliquePackages
Waukegan LDOStyle
1.000 2004 initial releaseCompany
LDO FontsAuthor
Luke OwensTrademark
Waukegan LDO Oblique is a trademark of LDO Fonts.Copyright
Copyright LDO Fonts, 2004. All rights reserved.License
This font is copyrighted 2004 by LDO Fonts and Luke Owens. All rights reserved. This software is the property of LDO Fonts and its licensors, and may not be reproduced, used, displayed, modified, disclosed or transferred without the express written approval of LDO Fonts. Distribution of this software is restricted without prior written permission. If you wish to distribute this software commercially, contact LDO Fonts for details on obtaining a license. You may not rent or lease the software, nor may you modify, adapt, translate, reverse engineer, decompile, or disassemble the software. You may not make derivative fonts from this software. THE SOFTWARE AND ANY RELATED FILES ARE PROVIDED AS IS AND WITHOUT WARRANTY OF ANY KIND.
Grim Natwick Betty Boop
Grim Natwick Betty BoopStyle
2008; 1.0, initial releaseCopyright
Copyright (c) Grim Natwick, as a gift to Reg Hartt. fonted by bobistheowl for Metaphase Brothel Graphix, 2008. All rights reserved.Description
Copyright (c) Grim Natwick, as a gift to Reg Hartt. fonted by bobistheowl for Metaphase Brothel Graphix, 2008. All rights reserved.
1.00 April 10, 2012, initial releaseCompany
max infeldCopyright
Typeface Xerographer Foundry. 2012. All Rights ReservedDescription
big spit is made in chico,californiaLicense
jigsaw pieces tfb
jigsaw pieces tfbStyle
1.00 November 22, 2012, initial releaseAuthor
truefonts.blogspot.com (kaiserzharkhan). 2012. All Rights ReservedCopyright
free for personal useLicense
free for personal use
Cotoris W01 Italic
Ryoichi TsunekawaAuthor
Ryoichi TsunekawaTrademark
Cotoris Italic is a trademark of Ryoichi Tsunekawa.Copyright
Copyright (c) 2006 by Ryoichi Tsunekawa. All rights reserved.Description
Flat-it type foundry. Standard License agreementLICENSING AGREEMENT 0. GeneralThis document is a legal agreement between you, the end user, and Flat-it type foundry. By using or installing this software, you agree to be bound by the terms of this Agreement.In this document the term gfont softwareh refers to any program or piece of code and/or computer information containing data that can be used to output letterforms or designs to any output or display device currently known or unknown. 1. License GrantNumber of Users: In consideration for the license fee paid, Flat-it type foundry grants to you only, the Licensee, the non-exclusive, non-transferable right to use and display the Flat-it type foundry Product purchased through this service on up to five (5) CPUs at your site connected to any number of printers at your own site. If you have paid for a multi-user license for more than five (5) CPUs, the maximum number of CPUs is equal to the number of users specified in the applicable receipt. If you are using this product for your work, this agreement applies to your employer.Backups: You may make one (1) copy of the Flat-it type foundry font data solely for backup purposes provided the copyright and trademark notices are reproduced in their entirety on the backup copy.Third Parties: You may send a copy of any Flat-it type foundry font along with your documents to a commercial printer or other service bureau to enable the editing or printing of your document, provided that such party agrees to delete the font or fonts from his/her/their system upon completion of your project.Portable Documents: You may gembedh Flat-it type foundry font(s) within PostScript-Language files, .PDF files, and .EVY files for distribution, viewing, and imaging to other parties.Distributions: You may not rent, lease, sublicense, give, lend, or further distribute the font software.Modifications: You may not modify, adapt, translate, reverse engineer, decompile, disassemble, or create derivative works based on the Flat-it type foundry Product without prior written consent from Flat-it type foundry.2. CopyrightThe Flat-it type foundry font software data is copyrighted; unauthorized copying of Flat-it type foundry is expressly forbidden. You may be held legally responsible for any infringement of Flat-it type foundryfs intellectual property rights that is caused or encouraged by your failure to abide by the terms of this Agreement.3. Termination This Agreement will immediately and automatically terminate without notice if you fail to comply with any term or condition of this Agreement. If this Agreement is terminated, you agreed to destroy all copies of the font data in your possession. To contact Flat it type foundry or Ryoichi Tsunekawa with any questions, comments, etc. Ryoichi Tsunekawa, Parace-Kurumada #103 2-33-2 Kurumada-cho Showa-ku Nagoya-shi Aichi-ken466-0001 JAPAN+81-52-733-0124or visit http://flat-it.com
CCJoeMad W00 Bold Italic
Active ImagesAuthor
John 'JG' RoshellTrademark
CCJoeMad is a trademark of Active Images.Copyright
Copyright (c) Active Images, 1999. All rights reserved.License
Active Images Font License AgreementPlease read this license agreement carefully before youinstall your fonts. By installing fonts sent to you byACTIVE IMAGES, you accept this license agreement. If you donot accept the terms and conditions of this license, do notinstall the fonts in question.1. LICENSE Active Images is the exclusive owner of theelectronically transferred computer lettering fonts. Wegrant you, and you accept, a non-exclusive license to usethe electronically transferred fonts, but you are not theowner of them. Your right to use the fonts under thislicense agreement is called the license. By fonts, we referto the electronically transferred fonts and any embodimentor use that you make of the fonts in any form.2. AUTHORIZED USER AND USE You alone, as the purchaser ofthe fonts, are authorised to use the fonts under the licensefor personal, business or distributed projects. With fewexceptions: fonts are licensed for use on 5 CPUs and/or 1output device. Additional licenses are available on a ratioof 5 CPUs to 1 output device. Call us if you are not clearon whether you require additional licenses. Any otherduplication or use by any other person or entity constitutessoftware piracy, a criminal offense.3. LIMITED WARRANTY Active Images makes no warranties,express or implied, regarding the electronically transferredcomputer software, its merchantability or its fitness forany particular purpose.Active Images warrants the magnetic media to be free fromphysical defects for 90 days, after purchase. Active Imageswill replace the disk free of charge should it fail withinthis time. These fonts are sold for use only on the computerplatform indicated on the packaging. Use of these fonts onany platform other than that for which it was originallyintended is not recommended and invalidates any warranty.Active Images reserves the right to terminate this Licenseat any time and void your license if you fail to comply withthe terms and conditions of this License.If you have any questions about the License Agreement, ourreturn policy, security and on-line ordering, or anythingelse, please feel free to contact us firstname.lastname@example.com, or at the telephone number below.ACTIVE IMAGES8910 Rayford DriveLos Angeles, CA 90045Tel: 310 215-0362Fax: 775 890-5787www.activeimages.com
Artimas W00 Bold
David Bergsland, Bergsland TypeAuthor
David BergslandTrademark
Artimas-Bold is a trademark of David Bergsland, Bergsland Type.Copyright
Copyright (c) David Bergsland, Bergsland Type, 2009. All rights reserved.Description
You can install this font on any two computers: Mac or PC. You are allowed to send copies of the font to service bureaus, printing companies, and the like for output use only.


