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Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway
- Eeshita G Dastidar†1, 2,
- Guillem Dayer†3,
- Zoe M Holland†4,
- Dominique Dorin-Semblat3, 4,
- Aurélie Claes1, 2,
- Arnaud Chêne1, 2,
- Amit Sharma5,
- Romain Hamelin6,
- Marc Moniatte6,
- Jose-Juan Lopez-Rubio1, 2,
- Artur Scherf1, 2Email author and
- Christian Doerig3, 4, 7Email author
© Dastidar et al; licensee BioMed Central Ltd. 2012
- Received: 27 December 2011
- Accepted: 31 January 2012
- Published: 31 January 2012
Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of Plasmodium falciparum CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2β1 and PfCK2β2.
We now show that the genes encoding both regulatory PfCK2 subunits (PfCK2β1 and PfCK2β2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2β1 or HA-PfCK2β2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2β1- and PfCK2β2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2β1 and PfCK2β2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α in vitro.
Our reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.
- Kinase Assay
- Parasite Line
- Recovered Protein
- Complementation Plasmid
- Nucleosome Assembly Protein
Malaria, a disease caused by parasitic protozoa of the genus Plasmodium (phylum Apicomplexa), is responsible for the death of one million people every year, mostly children living in sub-Saharan Africa [1]. The burden of morbidity and mortality inflicted by this disease presents a serious hindrance to socioeconomic development, causing up to a 1.3% decrease in the gross domestic product of countries with a high malaria rate [2]. The number of deaths due to malaria doubled in the last two decades of the 20th century, with one of the primary reasons being the spread of parasite resistance to anti-malarial drugs, especially chloroquine [3]. Artemisinin combination therapy (ACT) has helped to reverse this trend, but the worrying appearance in southeast Asia of parasite isolates with reduced artemisinin sensitivity suggests that this may be a short-lived respite [4, 5]. The development of anti-malarial drugs with novel modes of action remains an urgent priority.
Reversible phosphorylation is an integral part of many cellular processes, and perturbations in its regulation have been identified in many diseases, such as cancer, diabetes and rheumatoid arthritis [6, 7]. Protein kinases (PKs) are therefore attracting much interest as potential drug targets, and recent successes in cancer chemotherapy based on kinase inhibitors indicate that these molecules can perform as drugs with appropriate selectivity, potency and pharmacokinetic properties [8]. Investigations of the druggability of the genome identified serine/threonine PKs as one of the most promising groups for future small molecule inhibitors, comprising up to 20% of the druggable genome [9].
Protein kinase CK2 (thereafter called CK2) is a pleiotropic serine/threonine PK, with over 300 substrates identified in mammalian cells [10] and documented roles in multiple cellular processes, including differentiation, proliferation, survival, translation, apoptosis, transformation, tumorigenesis, RNA synthesis, cell cycle progression, cell morphology and polarity, cellular responses to stress and to DNA damage, and circadian rhythm [11, 12]. The enzyme phosphorylates a range of DNA-binding proteins, nuclear oncoproteins, and transcription factors [13], and several studies have highlighted the predominance of CK2 substrates involved in transcription, chromatin structure, and gene expression [10, 12, 14]. Taken together, these studies highlight transcriptional regulation as one of the major roles of the enzyme [15]. Controlled cellular compartmentalization is crucial for the function of a pleiotropic enzyme such as CK2, and substrate selection is presumably dependent on proper targeting [12]. Although predominantly nuclear [16], mammalian CK2 has also been found in the cytoplasm, in association with a number of organelles such as the Golgi apparatus, endoplasmic reticulum, and ribosomes [17], and plant chloroplasts [18]. The human genome contains two genes encoding catalytic (α and α') subunits, and a single gene encoding a regulatory (β) subunit. CK2 is present in human cells as a tetrameric complex comprising two identical β subunits and two catalytic subunits, which can be assembled in a α/α, α/α' or α'/α' configuration. CK2α has been found to be essential for viability in all organisms in which its role has been assessed, including P. falciparum [19]. The requirement for viability of the β subunits of CK2 varies across species: disruption of the single metazoan β subunit is lethal in mice [20] and Caenorhabditis elegans [21], whereas disruption of either or both of the β subunits in Saccharomyces cerevisiae is not lethal [14, 22, 23].
Phylogenetic analysis of P. falciparum PKs [24, 25] identified a CK2α ortholog and two putative CK2β subunits, PfCK2β1 and PfCK2β2 (see Holland et al. [19] for alignments with CK2 subunits form other organisms). The stoichiometry of the PfCK2 complexes is not known; by analogy with other systems, it is hypothesized that a homo- or heterodimer of β subunits is complexed with two catalytic subunits in the holoenzyme, but this still requires experimental demonstration. Using transcriptomics, it has been shown that all three subunits are expressed throughout the asexual erythrocytic asexual cycle, in gametocytes and in sporozoites [26, 27]. Proteomics data available for PfCK2α and PfCK2β1 indicate that both proteins are present in the asexual erythrocytic stages and gametocytes, and that the α subunit is also present in sporozoites [28]. We showed previously that PfCK2α is essential for completion of the erythrocytic asexual cycle [19], but neither the requirement of the β subunits for viability nor the cellular processes implicating the enzyme have been assessed.
