Previous attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under “classical” mouse ES conditions.We characterized two cell lines (C5 and B10) which were obtained from rat blastocysts in medium with serum and LIF. In the B10 cell line we found the expression of genes known to be expressed in trophoblast, Cdx-2, cytokeratin-7, and Hand-1. Also, B10 cells invaded the trophectodermal layer upon injection into rat blastocysts. In contrast to mouse Trophoblast Stem (TS) cells proliferation of B10 cells occurred independently of FGF4. Cells of the C5 line expressed traditional markers of extraembryonic-endoderm (XEN) cells, in particular, GATA-4, but also the pluripotency markers SSEA-1 and Oct-4. C5 cell proliferation exhibited dependence on LIF, which is not known to be required by mouse XEN cells.Our results confirm and extend previous findings about differences between blastocyst-derived cell lines of rat and mice. Our data show, that the B10 cell line represents a population of FGF4-independent rat TS-like cells. C5 cells show features that have recently become known as characteristic of rat XEN cells. Early passages of C5 and B10 cells contained both, TS and XEN cells. We speculate, that mechanisms maintaining self-renewal of cell lineages in rat preimplantation embryo and their in vitro counterparts, including ES, TS and XEN cells are different than in respective mouse lineages. Currently, the procedure of efficient production of chimaeras, transmission of the genetic changes to the germline and finally the formation of genetically modified animals works efficiently for mouse, but not for other mammalian species. Thus, for better understanding of the pluripotent state in different mammals, it is important to derive ES cell lines from different species and comprehensively describe the requirements for their pluripotency. For this purpose the comparison to mouse Embryonic Stem (mES) cells as a gold standard of pluripotent cells is necessary.Embryonic stem (ES) cells are pluripotent cells derived from preimplantation embryos and adapted to Several types of proliferating cells have been derived from the mouse blastocyst. Mouse ES cells are characterized in detail for the expression of genes maintaining their pluripotent state. Recent studies have suggested that Oct-4, Nanog and Sox-2, in concert with interacting proteins, constitute an autoregulatory, pluripotency network The rat is one of the mostly used laboratory animals for studying the cardiovascular system and many physiological and pathophysiological processes. Multiple laboratory rat lines are available, including outbred, inbred, and genetically modified strains, however, no gene targeting could be obtained due to the difficulties to derive truly pluripotent cell lines Recently germ-line competent rat ES cells were successfully derived by application of inhibitors of signaling pathways mediated by kinases ERK and GSK3β In this study we asked the question, why cell lines obtained from rat preimplantation embryos, namely C5 and B10, did not contribute to chimaeras. We characterized C5 and B10 cells by analyzing the expression of marker genes for embryonic stem cells and extraembryonic lineages. We showed that B10 and C5 cell lines consist of cells committed to 2 independent lineages: trophectoderm  and endoderm . Our data also show, that Cdx-2 and GATA-4 expressing lineages are formed very early during the process of outgrowth from rat blastocysts. Interestingly, the derivation and propagation of rat TS cells did not require the cell culture conditions used for mouse Trophoblast Stem (mTS) cells and occurred in the standard cell culture medium. Rat endodermal cells also revealed peculiar features, such as dependence on LIF and the expression of pluripotency markers, SSEA-1 and Oct-4. Presumably, the inhibitors of MEK1 and GSK3β kinases which have been successfully used to derive the pluripotent rat ES cells C5 early (passages 5–7) (C5E), C5 late (passages 20–30) (C5L), and B10 late (passages 30–60) (B10L) cell lines. C5L was obtained after expansion of a single cell, whereas B10L was derived by the expansion of individual clumps of tightly growing cells. B10L consisted of tight clumps and large flattened cells, whereas C5L consisted of rounded cells which were loosely attached and did not form tight clumps , cultivated for 5 days and then replated in equal density and cultivated