text:
Namibia has a generalized epidemic with HIV primarily transmitted through heterosexual means. The Strengthening Pharmaceutical Systems (SPS) Program, funded by USAID through PEPFAR, has made significant contributions in enabling the rapid scale up the ART through the strengthening of ART pharmaceutical systems in the country, including the country's adoption of WHO guidelines to treat patients at CD4 350 T-cell levels with anti-retroviral (ART) medication as opposed to 200 or lower. Unfortunately the rapid scale up has resulted in critical need to strengthen health systems including relevant sustainable pharmaceutical services delivery systems. This scale up, coupled with the vastness of the country, the sparse population, the shortage of qualified pharmaceutical personnel, has put added stress on an already over-burdened work force and system.
SPS's successor, SIAPS, is anticipating the need to support increased access of quality assured ART and other essential medicines to the remotest parts of the country.To accomplish this task, SIAPS is invoking a multi-faceted approach, identifying focus areas including the WHO Health Systems Strengthening (HSS) framework of service delivery, health workforce, information systems, financing, and leadership and governance. Within this strategy, SIAPS is practicing approaches of improving upon systems already in place and working with the people by integrating existing systems to maximize efficiency and supporting established action plans. The National Pharmaceutical Master Plan (NPMP), Partnership Framework, National Medicines Policy and National Strategic Framework for HIV and AIDS provide an over-arching vision under which SIAPS will help the Ministry of Health and Social Services (MOHSS) monitor the NPMP progress and use results to achieve plan goals and objectives. SIAPS supported interventions will have national coverage, helping to address the challenge of the vastness and sparseness of Namibia.
SIAPS will work with MOHSS and all key stakeholders in the pharmaceutical service delivery to develop sustainable capacity for assuring the long-term availability of quality pharmaceutical products and effective pharmaceutical services to achieve desired health outcomes in public and private sectors in Namibia.