In the current study, we used a reverse-genetics approach, and found that both regulatory subunits are also essential for intra-erythrocytic asexual proliferation. Reasoning that identifying interactors of the regulatory subunits might provide useful insight into the cellular functions of the PfCK2 holoenzyme, we analyzed the protein composition of complexes obtained by co-immunoprecipitation of PfCK2β1 or PfCK2β2 from transgenic parasite lines expressing tagged subunits. This allowed us to recover proteins involved in many cellular processes, consistent with our immunofluorescence assay (IFA) data localizing the kinase subunits to both nuclear and cytoplasmic compartments. To investigate the role of CK2 in gene regulation specifically, we focused validation experiments on nuclear proteins, and found that several proteins involved in chromatin dynamics, which were recovered in the β subunit pull-downs, are substrates for PfCK2α in vitro. Taken together, our data identify PfCK2 as an essential kinase involved in chromatin dynamics.
Both of the CK2β subunits play crucial roles in the erythrocytic asexual cycle
To ascertain whether pfckβ1 can be disrupted if the subunit is provided through expression of an episomal copy of the gene, we constructed a complementation plasmid (pCHD-PfCK2β1) containing the full-length pfckβ1 gene under the control of the PfHsp86 promoter and a 3' UTR. In parallel with the transfection of the pCAM-BSD-KOPfCK2β1 plasmid alone, a population of parasites was transfected with both pCAM-BSD-KOPfCK2β1 and pCHD-PfCK2β1. PCR (Figure 1B) and Southern blot (Figure 1C) analyses showed that disruption of the targeted locus occurred only in this doubly transfected, doubly resistant parasite culture. Probing the membrane with a BSD probe (Figure 1C, left panel) confirmed that integration had occurred only in the parasites transfected with both the knockout and complementation plasmids. The pfckβ1 probe hybridized to the 11.1 kb band, corresponding to the wild-type locus, in the lane that contained DNA from untransfected parasites and from parasites transfected with the knockout plasmid alone (lanes 1 to 3). This band was undetectable in DNA from parasites transfected with both the knockout and complementation plasmid (lane 4), indicating that the gene can be disrupted only when an additional cassette coding for the PfCK2β1 subunit is provided to the parasites. There are multiple possibilities for recombination of the knockout and complementation plasmids before or after integration, which presumably accounts for the additional bands. The crucial observation is the disappearance of the wild-type-specific band only in the doubly transfected parasite culture. Similar results were obtained for PfCK2β2 (see Additional file 2, Figure A1), indicating that both regulatory subunits play crucial roles in parasite proliferation in the red blood cell.
PfCK2 subunits localize to both cytoplasmic and nuclear compartments
Purification and mass spectrometry analysis of protein complexes involving the PfCK2β subunits
To gain insight into the functions of PfCK2 through identification of the proteins with which the holoenzyme interacts, we subjected protein extracts from the parasite line expressing HA-tagged PfCK2β1 to immunoprecipitation using the anti-HA antibody; the same analysis was performed on extracts from wild-type parasites as a negative control. The immunoprecipitates were separated by SDS-PAGE, and a western blotting was performed using anti-PfCK2β1 to verify the immunoprecipitation efficiency (see Additional file 2, Figure A4A). A parallel gel was loaded and slices excised (see Additional file 2, Figure A4B) for mass spectrometry analysis.
Potential interactors (see Additional file 3, Table A2) were identified based on the number of unique peptides recovered, and on the unweighted spectrum count of the number of detected peptides. We considered proteins as potential hits if they were present in the immunoprecipitate from the HA-tagged line but were absent (or present in trace amounts only) in those from the wild-type parasites. The same experiment was performed in parallel with the parasite line expressing HA-tagged PfCK2β2 (see Additional file 4, Table A3, for the list of recovered proteins), although in this case we were unable to perform western-blot verification of the immunoprecipitate because of the poor quality of the anti- PfCK2β2 antibody.
PfCK2α was pulled down with both HA-PfCK2β1 and HA-PfCK2β2, in line with the previously reported in vitro interaction between the catalytic subunit and both regulatory subunits [19]. Interestingly, PfCK2β1 and PfCK2β2 were present in similar amounts in both immunoprecipitates, suggesting that complexes containing both regulatory subunits may be relatively abundant.
A different distribution pattern was seen when the same protocol, using the same anti-HA antibody, was applied to parasites expressing other HA-tagged kinases, such as PfCK1 (data not shown); this result lends support to the specificity of the pull-down approach. For both PfCK2β subunits, the highest numbers of potential interactors were found in the processes of 1) post-translational modifications, and 2) mitosis and chromosome separation. As we were aiming to understand the nuclear function of PfCK2, we focused further investigations on the latter process.