for 5 days more. The propagation of mTS cells was achieved in conditions (2) only. In the absence of FGF4 or in the presence of PD173074 all mTS cells were differentiated , trophectoderm , and the marker of endoderm (GATA-6) in C5E, C5L, and B10L. Both trophectoderm-specific genes were expressed in C5E and B10L, but were completely absent in C5L  (C5E), C5 late (passages 20–30) (C5L), and B10 late (passages 30–60) (B10L) cell lines. C5L was obtained after singe cell cloning. B10L was obtained by amplification of a single tight clump. The B10 cell line was cultivated without feeders. C5 cells were cultivated with or without feeders, as indicated. As feeder cells, mitomycin C-treated mouse embryonic fibroblasts (MEF), passages 3–5, isolated from 14-day old mice (NMRI strain) embryos were used. Mouse feeder-free ES cell line E14tg2a (passages 30–60) was used as positive control in several experiments. B10 cells were grown in the medium, consisting of DMEM (Sigma) with 15% fetal calf serum (Invitrogen) supplemented with nonessential amino acids, glutamate, sodium pyruvate, β-mercaptoethanol, penicillin-streptomycin. C5 cells were grown in the same medium supplemented with ESGRO (Chemicon), 1000 U/ml. C5 and B10 cell lines were replated every 2–5 day after 5 minutes of trypsinization. The mouse trophoblast stem cell line, GFP-EXE, was a kind gift from the lab of Janet Rossant and was cultivated according to the conditions published on the web site: http://www.sickkids.ca/rossant/protocols/TScells.asp. For alkaline phosphatase staining cells were fixed in 4% paraformaldehyde, washed with PBS, and then incubated for 30 minutes in Tris-Maleate buffer containing FAST-RED TR, F2768 (Sigma) and Naphtol-AX-MX, N5000 (Sigma).B10 and C5 cell lines were derived from the inbred WKY and outbred SD rat strains respectively. B10 cell line was described Female Sprague-Dawley (SD) rats (60-day-old) were obtained from a commercial animal breeder (Charles River/Sulzfeld). The rats were kept at a temperature of 21±2°C in a 12 hr light/dark cycle (lights on 6.00 a.m.–6.00 p.m.) with a humidity of 65±5%.For production of embryos at blastocyst stage females SD rats were mated with males of the same strain and on the following morning (day 1 of pregnancy) examined for a plug. Pluged females were sacrificed at 12 a.m. on day 5 of pregnancy and blastocyst stage embryos were recovered by flushing the excised uterine horns with M2 medium (Sigma) in vitro overnight in M16 medium in 4-well culture dishes (Nunc) under 5% CO2 in air at 37°C B10 cells were labelled with fluorescent substances (5 µM CFSE) or by infection with lentivirus, containing EGFP . For the enrichment of the EGFP-positive population B10L-EGFP cells were additionally FACS-sorted on BD FACSAria sorter. Blastocyst injection was carried out by injecting 5–7 B10L cells into cavity of host blastocysts of SD rats. After injection, blastocysts were cultured Blastocysts of SD rats of day 4.0–4.5 of development were plated in 4-well dishes (Nunc) on coverslips covered with MEFs in ES medium containing ESGRO (1000 U/ml). Embryos were undisturbed until attachment (2–3 days) and afterwards the medium was changed daily. After 6 days the outgrowths were fixed with 4% paraformaldehyde and subjected to immunostaining.5–106 cells were harvested, washed with PBS, resuspended subsequently in 100 µl of solution A , and incubated for 15 min at 37°C. Probes were analyzed by flow cytometry on a BD Calibur.For cytometric analysis of DNA content, 5×10Cells were cultured on MEF or gelatine-coated coverslips, fixed in 4% paraformaldehyde (Sigma), permeabilised in 0.5% Triton-X-100 (Sigma), incubated in blocking solution (5% BSA in PBS) for 1 hour. Blocked samples were incubated for 1 hour at room temperature or overnight at 4°C with primary antibodies diluted in blocking solution 1∶50 , diluted 1∶1000 in blocking solution. Between incubations samples were washed with PBST (PBS with 0.05% Tween-20 [Sigma]). For DNA staining samples were incubated with 10 µg/ml Hoechst 33342 (Sigma). Samples were mounted with DAKO Cytomation, S3023 (DAKO). Immunostained cells were examined on a confocal Leica TCS SP5 microscope with 20× air and 63× oil immersion objectives. UV-laser (405), Ar-laser (488) and HeNe-laser (633 nm) were used to excite the fluorophores. Images were acquired using the Leica TCS SP5 software.Trypsin-EDTA-dislodged cells were lysed in RIPA buffer  supplemented with Protease Inhibitor Cocktail, # 11836170001 (Roche). Protein concentrations were determined with Bradford reagent, B6916 (Sigma), and equal amounts of total protein were resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred to PVDV membranes (Amersham), probed with primary antibodies , anti-STAT3, 9132, anti-phospho-tyrosine 705 STAT3, 9131, GAPDH, 2118, [Cell Signaling], secondary goat anti-mouse HRP-conjugated, #1858413 (Pierce), goat anti-rabbit HRP-conjugated, #1858415 (Pierce), and detected by chemiluminescence using the ECL kit (Pierce) and Kodak film.