text:
Guru Dutt Collection: Sahib Bibi Aur Ghulam / King, Queen and Slave / Master, Mistress and Servant (1962), Kaagaz Ke Phool / Paper Flowers (1959), Chaudhvin Ka Chand / The Moon of the Fourteenth / Full Moon (1961), Mr. & Mrs. '55 (1955), Aar-Paar / From One Side to the Other / Across the Heart (1954), C.I.D. (1956) 2 x DVD9on June 9th, 2016 at 09:54
Vasanth Kumar Shivashankar Padukone (9 July 1925 – 10 October 1964), popularly known as Guru Dutt, was an Indian film director, producer and actor. He made 1950s and 1960s classics such as Pyaasa (Thirsty), Kaagaz Ke Phool (Paper Flowers), Sahib Bibi Aur Ghulam (The King, the Queen and the Jack) and Chaudhvin Ka Chand (The Fourteenth Day Moon in Muslim calendar but actually means full moon, a metaphor for beauty). In particular, Pyaasa and Kaagaz Ke Phool are now included among the greatest films of all time, both by Time magazine's "All-TIME" 100 best movies and by the Sight & Sound critics' and directors' poll, where Dutt himself is included among the greatest film directors of all time. He is sometimes referred to as "India's Orson Welles". In 2010, he was included among CNN's "top 25 Asian actors of all time". He is most famous for making lyrical and artistic films within the context of popular Hindi cinema of the 1950s, and expanding its commercial conventions, starting with his 1957 film, Pyaasa. Several of his later works have a cult following. His movies go full house when re-released; especially in Germany, France and Japan.
2 x DVD9 | PAL 4:3 | 7.42 Gb + 6.83 Gb + 3% rec
Language: Hindi
Subtitles: none
Genre: Action, Drama, Musical, Romance, Thriller, Mystery
Related posts:
- Il mondo perduto: I cortometraggi di Vittorio De Seta 1954-1959 (2008): Lu tempu di li pisci spata (1954), Isole di fuoco (1955), Surfarara (1955), Pasqua in Sicilia (1956), Contadini del mare (1956), Parabola d'oro (1955), Pescherecci (1958), Pastori di Orgosolo (1958), Un giorno in Barbagia (1958), I dimenticati (1959) DVD9
- Weird-Noir – Six B-Movies: Girl on the Run (1953), The Naked Road (1959), The 7th Commandment (1961), Fear No More (1961), Fallguy (1962), Stark Fear (1962) 2 x DVD9
- Khoya Khoya Chand / Lost Moon (2007) DVD9
- Les nuits de la pleine lune / Full Moon in Paris (1984) 2 x DVD9
- The Apu Trilogy: Pather Panchali / Song of the Little Road (1955), Aparajito / The Unvanquished (1956), Apur Sansar / The World of Apu (1959) 3 x DVD9 and 3 x Blu-Ray Criterion Collection


text:
Social Media Marketing In Seattle Helps Businesses "Talk" To Customers
Posted on 04. Jun, 2014 by Jay in Industry News
Social media has drastically changed the way people interact with one another. Businesses have seen the potentials of this technology to spread awareness about their products and services. Even the Catholic Church has conceded that they can no longer ignore how strong a medium social media is to reach millions of their flock worldwide, as reported by David Gibson of The Huffington Post:
"In our church we are always fishing inside the aquarium," Archbishop Claudio Celli, head of the Pontifical Council for Social Communications, told a gathering of journalists on Thursday (May 22). "And we forget that most fish are outside the aquarium."
Unless the church engages social media, he said, "we will wind up talking to ourselves."
…Celli said there has been a notable shift in tone under Francis, which he attributes in part to the new pope's own eagerness to communicate by any means possible. He said that openness to media is a direct reflection of Francis' view of the church.
Businesses can learn from Archbishop Claudio Celli when it comes to social media marketing in Seattle and in other parts of the United States. They must realize that social networking is most effective when used to "talk" or engage people, rather than simply making announcements that are ultimately ignored.
A lot of social media campaigns have failed because they didn't consider that the public wants to be involved with companies and businesses online. Unlike advertisements in traditional media, social networking is designed to be more intimate as it was first designed to connect with family members and friends. Enterprises must therefore adopt a more conversational tone in their posts and keep status updates reader-friendly.
Companies must also avoid spamming to help integrate product and service value proposition into what people are truly interested in. Content must be provided in a way that it's entertaining and socializing, as these are the main reasons why users go to Facebook and Twitter. Do not oversell, and remember to always monitor social network accounts for inquiries and feedback, and be interactive and timely in engaging the public.
Many consumers also appreciate it when businesses take the time to personalize their responses and those that develop a unique way of connecting with fans and followers. Understanding people is the true nature of social media sites and establishments can certainly reap the benefits of their online presence when they know how to properly write a status update.
Seattle social media marketing consultants, such as OnPage1.net, know how to interact with users of Facebook, Twitter and other major social networking sites. Businesses can rely on them for guidance to become responsible and likeable online citizens who know how to really "talk" to people.
(Source: Vatican Communications Chief Says Church Can't Ignore Social Media, David Gibson, June 2, 2014)