Potential interactors in the nucleosome assembly and regulation pathway.a
PfCK2β1HA (3D7/tagged)c
PfCK2β2HA (3D7/tagged)c
Histone H2A
Histone H2A variant
Histone H2B
Histone H2B variant
Histone H3
Hhistone H4
High mobility group protein
Chromodomain helicase DNA-binding protein
Smarca-related protein
Structure-specific recognition protein
P. falciparum Naps, histones and Alba proteins are substrates for PfCK2α in vitro
We next examined the ability of histones to act as substrates for recombinant GST-PfCK2α. Recently, several phosphorylation sites in plasmodial histones have been identified ([39]; Dastidar et al., unpublished data), and because several histones were recovered in the PfCK2β precipitates (Table 1), we thought that PfCK2α might be one of the kinases that can phosphorylate histones. When purified parasite native histones were subjected to a GST-PfCK2α kinase assay, preferential phosphorylation of histone H2B (13 kDa) was seen, whereas the dead-mutant kinase failed to phosphorylate any of the histones (Figure 5B). We also tested in PfCK2α kinase assays all four P. falciparum Alba members expressed as GST fusion proteins [35], as well as the nuclear protein PfSir2, which is implicated in establishment of heterochromatin in sub-telomeric region and hence represses var gene expression [40–42]. PfAlba3, PfAlba4, and Sir2 were not phosphorylated by GST-PfCK2α (Figure 5E). By contrast, PfAlba1 (55 kDa) and PfAlba2 (52 kDa) gave signals on the autoradiographs after a kinase assay was performed with GST-PfCK2α, whereas none of the substrates subjected to the GST-PfCK2α dead mutant was phosphorylated (Figure 5C).
Our reverse-genetics data strongly suggest that both PfCK2β subunits are essential for parasite viability, indicating that they have non-redundant functions in the parasite. This was unexpected given the dispensability of the β subunits for viability in S. cerevisiae (which has two CK2β subunits that can be knocked out individually or simultaneously [14, 22, 23]) and ib Schizosaccharomyces pombe (which had a single β subunit) [43]. However, the single β subunit present in C. elegans [21] and in mice [20] is essential. The different interactomes for the CK2β subunits in S. cerevisiae [44] are consistent with functional specialization of each β subunit. The immunoprecipitates obtained with PfCK2β1 and PfCK2β2, despite displaying significant overlap in the identity of recovered proteins, also contained hits that were found in only one of the two immunoprecipitates (see Additional file 5, Table A4). This would be consistent with the non-redundancy in function suggested by reverse genetics; however, we do not have sufficient data to draw any firm conclusion in this respect. The dataset presented in the supplementary material (see Additional file 3, Table A2; see Additional file 4, Table A3) is from one of three experiments. Although several hits obtained in the first dataset were confirmed in two further experiments, fewer proteins were recovered in the latter, pre-empting statistical analysis, More work is needed to confirm that individual proteins are truly absent from one of the other PfCK2β interactome. However, the data (see Additional file 3, Table A2; see Additional file 4, Table A3) nevertheless enabled us to propose assignment of specific functions to PfCK2.
Using recombinant proteins in pull-down experiments, we previously found that PfCK2α is able to interact with both β subunits in vitro [19]. MS analysis of immunoprecipitated subunits (this study) indicates that these interactions are also found in parasite extracts. CK2 is known to have several functions in various organisms [11, 12], and the glimpse into the interactomes of the P. falciparum CK2 regulatory subunits provided by our study is consistent with their involvement in a number of cellular processes. Implementing the same protocol with different HA-tagged kinases such as PfCK1 (data not shown) yielded protein repertoires that were clearly distinct from those described here for the PfCK2β subunits, lending support to the specificity of the approach. Potential PfCK2 interactors were found in 17 out of 18 metabolic processes described on the MPMP website. Many processes displayed only a very small number of hits; whether or not these represent genuine interactors will require in-depth investigations of each individual case.
By contrast, other processes and pathways exhibited a large proportion of their proteins recovered in the immunoprecipitates, suggesting that a number of distinct functional complexes had been pulled down. A striking example is the 'nucleosome assembly and regulation' pathway, many components of which were found in both PfCK2β1 and PfCK2β2 immunoprecipitates (Table 1). This included both PfNAPs, many histones, and several proteins involved in chromatin remodelling (ISWI, CHD1, high mobility group protein, and structure-specific recognition protein). Interestingly, all recovered proteins were members of the so-called 'activating factors' in this pathway, whereas none of the 'inactivating factors' were present in the immunoprecipitates (see Additional file 2, Figure A5), which again is consistent with the specificity of the approach. Some of these interactions were validated by coupled co-immunoprecipitation/western-blot analysis (see Additional file 2, Figure A6) and in vitro kinase assays (Figure 5).
Gene name
PlasmoDB ID
CK2 scorec
We found that PfCK2α preferentially phosphorylated histone H2B in vitro in a mixture of native histones extracted from the parasite. Histone post-translational modifications such as methylation and acetylation play key roles in the control of expression of virulence genes (for example, the var family) in malaria parasites [48]) Similarly, histone phosphorylation is known to play an important regulatory role in other eukaryotes [49], and in yeast, CK2 phosphorylates histone H4 at serine 1 as part of a DNA damage-signaling pathway [50]. Although previous studies have identified few phosphorylation marks in P. falciparum histones, we recently characterized several novel phosphorylation marks on the parasite's histones (Dastidar et al., unpublished). Whether PfCK2 is responsible for phosphorylation of any of these sites in vivo, and the functional consequences of these phosphorylation events, remain to be investigated. Nevertheless, taken together, our data strongly suggest that PfCK2-dependent phosphorylation may play a role in chromatin dynamics.