β-actin mRNA expression. The method of Livak and Schmittgen −ΔΔCT. The primers used are listed in Supplemental RNA was extracted by GeneElute Total RNA purification Kit (Sigma), and residual genomic DNA was removed by DNase I treatment . RNA was reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase . The real-time PCR approach used the SYBR green method in a 96-well plate format using a iQ5 BioRad cycler. Reactions contained 7.5 µl 2× SYBR MasterMix (Qiagen), primer (100 nM), and template in a total volume of 15 µl. The thermal profile used for amplification was 95°C for 8 min followed by 50 cycles of 95°C for 15 sec, 58°C for 20 sec, and 72°C for 40 sec. At the end of the amplification phase, a melting-curve analysis was carried out on the products formed. Gene expression was normalized to Figure S1(A): Immunofluorescence analysis of Oct-4 expression in B10 and mES cells, scale 25 µM; (B): Immunofluorescence analysis of E-cadherin expression in B10L cell line, scale 25 µM; giant trophoblast cells (asterisk) (C): Western blot analysis of Sox-2 expression in mES cells (1), Mouse Embryonic Fibroblasts (2), C5E passage 5 (3), C5E passage 7 (4), B10L (5), Actin was used as internal control.(0.49 MB TIF)Click here for additional data file.Figure S2Rat preimplantation embryos after injections of labelled B10L cells. The positions of cells integrated into the trophectoderm are shown by arrrow. Images were taken at confocal (A-G) and epifluorescence  microscopes.(0.60 MB TIF)Click here for additional data file.Figure S3Immunofluorescent analysis of Cdx-2 expression in B10 cells, cultivated in the indicated conditions, merge images of Cdx-2 with Hoechst 33342 are shown, scale 50 µM.(0.27 MB TIF)Click here for additional data file.Figure S4(A): Morphology of C5L cells grown on feeders (MEF) or on plastic with and without LIF; (B): Western blot for phospho-tyrosine 705 STAT-3 in C5L cells after stimulation with LIF; C5L were starved for 12 hours without serum and LIF, and stimulated by 1000 U/ml LIF for 15 minutes, levels of total STAT-3 and GAPDH are shown in the same lysates.(0.65 MB TIF)Click here for additional data file.Figure S5Immunofluorescent analysis for Nanog in B10L and C5E cells. mES cells and rat SD blastocysts are shown as positive controls, nuclei were stained by Hoechst 33342, scale 25 µM.(0.43 MB TIF)Click here for additional data file.Figure S6Morphology and expression of trophoblast and extraembryonic endoderm specific markers in cell lines derived from rat preimplantation embryos. (A): Clump formed by blastocyst (WKY + Fisher) after 8 day cultivation on feeder; outgrowing cells with XEN morphology are shown with a white arrowheads. (B): Morphology of rat cell line derived from preimplantation embryo at passage 4; giant trophoblast cells (asterisk), XEN-like cells (arrow) and tight clumps (arrowhead) can be seen. (C): the expression of Cdx2 and Cytokeratin 7 in a cell line derived from a Fisher rats. (D): expression of laminin B2, collagen 4 in a XEN-like cell line derived from a Fisher rat preimplantation embryo.(1.31 MB TIF)Click here for additional data file.Figure S7Morphology of rat TS-like cell line derived from C5E cell line. (A): phase contrast (left) and differential interference contrast  photos of rat TS-like cells, photos were taken with 5× objective, arrow indicates the characteristic rat TS clump. (B): Immunofluorescence analysis for Cdx-2 expression (right) and corresponding phase contrast image (left) (C): Immunofluorescence analysis for Cdx-2 expression (left) and corresponding merged image of nuclei staining with Hoechst 33342 (left). Photos were taken with 40× objective.(1.17 MB TIF)Click here for additional data file.Table S1Summary of real time RT-PCR primers.(0.01 MB TIF)Click here for additional data file.