text:
Holiday Rentals Spain
Welcome to Holiday Rentals in Spain website, we list information and links to Holiday Rentals in Spain. From North Spain to South Spain we have direct links to them all.
By using the Holiday Rentals in Spain site you can get direct access to the great Apartments and other properties, thus cutting out the middle man
and saving you £ $ , when buying your luxury Holiday Rental.
Affordable Prices
Book now and sav save save, upto 30% off the value of great Spanish Holiday Rentals, thats because we also list Holiday Rentals direct from the owners
by giving you direct access to them, ExampleAn Apartments let in Marbella could have cost 600pw but booking your holiday apartments might be 30% less, saving you 's to treat yourself to a nice meal or an other holiday with the money you saved.
(Please use quick Apartments search links below)


text:
Skip to comments.Florida studying a possible universal ID
Posted on 09/06/2011 6:14:58 AM PDT by TheBombshellProject
Gov. Rick Scott is on the prowl for new ways to reduce the cost and size of government.
He has a group patrolling for duplication in law enforcement. He wants agencies to scrutinize their budgets.
And he helped create a Government Efficiency Task Force of 15 business leaders and legislators, who will suggest cuts in state spending by up to $3 billion.
Your input is welcome at floridaefficiency.com.
Now, see what you think of this cost-cutting idea:
Issue Floridians a single ID card that would hold several kinds of vital information: your driver's license, car insurance, health insurance and voter registration.
Good idea?
(Excerpt) Read more at tampabay.com ...
What they're trying to "accomplish" isn't saving taxpayers money. It's keeping tabs on the troublemakers.
Myself included :-)
No because my voter ID card is paper and we don't even need to bring it to the polls as we still have to provide photo ID.
No because my insurance companies provide my cards and there's no cost to the state.
Sounds like an effort to consolidate all your personal information. I don't think it will save any money at all!
State of Florida makes about $60 million a year selling our information. Most of it comes from the drivers license data.
They could probably make more if they included the health and insurance data.
this idea will die an agonizing death faster than a Sunday Morning Preacher can say "Mark of the Beast".
Such a card is a very bad idea in the US for a thousand reasons. However, what is bad for us could be a very good thing in future military operations in foreign countries.
That is, one of the biggest problems our military had in the Iraq occupation was that their government was effectively worthless. Those parts of it we tried to save were disasters, and only those parts that were replaced with modern, efficient, western equivalents were worth a darn.
But once they had been replaced, by J. Paul Bremer, who in future should be remembered as "the man who built Iraq", despite some setbacks, what the country needed more than anything else, was an individual identification system, run by the US military.
That is, every military unit in Iraq needed an off the shelf system to identify each and every Iraqi into a US military run database, and issue them a hard to counterfeit photo ID card.
Because personal information in Iraq could be deadly, all anyone could see on the card would be the face of its owner. But on the backside of the card, in encrypted data matrix bar code that can hold up to 1000 characters of information, would be their name, address, home district, fingerprints, religion, age, weight, sex, height, etc., etc.
From that point, the very first thing that would happen when the US soldier met an Iraqi would be to scan their card, which could be done with an ordinary cellphone. It would verify their picture and check the database to see if they were criminally wanted, and with different passwords, the soldier could find out information about them he needed to know.
If an Iraqi lost their card, it would mean that they would be detained until their identity was verified. This would make it incredibly hard for a foreigner to pretend that he was an Iraqi, or for a villain to explain why he was far from home.
Such cards would make governmental tasks in Iraqi far more easy and fair. A Shiite bureaucrat would only see a Sunnis face, so would not know they were a Sunni, not having a password to identify them by religion. Thus the card could be used as a ration card, for voting and any other government service.
This would have helped Iraq recover far more quickly.
But that is the point. In chaos, it helps recovery. But when there is no chaos, like in the US, such cards would be a disaster, again, for a thousand reasons. It would not solve chaos, but create chaos.
Check with the big advocate- Marco D'Beast...
Re my #7- you beat me to it!
And to monitor what medications you're taking, what guns you're buying, what food you're eating...
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