We also tested other known nuclear proteins with a potential role in gene regulation to see if they could be substrates for the kinase. Of all the proteins tested, recombinant PfAlba1 and PfAlba2 were found to be substrates for the PfCK2α in vitro (Figure 5), and PfAlba1 was found to be phosphorylated in parasite extracts on several residues [46, 47], including one in an acidic stretch consistent with CK2 substrate preferences (Table 2). By contrast, recombinant PfAlba3, PfAlba4, and PfSir2 were not phosphorylated by the kinase (Figure 5E). PfAlba1 and PfAlba2 belong to the same phylogenetic cluster, which is distinct from that containing PfAlba3 and PfAlba4 ([51, 52]), consistent with their different ease if phosphorylation by PfCK2. Moreover, the former two PfAlbas possess an arginine- and glycine-rich RGG domain in addition to the Alba domain. It has been shown in S. cerevisiae that phosphorylation of the RGG domains affects protein localization and transport between the nucleus and cytoplasm [53]. Given that PfAlba1 and PfAlba2 concentrate at the perinuclear space in ring-stage parasites, but expand to the cytoplasm of trophozoites and schizonts [51], it is possible that phosphorylation by PfCK2 may contribute to the regulation of their nucleocytoplasmic transport.
Several proteins involved in mitosis and chromosome separation and in DNA replication and DNA damage pathways (for example, several replication factor C subunits, ATP-dependent DNA helicase, DNA ligase 1) were also pulled down. Implication of CK2 in the DNA damage response is well documented in other systems [12], and a DNA repair pathway has been characterized in P. falciparum [54], of which the DNA polymerase and DNA ligase components can be retrieved in the CK2β immunoprecipitates (see Additional file 3, Table A2; see Additional file 4, Table A3; see Additional file 5, Table A4). Because DNA repair is markedly reduced in the highly drug-resistant W2 isolate, which displays a higher rate of drug-resistance acquisition than do drug-sensitive strains [55], it might be of interest to compare CK2 activity in the W2 versus drug-sensitive clones.
Although many of the proteins identified in the immunoprecipitates were of nuclear origin, a number of potential interactors were associated with other cellular compartments. Some of the recovered proteins were putative or established Maurer's cleft proteins (for example, Maurer's cleft 2 transmembrane protein; Pfmc-2TM), proteins involved in invasion and motility processes (for example, glutamate-riche protein, merozoite surface protein 2 precursor, RhopH3, and other rhoptry-associated proteins) or in cell-cell interaction processes (for example, mature parasite-infected erythrocyte surface antigen, PfEMP2 and rifins). Likewise, several proteins and enzymes involved in redox metabolism were recovered (thioredoxin, thioredoxin-related protein, thioredoxin peroxidase, glutathione peroxidase, glutathione S-transferase, ribonucleotide reductase). This diversity is consistent with our IFA, western blot, and immuno-EM data localizing all the three subunits to both the nuclear and cytoplasmic compartments. Although these results require confirmation by other approaches, and despite the fact that we cannot exclude false positives in our interactome list, they are consistent with an involvement of PfCK2 kinase in varied cellular functions in the parasite.
The availability of parasite lines expressing individual tagged PKs for the almost entire P. falciparum kinome [46] now allows implementation of a kinome-wide investigation of kinase function based on the interactomics approach used here for PfCK2. Preliminary data obtained with other PKs suggest that distinct functions can therefore be assigned to different enzymes on the basis of specifically pulled-down protein repertoires.
In conclusion, we have identified the PfCK2 complex as a kinase distributed to different cellular compartments. Our data point to PfCK2 as a pleiotropic kinase with various cellular functions. In this study, we explored potential nuclear functions of the kinase, and we propose a role for PfCK2 in modifying a number of chromatin-related proteins, several of which are also substrates for the catalytic subunit of PfCK2 in vitro. Furthermore, reverse genetics data show that all three subunits of the kinase are essential for the viability of the blood-stage parasite, confirming its potential as target for chemotherapeutic intervention. This study paves the way for a kinome-wide interactomics-based approach to elucidate PK function in malaria parasites.
Construction of the knockout plasmids pCAM-BSD-KOPfCK2β1 and pCAM-BSD-KOPfCK2β2
Fragments from central region of the PfCK2β1 and PfCK2β2 ORFs were amplified by PCR and inserted between the BamHI and NotI sites of the pCAM-BSD plasmid [31], which contains the Aspergillus terreus blasticidin-S-deaminase gene, whose gene product confers resistance to blasticidin (see Additional file 1, table A1, for cloning primers).
Construction of the complementation plasmids pCHD-PfCK2β1 and pCHD-PfCK2β2
A plasmid for in vivo episomal expression of PfCK2β1 and PfCK2β2 subunits were constructed as follows. The full-length coding sequences were first inserted between the BglII and NotI sites of plasmid pHGB [56], and then transferred into plasmid pCHD-1/2 [56] by a clonase reaction (Gateway system, Invitrogen) according to the manufacturer's instructions. Plasmid pCHD-1/2 includes a cassette encoding human dihydrofolate reductase, conferring resistance to the antifolate drug WR99210. Parasites that were transfected with both a KO plasmid and a complementation plasmid were cultured under double-drug selection (see below).