The T-cell receptor (TCR) repertoire expression of tumour-infiltrating lymphocytes (TILs) from 19 nasopharyngeal carcinoma (NPC) biopsies was compared with those of lymphocytes from 18 control nasopharyngeal biopsies. mRNA was extracted from these lymphocytes and the cDNA transcribed. A panel of 18 V alpha- and 21 V beta-specific primers was used to detect the TCR gene use from cDNA. The use of V alpha and V beta genes was restricted in TILs compared with lymphocytes from biopsies. The frequencies of V alpha 2, V alpha 3, V alpha 9, V alpha 10, V alpha 11, V alpha 13, V alpha 14, V alpha 15, V beta 11, V beta 15 and V beta 20 were decreased and the frequencies of V alpha 10 , V alpha 11 , V alpha 13 , V alpha 14 , V beta 14  and V beta 20  remained significantly reduced after correction for the number of families typed. The frequency of V alpha 17 was higher in NPC biopsies than in NPC PBLs (P = 0.05), and the frequency of V beta 15 was lower in NPC biopsies than in NPC PBLs (P = 0.02). The frequencies of V alpha 17 and V alpha 18 in HLA-B46+ patients were significantly lower  than in B46+ controls. The results suggest that the restriction of TCR gene use in NPC patients may be important in NPC pathogenesis.


The retinoblastoma (Rb) tumor suppressor protein can function as a DNA replication inhibitor as well as a transcription factor. Regulation of DNA replication may occur through interaction of Rb with the origin recognition complex (ORC).Drosophila Rb, Rbf1, with ORC. Using expression of proteins in Drosophila S2 cells, we found that an N-terminal Rbf1 fragment (amino acids 1–345) is sufficient for Rbf1 association with ORC but does not bind to dE2F1. We also found that the C-terminal half of Rbf1 (amino acids 345–845) interacts with ORC. We observed that the amino-terminal domain of Rbf1 localizes to chromatin in vivo and associates with chromosomal regions implicated in replication initiation, including colocalization with Orc2 and acetylated histone H4.We characterized the interaction of Our results suggest that Rbf1 can associate with ORC and chromatin through domains independent of the E2F binding site. We infer that Rbf1 may play a role in regulating replication directly through its association with ORC and/or chromatin factors other than E2F. Our data suggest an important role for retinoblastoma family proteins in cell proliferation and tumor suppression through interaction with the replication initiation machinery. During the cell cycle each chromosome must be faithfully replicated before cell division. Numerous mechanisms exist to ensure the appropriate replication of chromosomes, including precise control of replication initiation Specific locations in the genome, called origins of replication, are sites of DNA replication initiation during S phase. A heterohexameric protein complex called the origin recognition complex (ORC) binds to origins of replication and becomes a stage upon which the replication initiation machinery assembles. Cdc6 and Cdt1 associate with ORC and help recruit the MCM helicase complex. Many other factors are recruited, including DNA polymerase, which allow DNA replication to begin Rb deficient cells can re-replicate their DNA to give polyploid cells Drosophila Rb homologue, rbf1, results in inappropriate replication and mislocalization of Orc2 in follicle cells Drosophila replication origin is altered in rbf1 mutant follicle cells, also suggesting a role for this protein in chromatin-mediated origin activity