text:
How many of us read—in fact, poured over every word of the famous pregnancy handbook, What to Expect When You're Expecting? I know many people whose copies are well-used and worn. Despite some of the worry it created—"Why isn't this happening on Day 94 like the book says it should?!"—I still appreciated being able to check the book for what might happen next.
If only we had such a guide for adulthood. Much like a baby guide, it could never be completely accurate for everyone. We all have unique experiences. Still, it is sometimes comforting to know that you're pretty normal. So many times, I've had a freak-out over a life change only to realize later that most of my friends were experiencing the same thing.
Well, I'm here to help with What To Expect When You're Adulting.
Hopefully, our parents and education covered a lot of the basics—cooking, laundry, driving, maintaining a positive bank account balance, developing marketable skills. But there is so much more to being an adult, isn't there? Adults still do an amazing amount of learning and developing. No one coasts unchanged from high school graduation to retirement and grandparenthood. Looking back at the last twenty-five years of my life, I noticed clear themes that evolved over time.
The Twenties: Community Building My twenties were largely devoted to creating a community of friends. Right after college, my husband and I moved to a city where we had no family. During that time, we made our own. Much like college, there were parties and fun and adventures, but in the process friends were becoming our chosen family. They would soon bring us food when a baby was born. They would be the ones who answer midnight phone calls and come stay with sleeping children in emergencies. Theirs are the names now listed on medical authorization forms for summer camp. Even though I wasn't a parent yet, the bonds created during my twenties are one of the foundations upon which we built our family.
The Thirties: Growing a Family The thirties focused on becoming a parent. My first baby was born when I was twenty-nine. With two more arriving right behind him, it felt like I didn't come up for air for an entire decade. "Becoming a parent" sounded as simple as a trip to the hospital and coming back with a baby. Of course, it turned out to be so much more.
Instead, the skills of motherhood took years to learn. Simply making sure everyone's basic needs were met—healthy food, clothing that fit, baths, walking, talking, going potty, manners, playing, socializing. Figuring out how to still be a wife when tiny humans seemed to take all my energy and focus. Finding someone to care for our children when I wanted to do ANYTHING but take care of them—meet a friend for coffee, shop for new jeans, work. Having small children was all encompassing in a way that wasn't clear until the fog started to dissipate. (For those of you engulfed in that fog right now, let me just tell you that it does clear. The load lightened significantly around the time the children could all dress themselves, get in and out of the car independently, and make themselves a sandwich.)
The Forties: Designing My Life's Work This is where I am now, and there's a lot going on. As a stay-at-home/work-from-home mom, I thought my work life was pretty settled during my thirties, but the forties have surprised me and provided new clarity. Whether I am working for money or working at being a mom, aligning my personal goals and my career goals is more important than ever.
Home schooling has transformed my "job" as a mom. Around the time I turned forty, home schooling started nagging my mind. When my kids were in school, I struggled to balance the value of family with the value of education. Too many committees, meetings, clubs and homework meant our family time suffered. Too often, personal values and the school's values conflicted. I struggled to explain why, all too often, kindness was not reciprocated, honesty was not appreciated, and hard work was not rewarded. Now, my work makes more sense. I have less internal conflict, the kids love learning, and we have more time, less stress, and closer relationships. That's a job I can love.
The work I do for money also demanded some changes. As someone with my own business, I have a lot of choices—when I work, how I work, who I work with. Yet, I hadn't taken full advantage of those choices. So at age forty, I decided to do just that. My work may not look a lot different from the outside, but just like with home schooling, it is a better fit for me and my life.
The forties are also reminding me of some interests that I had pushed aside. With babies to care for, I simply didn't have time or energy for the political causes that are important to me or for the writing that I love. Even if they are unpaid, these jobs still hold an important spot in my life's work. The forties are turning into a decade about fitting my many jobs into the shape of my life.
The Fifties: New Roles, New Freedom Since I am not in my fifties yet, I asked for opinions from friends who are. Answers included concerns about aging parents, questions about their life's work and legacy, retirement worries, and their changing role as mothers. Children are busy but (at varying degrees) more independent. While they may need less hands-on time, kids still need plenty of support, not to mention money as college expenses approach.
One major theme stood out with every fifty-something woman I talked to: freedom. Over and over, everyone mentioned an increased sense of freedom at this age, whether by circumstance or choice. For some, it is the freedom of time as family duties decrease. Others felt menopause brought sexual freedom and ended worries of unwanted pregnancy. Many have freed themselves from the opinions of others, concern over their appearance, "putting up with people's crap", and holding their tongues. The fifties are sounding pretty awesome to me.
So what about you? Have you noticed themes in your life? Are they similar to or different from those I've found? And what about The Sixties? Anyone want to tell us what to expect in our sixth decade? No matter what, I am sure some interesting surprises await!


text: Round Hotel is steps from Xiaolongwan subway station (Line 1 South Extension), which provides an easy access to main attractions of Nanjing. It has a business center and accommodations with free internet access. Round Hotel is 5 miles from Nanjing South Train Station and 10 miles from Xinjiekou shopping area. Lukou International Airport is 19 miles away. Simply decorated, each guest room is equipped with a flat-screen cable TV, a desk and an electric teakettle. The attached bathroom has shower facilities and free toiletries. The staff at the 24-hour front desk can assist with luggage storage service. Fax, printing and photocopying facilities are available. The on-site Chinese restaurant serves a variety of local dishes.