3'-tagging of plasmids
The 3' end of the PfCK2β2 coding sequence (573 bp, omitting the stop codon) was amplified by PCR using primers incorporating PstI and BamHI restriction sites and inserted between the same sites of the pCAM-BSD-HA plasmid [57]. For pfCK2β1, the 3' end of the gene (540 bp) was amplified, then the PCR product was treated with the restriction enzymes PstI and BglII and inserted between the PstI and BamHI sites of pCAM-BSD-HA.
Parasite culture and transfection
Four P. falciparum strains (3D7A [58] and three HA-tagged strains of PfCK2 subunits: PfCK2αHA, PfCK2β1HA and PfCK2β2HA) [19] were grown in human erythrocytes as described previously [59]. The medium for the HA-tagged lines was supplemented with blasticidin (2.5 μg/ml; VWR International Ltd., West Chester, PA, USA). Cultures of the P. falciparum clone 3D7A were maintained at 37°C in RPMI 1640 medium (Gibco) supplemented with 25 mmol/l sodium bicarbonate, 2 mmol/l L-glutamine, 300 mmol/l hypoxanthine, 10 μg/ml gentamicin, and lipid-rich bovine serum albumin (BSA) (AlbuMAX II; Sigma-Aldrich, St Louis, MO, USA). Cultures were seeded at 5% hematocrit volume and maintained at a parasitemia of 1 to 10% with daily changes of medium. The incubator was flushed with a gas mixture containing 5% CO2. For transfection, asexual blood-stage parasites were synchronized by sorbitol treatment [60] so as to result in the majority being ring-stage parasites. Forty-eight hours later, the ring-stage parasites were transfected by electroporation with 100 μg of purified plasmid DNA in buffer (Cytomix; Gibco-BRL, Grand Island, New York, NY, USA) as described previously [61]. Blasticidin (2.5 μg/ml) was added to the culture medium to select for transformed parasites. Parasites under double selection had 5 nmol/l WR99210 added to the culture medium in addition to blasticidin. Parasites were maintained in this supplemented medium from 2 days post-transfection.
DNA extraction and Southern blotting
Parasite cultures were lysed in 0.15% saponin. The parasite pellets were resuspended in cold phosphate-buffered saline, and treated with 150 μg/ml proteinase K and 2% SDS at 55°C for 2 to 3 hours. Total DNA was extracted in phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated in ethanol and 0.3 mol/l sodium acetate. DNA was digested with ClaI and EcoRI for PfCK2β1, and with ClaI and NcoI for PfCK2β2.
Western-blot analysis
Cytoplasmic and nuclear extracts were prepared as described previously [41, 62]. Using standard procedures. SDS-PAGE was performed and DNA was transferred to nitrocellulose membranes, which were probed using either commercially available antibodies against HA-tag (ab9110; 1:1000 dilution), histone H3 (ab1791; 1:2500 dilution), P. falciparum aldolase (ab38905; 1:1000) (all Abcam, Cambridge, MA, USA) or subunit-specific antibodies generated in rabbits against the PfCK2β1-derived peptide DSNKDLQDSKSDKS (1:500 dilution) or the PfCK2β2-derived peptide DEINRDSEEMYKNK (1:1000 dilution (both BioGenes GmBH, Berlin, Germany) After probing with appropriate secondary antibodies conjugated to horseradish peroxidase (GE Healthcare Life Sciences, Princeton, NJ, USA), the membranes were developed with a commercial substrate (Super Signal West Pico Chemiluminescent Substrate; Thermo Fisher Scientific Inc., Rockford, IL, USA) in accordance with the manufacturer's recommendations.
Immunofluorescence assays
IFAs were performed on synchronized stages (ring, trophozoite, and schizont) of the three HA-tagged strains of the PfCK2 subunits as described previously [56]. The fixed cells were incubated with primary antibodies anti-HA (ab9110; 1:2000 dilution) and anti-Exp2 [32] (1:800 dilution) (both Abcam). After incubating with appropriate secondary antibodies, the cells were examined under a microscope (Nikon Instruments, Tokyo, Japan).
Immuno-gold electron microscopy
Infected erythrocytes were fixed in 1% glutaraldehyde in RPMI-HEPES buffer for 1 hour at 4°C. After washing, polymerization in agar (Type IX; Sigma-Aldrich), and dehydration with ethanol, the samples were transferred into embedding resin (LR-White; London Resin Company Ltd, Reading, Berkshire, UK), and polymerized for 12 hours at 4°C, and cut. Ultrathin sections of the sample were collected and mounted on Cu/Pd grids. Free aldehyde groups were blocked by incubating the samples for 5 minutes in 50 mmol/l NH4Cl. Sections were blocked in phosphate-buffered saline containing 5% (v/v) normal goat serum, 1% (w/v) BSA and 0.01% (v/v) Tween-80. The washed grids were incubated for 2 hours in antiserum diluted in phosphate-buffered saline containing 1% (v/v) normal goat serum, 1% (w/v) BSA and 0.01% (v/v) Tween-80 and anti-HA antibody (ab9110; Abcam). Samples were washed and incubated for 45 minutes with 12-nm gold-labeled goat anti-rabbit IgG (111-205-144; Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA). Washed sections were stained for 15 minutes with aqueous 4% uranyl acetate followed by staining for 2 minutes with 1% lead citrate. Sections were analyzed using an electron microscope (JEM-1200EX; JEOL Ltd, Tokyo, Japan) at 120 kV.