text:
Pepper Hamilton Sues FDIC Over Bank's Legal Fees
Philadelphia-based Pepper Hamilton lodged its complaint Friday in the U.S. District Court for the District of Columbia, saying that it provided an extensive range of legal services to Advanta Bank Corp. in the weeks surrounding its placement into receivership and alleging that the FDIC is now not properly addressing the claims....
To view the full article, register now.
Try Law360 FREE for seven days
Already a subscriber? Click here to login


text:
Interview: H.E. Tahir Taghizadeh
Interviewed on the sidelines of the TEAS Business Forum London on 5 November 2014, H.E. Tahir Taghizadeh, Azerbaijani Ambassador to the UK, outlines the measures currently being implemented in his country to increase the level of overseas investment, the development of SMEs and private business in the country, the effect of the Southern Energy Corridor, and the evolution of the financial services sector.


text:
It's 2:23 pm. You're at the Quad lawn, waiting for your project group to turn up for an assignment meeting that was supposed to start at 1:30 pm. "Oh well", you think to yourself, "it's just a group assignment, I'll survive".
It's 2:24 pm. Finally, your group has finally arrived. Unfortunately, they have also turned into zombies.
You run to the top of the Basser Steps, knowing that even the undead would rather starve than walk the stairs of death. You look down and realise that the entire campus has now become infested with zombies. What do you do now?
Here are some quick tips on how to survive a zombie apocalypse at UNSW!
Arc Legal & Advocacy
Just because the world has descended into chaos, doesn't mean that your WAM has to as well! Book an appointment with the lawyers at Arc. They'll let you know whether you can get special consideration for your Week 9 Assignment and whether you still have to pay off HECS in the zombie apocalypse!
Arc Reception
You watch on in dismay while the world you knew crumbles before your very eyes, you realise that your phone battery is dying. What a bummer of a day! Head to Arc reception to charge your phone at one of the FREE charging stations, and talk to one of the friendly Arc receptionists if you haven't signed up to be a 2017 Arc member (it's FREE). You never know, they might have a crossbow discount and you can learn to be the next Daryl Dixon!
Campus Security
Get the Campus Security App to get a shuffle bus or safety escort from one of these specially trained zombie killers.
As Shaun from Shaun of the Dead would say: Head to the Greenhouse, have a nice cold pint, and wait for this all to blow over!


text:
This colorful calendar for 2015 takes inspiration from paint swatches, infusing color into each and every day of the year. The Color Swatch Calendar has a shape that fans out like a deck of color cards. When creating this unique paper calendar its creator Peter Von Freyhold notes that the inspiration came to him while thinking of the lack of color that comes with the dreary winter months.
As well as being able to help you keep track of the months and days, the calendar also shows off the CMYK values for each color, which makes it also pretty handy as a color reference guide. If you know a graphic designer or someone who is just obsessed with color, this is a fantastic calendar gift for the new year.
Paint Swatch Calendars
More Stats +/-
Boozy Advent Calendars
Uplifting Beauty Calendars
Paint-Dripping Cityscape Calendars
Christmas Cosmetic Calendars
Playing Card Calendars
This Colorful Calendar Doubles as a CMYK Reference Tool
- By: Laura McQuarrieNov 12, 2014
References: typografie.de & psfk
References: typografie.de & psfk


text:
Status: Closed
Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Hantzmon Wiebel serves local, state-wide, national and international clients. We serve "mom-and-pops", professional firms, major nonprofit organizations, foundations, and international business clients with revenue in the hundreds of millions. We audit, we account, we advise, we plan, we report - most of all, we care.
For more than 85 years, Hantzmon Wiebel LLP of Charlottesville, VA, has been a leading accounting and tax firm. With over 67 professionals on staff, we have the expertise to guide any business, individual or non-profit client. As we work together, our focus is two-fold: we aim to provide solutions to your tax and financial planning questions, and we strive to create value with all of our...
Hantzmon Wiebel & Co is located at the address 818 E Jefferson St in Charlottesville, Virginia 22902. They can be contacted via phone at (434) 296-2156 for pricing, hours and directions. Hantzmon Wiebel & Co specializes in Antiques, Child Support, Social Security.
Hantzmon Wiebel & Co has an annual sales volume of 2M – 4,999,999. .For more information contact Gary Vickers, President or go to...
Be the first YellowBot user to add photos or videos of Hantzmon Wiebel LLP
5 star
4 star
Since 1956, Hantzmon, Wiebel & Co has been providing Certified Public Accountant from Charlottesville.
Certified public accountants & advisory services Tax accounting, Bookkeeping services, Payroll accounting services, Billing services, Inventory accounting, Audit services, Corporate finance, Taxation issues and preparation, Accounting and auditing
If you don't see your business listed on YellowBot, please add your business listing.
YellowBot wants to get your input! If you have a comment, find a bug or think of something neat we should do, let us know by emailing us.
© 2007-2018 Solfo, Inc. – All rights reserved


text:
Things to Do in GuimaraesRead about the top 3 things to do in Guimaraes, Portugal. Get ideas for Guimaraes sightseeing and tourist attractions in Guimaraes.
Castelo de Guimarães
#1 of 3 things to do in Guimaraes
The castle was built in the tenth century in order to defend the monastery from …
Paço dos Duques de Bragança
#2 of 3 things to do in Guimaraes
Built in the fifteenth century for the Duke of Braganza, the palace is a wonderf…
Sao Miguel Chapel
#3 of 3 things to do in Guimaraes
The chapel is located just outside the castle of Sao Miguel, and is often dismis…


text:
Fully Licensed & Bonded
The NH Amsterdam City Centre Hotel offers guests a gateway to the historic and beautiful Amsterdam. Located in the heart of the city and overlooking the Singel Canal, the hotel is only a very brief walk to the famous floating flower market, the powerfully moving Anne Frank House, and the bustling shops of Dam Square. A tram connects travellers to the myriad experiences that Amsterdam has to offer, from Museum Square to the magnificent Rai Congress Centre. After a day spent absorbing the sights and sounds of Amsterdam, indulge in a world-class meal in a historic setting at the Restaurant d'Vijff Vlieghen. Famous for its mastery of New Dutch cuisine, the restaurant consists of eight individual rooms, each with its own particular feel and atmosphere, spread across five authentic 17th century buildings. The restaurant's bar, Gerzonis, provides the perfect place to unwind and connect over cocktails after dinner. In addition to its 209 comfortable guest rooms with impressive views of downtown Amsterdam or the Singel Canal, the NH Amsterdam City Centre Hotel offers a peaceful winter garden for all guests to enjoy as well as an elegant boardroom for those traveling on business. Guests of the NH Amsterdam City Centre Hotel will find the hotel within easy reach by all major modes of transportation. An airport shuttle bus will carry you from the hotel directly to the airport in 45 minutes for a small fee. The tram allows easy access to Amsterdam's attractions, while bicycles can be rented for invigorating exploration on two wheels.


text:
Color has a way of affecting your mood, freshening a room, and bringing your home up-to-date with current trends. Looking back at my parents' choice of colors in our home growing up will tell you that trends don't usually last.Continue reading… Roses are Red, Nurseries are…
Month: November 2012
Carousel Designs is in the Houzz
Eeny, meeny, miny, moe how do I know which way to go….to design a beautiful nursery? I was recently referred to Houzz to get amazing ideas to redecorate my home. Now I'm addicted. For every nook and cranny of yourContinue reading… Carousel Designs is in the Houzz


text:
Apple TV "Barely Watchable" 424
lpangelrob writes "Peter Svensson of the Associated Press reviews the Apple TV, and comes away less than impressed.While the Apple TV gets solid marks for "a very iPod-like interface, commendably clear and easy to use", the Apple TV experience falls apart on an HD television. The reviewer notes that "videos from Apple's online iTunes store look horrible on an HDTV set. The movies and TV shows have the same nominal resolution as DVDs, but look much blurrier, approaching the look of standard-definition broadcast TV.'"


text:
There are absolutely sufficient should go to Sedona, nonetheless great deals of individuals refer to the place for something that can not be offered in a shop or labeled on a map peru iowaska. Because it wased at first started by westerners in the 1800's, individuals acknowledged "a certain something" concerning Sedona that went a lot past its charm. It had not been till intuitives concerned the area that its true destination was uncovered. They determined that Sedona launches energy unlike other location on earth. This power raises plain senses, awakens dormant vitality, as well as promotes spiritual advancement. Word spread, or even today Sedona has actually wound up being a center for much of one of the most transformative spiritual retreats worldwide.
If you are interested in worrying Sedona for a spiritual refuge, there are many indicate take into consideration before making a choice which pull back to do. Many traditional hotels supply group workshops. While these are a wonderful method to call into play the cumulative power of the group, individuals could really feel that the subject issue does not continually placed on them. They may furthermore really feel omitted, as teachers could only provide a very little amount of in person focus. For clients that require more personalized passion, "individual spiritual resorts" are regularly a so a lot a lot more appealing choice. These hotels are a far more individual experience. They normally last 3-5 days, in addition to you reach feature one on one with different professionals of various healing modalities.
Specific spiritual refuges are merely that, they focus not on teams, however on solitary individuals. They allow you to select from one of the most reliable recovery methods to deal successfully with your troubles. You might work together with certain educators that can appear your spiritual experience as well as development your growth as high as possible. The most essential elements of these refuges are development as well as recovery. It is a time to be alone and even mirror on specifically just what your life has actually been or even will other in the future, as well as a time to come deeply right into the presently and consider life's deeper significance. If you take a specific spiritual retreat as well as quit to your impulses, you'll see that everything will merely click right into area.