Parasite proteins extraction and immunoprecipitation
Parasite cultures were lysed in 0.15% saponin and pellets were stored at -80°C. Proteins were extracted on ice using mammalian protein extraction reagent (M-PER) (Profound™ Mammalian HA-tag IP/Co-IP application; Thermo Fisher Scientific) with protease inhibitors (1:25; Complete Protease Inhibitor Cocktail; Roche Diagnostics, Basel, Switzerland) and benzonase nuclease (1:500; Novagen, Darmstadt, Germany). The lysates were cleared by centrifugation at 11,180 g for 15 minutes at 4°C, and the concentration of proteins measured by the Bradford assay. Proteins were loaded on a spin column with a pre-inserted frit, preventing bead loss between washes, and 6 μl (50% v/v_ of anti-HA crosslinked onto sepharose beads were added (both included in the kit used for protein extraction; Thermo Fisher Scientific). The columns were then transferred to a rocker platform and incubated overnight. The flow-through was collected by centrifugation at 300 g for 10 seconds at room temperature. The columns were washed three times using the Tris-buffered saline (TBS) provided in the kit, followed by a short burst of centrifugation (300 g for 10 seconds). Finally, any proteins bound to the beads were eluted using boiled 4× Laemmli buffer, and recovered by centrifugation (15,600 g for 3 minutes). Aliquots of protein extracts and flow-through liquid were taken before and after immunoprecipitation to monitor the yield.
Mass spectrometry analysis
Samples were separated by SDS-PAGE on 10% polyacrylamide gel, which was then stained with Coomassie blue (Biosafe; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and later placed in 10% ethanol and 1% acetic acid. Each lane was removed from the gel and cut into eight slices, then the proteins were digested in the gel using trypsin As follows. Briefly, the samples were reduced in 10 mmol/l dithioerythritol (DTE) and alkylated in 55 mmol/l iodoacetamide (IAA), the gel pieces were dried. The samples were incubated with 12.5 ng/ml trypsin overnight at 37°C. The tryptic peptides were extracted from the gel slices, dried, and resuspended in 2% acetonitrile (ACN):0.1% formic acid (FA) for liquid chromatography/tandem mass spectrometry (LC-MS-MS) analysis
A mass spectrometer (LTQ Orbitrap XL (Thermo Fischer Scientific) equipped with an ultraperformance LC (UPLC) system (NanoAcquity; Waters Ltd, Elstree, Hertfordhshire, UK) was used. Peptides were trapped in a custom-made precolumn (Magic C18 AQ stationary phase, 5 μm diameter, 200Å pore, 0.1 × 20 mm, Michrom Bioresource) and separated in a custom-made main column (Magic C18 AQ, 3 μm diameter, 100Å pore, 0.75 × 150 mm), using a run of 53 minutes and a gradient of H2O:ACN:FA 98%:2%:0.1% (solvent A) and ACN:H2O:FA 98%:1.9%:0.1% (solvent B). The gradient was run at a flow rate of 250 nl/min as follows: 100% A for 3 min, 30% B within 36 min, 47% B within 14 min, 90% B within 5 min held for 5 min and 100% A for 17 min. The MS/MS was operated in an information-dependent mode, in which each full MS analysis was followed by 10 MS/MS acquisitions, during which the most abundant peptide were selected for collision-induced dissociation (CID) to generate tandem mass spectra. The normalized collision energies were set to 35% for CID.
Data search was performed using Mascot software (version 2.3; Matrix Science Inc., Boston, MA, USA) and Proteome Discoverer (v.1.1; Thermo Fisher Scientific), and the sequences searched against a concatenated database consisting of non-redundant Plasmodium database (PlasmoDB, version 6.4; http://plasmodb.org) and the reversed-sequence version of the same database and common contaminant proteins. Finally, results were imported into Scaffold (version 3_00_8 Proteome Software; http://proteomesoftware.com/) for validation of protein identification, normalization, and comparison of spectral count. Peptide identifications were accepted if they could be established at a probability of greater than 95% as determined by the ProteinProphet algorithm [63]. Protein identifications were accepted if they were assigned at least two unique, validated peptides, and could be established with at least 99% probability as determined by the ProteinProphet algorithm [64].
Kinase assays
The GST-PfCK2α and GST-PfCK2α-dead K72M mutant fusion proteins [19] were purified from E. coli (BL21 Gold strain) as described previously [65]. PfCK2α kinase activity was determined by measuring 33P or 32P incorporation using γ[33P]-ATP or γ[32P]-ATP. All the substrates (Alba1, Alba2, Alba3, Alba4, PfSir2; kindly provided by C. Scheidig-Benatar), and extracted P. falciparum histones were treated with active PfCK2α and dead mutant as described previously [65]. Samples were run on a 16% gel, which was dried and exposed to film for autoradiography.