text:
Comments from TLSLOEWS
Showing 251 - 275 of 4,654 comments
Another great Vintage photo.
Nashville Flood May 2010.
This is great!
This was a Loews United Artist theatre when first built.
The auditorium of this theatre has been torn down and replaced with a Harris Tetter Store.
From this weeks Nashville Scene.Darrel Scott with String Quintet Sat.Aug 13 8.P.M.August 18-28 Studio Tenn Presents:Guys and Dolls. Nanci Griffith Sat. Sept.3 at 8 P.M.Happy Birthday,Patsy Cline with Mandy Barnett Thursday,Sept.8 at 8 P.M.Loudon Wainwright 111 Sat. Sept.10 at 8 P.M.Nitty Gritty Dirt Band Friday,Sep. 23 at 8.P.M.
This theatre was A.K.A Loews Madison when i worked there.It is a church now.
That is a nice shot,Thanks for posting btkrefft.
The Lobby and concessions area with the carpet pulled up.AKA Loews Madison,this theatre is a church now.
These videos are great,does not look like many people check them out.Or maybe they just do not leave comments.
I agree Mike in Nashville we went from $2.50 to $3.00 in the late 70,s same result.
Yes that is a nice auditorium shot,thanks for posting.
Thanks again Will,great find.
Thanks again Will,great find.
Wow what a piece of history,Thanks Will.
I agree Ron,if it will help them stay open give it a try.The Franklin Theatre near me has a full bar and beer too,As far as I know little or no trouble,the patrons want to see a movie or show and have a few drinks,most are not there to get wasted.
Cool photo of the Loews State and Loews New York right next each other.
What a great photo,love those large signs.
What a great piece of history,cool little film.
Pretty neat showing 2001 in the year 2001.
What a great shot.
What a sad photo.
Thanks for the info Mike.
You can find 1 or more photos of the Premier on my page.Just click on the photo to enlarge and then click on the favorites heart sign and it show upload to your profile page.You can also leave a comment if you wish.


text:
New 2018 Chevrolet Silverado 1500 Crew Cab Standard Box 4-Wheel Drive LTZ
Drive It Now Price $47,187
Heated Leather Seats, 4x4, Alloy Wheels, Bed Liner, CD Player, Satellite Radio, Back-Up Camera, Tow Hitch, SPORT PACKAGE. DEEP OCEAN BLUE METALLIC exterior and JET BLACK interior, LTZ trim. FUEL EFFICIENT 20 MPG Hwy/15 MPG City! CLICK NOW!
Back-Up Camera, Satellite Radio, CD Player 4x4, Onboard Communications System, Aluminum Wheels, Privacy Glass, Keyless Entry, Steering Wheel Controls, Electronic Stability Control.
SPORT PACKAGE includes (RD2) 20 x 9 chrome wheels and (UQA) Bose Sound speakers. LTZ PLUS PACKAGE includes (JF4) power adjustable pedals, (UQA) Bose premium audio system, (UD5) Front and Rear Park Assist and (UVD) heated leather steering wheel, SEATING, HEATED AND VENTED PERFORATED LEATHER-APPOINTED FRONT BUCKET, LPO, ASSIST STEPS - 4 BLACK ROUND, SEATS, FRONT FULL-FEATURE LEATHER-APPOINTED BUCKET WITH (KA1) HEATED SEAT CUSHIONS AND SEAT BACKS includes 10-way power driver and front passenger seat adjusters, including 2-way power lumbar control, 2-position driver memory, adjustable head restraints, (D07) floor console, storage pockets and (K4C) Wireless charging. BED LINER, SPRAY-ON, PICKUP BOX BED LINER with bowtie logo. Liner is permanently bonded to the truck bed providing a water tight seal. The textured, non-skid surface is black in color and robotically applied. Spray-on liner covers entire bed interior surface below side rails, including tailgate, front box top rail, gage hole plugs and lower tie down loops. ENGINE, 5.3L ECOTEC3 V8 WITH ACTIVE FUEL MANAGEMENT, DIRECT INJECTION AND VARIABLE VALVE TIMING includes aluminum block construction (355 hp [265 kW] @ 5600 rpm, 383 lb-ft of torque [518 Nm] @ 4100 rpm; more than 300 lb-ft of torque from 2000 to 5600 rpm) (STD)
Plus sales tax, title and license. A negotiable documentary service fee up to $150 may be added. Price contains all applicable dealer incentives and non-limited factory rebates. You may qualify for additional rebates; see dealer for details.