This research received funding from Inserm, EPFL and the European Community's Seventh Framework Programme (FP7/2007-2013) (MALSIG project to CD and ASh and EviMalar network of Excellence to CD and ASc), by Inserm, EPFL, the FP6 (SIGMAL and ANTIMAL projects to CD and BioMalPar network of Excellence to ASc), and by a European Research Council Executive Agency Advanced Grant (PlasmoEscape 250320) to ASs. EGD benefits from a PhD fellowship funded by the European Union Framework Program 7 Marie Curie Initial Training Network 'Intervention strategies against malaria (InterMalTraining)', contract number 215281. ZMH benefitted from a PhD Studentship form the Wellcome Trust. A postdoctoral fellowship from the Conseil Régional d'Île-de-France (DIM Malinf) to AC is gratefully acknowledged.
Authors' Affiliations
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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I am a human-centered designer who is passionate about developing innovative products and experiences, especially through the application of emerging technology. I desire to improve user's lives by following my passion of creating joyful interactions.
Currently I am an Experience Designer at Pillar Technology, a part of Accenture Industry X.0. I am also a Venture for America fellow, doing my fellowship in Detroit, MI.
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subset: commoncrawl


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Search the Community
Showing results for tags 'jehovahs witnesses and racism'.
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Jehovah's Witnesses and Race
Jack Ryan posted a topic in Jehovah's Witnesses's Topics"Color and Physiognomy.....a man whose nose upturns can no more be expected to administer justice than a pug dog can be expected to act as a shepherd." The Golden Age January 19, 1921 pg. 224 Â


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text:
I have been thinking about writing a series on "notes from the life of a COW (Career Oriented Woman)" (Copyright mine!). I do not have as many readers as I had in the past, not that there were too many in the past, but even those have moved on. Don't see the point of writing anything on this blog. But I always wrote for myself, sometimes as my record, sometimes when I wanted to vent out, sometimes when I wanted to share … So, notes from a COW may be just a record for now, which I might find boring myself or may not find time to write (which is the point of this COW series!).. Let us see!
upcoming series
26 Feb 2013 2 Comments
2 Comments (+add yours?)
just delurking to let you know there is a reader! 🙂


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2-6 May, 2018
Indonesia Convention Exhibition (ICE)
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10 July 2015


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The third workshop in a three part women only journey, centred on Transformational Breathing and Yin Yoga.
Workshop three will represent healing, influencing our self-esteem and realising our power for transformation focusing on the third Chakra, Manipura. In this workshop, we will energise our inner fire, restore vitality so we are able to move from the strength of our core.
The Yin Sequence in workshop three will focus on the upper abdomen and upper back with reference to the heart. Through abdominal strengtheners, twists and passive backbends we will gain a greater understanding of our true personal power .


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Second Look: Aaron Sorkin's Newsroom
May 3, 2012
We're another day closer to the newest introduction to the Aaron Sorkin TV canon and the marketing team at HBO has blessed us with a new teaser for The Newsroom.
From the looks of it, Jane Fonda, playing a Ted Turner-type, will be a real draw; she's my favorite part of the tease. But, with idiots running the control room – one girl confuses Georgia the country and Georgia the state – there should be plenty of drama to tell.
The Newsroom premieres Sunday, June 24th on HBO.
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Thank You From Pelham Cares
Thank you to our generous community, from Pelham Cares!
As our volunteers work their way through the many boxes and bags of donations received during this past holiday season, we are reminded of the generosity and kindness that abounds in the community of Pelham! Throughout the year, and especially during the Holiday Season, the donations of food, personal care items, household products, toys, and money, grants, the contributions of hundreds of volunteers, and the support of local media, allow us to continue to assist families in our community.
The need continues for our various programs including Food Bank, emergency food services, holiday hampers, back-to-school backpacks & snacks, transportation to medical appointments, sponsorship of youth in sports, recreation and educational activities, and referrals to other local services.
As they say 'It takes a village…' , it is never more true than in this case. It is contributions from donors and volunteers; individuals, businesses large and small, social clubs, schools, churches and granting foundations, which allow Pelham Cares to continue to meet this need in Pelham.
On behalf of the Board of Directors of Pelham Cares, staff, volunteers and clients; a heartfelt thank you goes out to the Pelham community for your support again this past year!
Tracy Holmwood, President Board of Directors


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The motorcycle gods are conspiring against me this summer. My social calendar has been packed (for me) lately, and the combination of that with a) trying to get back in shape and b) fairly regular afternoon thunderstorms (or at least the threat thereof), and c) projects around the house has kept me off my Bonneville for the most part. I've only had the poor thing out twice this year and that was in the Spring.
I'm not too worried, though. I'll get my chances eventually. Plus, I much prefer riding in the transition seasons than in the blast furnace that is Summer in the Denver metro.
In other news, I'm going in to talk to Fish about my next tattoo in a couple weeks.


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|» Products
|» Induction Pressure Cooker
|» 5 Ltr. Stovetop Pressure Cooker
5 Ltr. Stovetop Pressure Cooker
Product Code : SI 16
Brand Name : SUNRISE INDIA
Price And Quantity
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- Unit of Measure
- Piece/Pieces
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Our ethical and fair business practices, we are able to manufacture, export and supply quality checked 5 Ltr. Stovetop Pressure Cooker. It is manufactured with the implication of sophisticated tools and techniques by a team of experienced professionals. In order to tenderize meat and vegetable, the provided cooker is ideal to prepare foodstuff in few minutes along with same nutritional values. We offer this 5 Ltr. Stovetop Pressure Cooker to the clients at competitive prices.
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Key points:
- Long and heat resistant handles
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- Payment Terms
- Cheque
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- 100piece Per Day
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- 1 Week
- Packaging Details
- Cartons
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- Asia, Australia, Central America, North America, South America, Eastern Europe, Western Europe, Middle East, Africa
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Other Products in 'Induction Pressure Cooker' category
Plot No-12/10, Gali No 9, Near Shagun Restaurant, Main Raliway Road, Samaypur Badli, Delhi - 110042, India
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SUNRISE INDIA All Rights Reserved.(Terms of Use)
Developed and Managed by Infocom Network Ltd.


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Body Baton
$29.00 $38.00
Body Bâton is a luxurious, go-anywhere moisturizer. The solid, push-up applicator is mess-free. Apply to the back of your hands and keep your palms slip-free for your daily activities. Apply to lips, elbow, knees or anywhere you need a quick-absorbing moisturizer. Body Bâton's compact size makes it the perfect addition to your desk, purse, bedside table, sink or travel companion.
Remove cap and push up from bottom. Apply to hands, feet and body as needed.
beeswax, mango butter, lavender oil, evening primrose oil, calendula oil, arnica oil (all ingredients are organic)


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Poetry Countdown will do just that: count down from the 64th-greatest poem in the English language to #1. My inspiration for this project comes from various MTV countdown shows, from Casey Kasem's American Top 40 on the radio over the years, and from a Rolling Stone feature several years ago that chose the 500 greatest songs of all time, or at least of their time.
I have a few rules, not many, to guide me in choosing the Top 64.
The poems must come from the year 1798 or later. In 1798, William Wordsworth and Samuel Taylor Coleridge published the first edition of their book Lyrical Ballads, which some people see as the first book of truly modern English poetry. Whether it was or not, it was very important in promoting the "Romantic" approach to poetry, using ordinary language and subjects, stressing nature and the imagination. Any earlier than 1798 and we're in a somewhat different kind of English and a somewhat different approach to poetry than we still employ today.
Eligible poems must have less than 100 lines. That was Edgar Allan Poe's idea in his essay "The Philosophy of Composition": to have a single effect on the reader, a poem must be short: 100 lines or fewer. So you won't see "The Rime of the Ancient Mariner" or "The Ballad of Reading Gaol" or "Crossing Brooklyn Ferry," great as they are. Maybe I'll do another course next year on The Slightly Longer Poem.
The poems will mostly have universal themes. Some will be difficult, but none will be esoteric: that is, they won't demand that you have special knowledge. "Easter 1916" by William Butler Yeats is a very great poem, for instance, but to work it requires that you know a lot about the 1916 Easter Rising rebellion in Dublin. So it isn't included here.
In choosing the Top 64, I have tried not to rely too much on my personal favorites. I have also tried not to choose poems just because they are of historical importance. Basically, they have to be good. Matthew Arnold, another inspiration for this project, advises against evaluations that are too personal or too historical. He wanted people to study "the best that has been thought and said," and for our purposes, I agree.
Some of these poems will be very famous and familiar: the "usual suspects" of English literature. But some usual suspects will be missing, and some little-studied poems will make the list. Ultimately nobody's Top 64 will correspond exactly with anyone else's. I have taken a lot of hints from scholars, poets, and anthologists. But I can't abdicate my own critical judgment, either.
An awful lot of these poems are by white guys. In a sense, much of English-language literary culture till recently was a white guys' culture, as well as an upper- or upper-middle class culture. But people are still people, even if they're privileged; all these poems speak to what's human in all of us. And some are by women; some are by black writers. Some are by English poets, some by Americans, a few by Irish writers, and there's the occasional poem by someone of another nationality, but all were originally written in English. Some poets included here are gay, others straight. Most of the authors, though not all, are dead, and the poems by living writers tend to be from early in their careers. It's hard to be certain that a poem is great if it's not a few years old. I have no requirements for "coverage" or "distribution" or "representation." If a single poet happened to write six or eight of the greatest 64 poems, so be it.
When I was initially casting about for the Top 64, I thought most of the poems would be about "love and death," what Emily Dickinson said were the only real themes for poetry. And indeed, there's a lot of love and death here. But there's also nature, and childhood, and language itself. And there's a surprising (to me) amount of poetry about faith. The last 200 years or so are often seen as a secular age. Maybe just because they've been secular times, a lot of the best poetic thought has been about faith: keeping, losing, reacquiring, or questioning faith, but faith nonetheless. We will study these poems in a secular context, but we will treat their ideas seriously.
A poem must be poetic: "equal to / not true," said Archibald MacLeish. It is not just a dressed-up message. The language must be beautiful, but there must also be a poetic idea, something fused and perfect that can't quite be paraphrased or condensed. "Why did they use all those words," students sometimes say. But if the poet didn't use those exact words, the poem would mean something different, something less coherent, something less exciting.
None of the poems here has a "hidden meaning." If a meaning can't come out into the open, I say the hell with it.


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