The retinoblastoma tumor suppressor (Rb) regulates DNA replication and is important for maintaining proper ploidy. Rb has been detected at sites of DNA replication The canonical function of Rb is to restrict cell proliferation by binding and suppressing members of the E2F family of transcription factors, which results in downregulation of genes required for DNA synthesis and S phase progression in vitro replication assays have shown that the Rb amino-terminus can bind and inhibit MCM7, a component of the replicative helicase that is important for replication initiation and elongation Drosophila Rbf1 interacts with ORC in an E2F independent manner through multiple domains that are outside of the E2F binding domain. The Rbf1 amino-terminal domain associates in vivo with chromosomal regions implicated in replication initiation, including colocalization with Orc2 and acetylated histone H4. Significantly, our work illustrates novel interactions of Rb with the replication initiation machinery that have important implications for our understanding of cell proliferation and tumor suppression.The amino-terminal domain of Rb may play a role in regulating DNA replication initiation. Some Drosophila Orc1 and Orc2 proteins interact with the dDP/dE2F1/Rbf1 complex in ovarian extracts, and we wished to further characterize this interaction i2e2f1 mutant flies Drosophila S2 cell culture to test the association of transiently transfected Rbf1 proteins with endogenous ORC proteins. We expressed V5 epitope tagged Rbf1 deletion fragments in S2 cells under the inducible metallothionein promoter Drosophila, Rbf1 and Rbf2, we tested whether ORC also interacts with Rbf2. We had previously shown that endogenous Rbf1 from Drosophila ovarian extracts interacts with ORC We showed previously by coimmunoprecipitation that Drosophila Rbf1 might be tethered to chromatin independently of dE2F by association with other nuclear proteins. To examine the intracellular localization of the amino-terminus of Drosophila Rbf1, we transfected S2 cells with Rbf1(1–345)-V5, hereafter referred to as Rbf1N. Immunostaining of V5 shows Rbf1N localizes strongly to the nucleus, with some cytoplasmic staining . Flow cytometry analysis of follicle cell nuclei also did not reveal any significant differences in ploidy content . RBF1 cDNA fragments 5′-CTTGGTACCTATGAGCGAGCCTGACCCGCAG-3′ and 5′-TCCTCCCCGCGGGGCAGTGTGTTCCCCCGCATC-3′. For RBF1 345–797 (Rbf1C) the primers used were 5′-CTTGGTACCTATGGCCCTCAACGACCAGTCCCTG-3′ and 5′-TCCTCCCCGCGGCTAGTCCGGCTCGTCGCCAAAGCT-3′. Subsequent restriction digestion and cloning of PCR products was done directly into the vector. All clones were validated by sequencing. To generate the UAS>Rbf1N-RFP vector, the mCherry RFP 5′-ATAGTAGTATGGTGAGCAAGGGCGAG-3′ and 5′-GCTCTAGATTACTTGTACAGCTCGTCCAT-3′. The mCherry PCR product was digested and cloned into pUASP 5′-ATAGGTACCATGAGCGAGCCTGACCCGCA-3′ and 5′-CGCACTAGTCGTAGAATCGAGACCGAGGA-3′. This PCR product was digested and subcloned in-frame with mCherry in pUASP, resulting in Rbf1N with a C-terminal V5 and mCherry tag. Sequencing of this construct revealed a missense mutation converting amino acid 241 of RBF1 from Lys to Glu. However, this change retains the ability to coimmunoprecipitate with ORC (data not shown). The UAS>Rbf1N-RFP construct (pJA024) was used for embryo injections to create transgenic fly lines. The pUASP and mCherry plasmids were a kind gift from Marc Brabant and Hanna Fares, respectively.All C-terminal V5-tagged RBF1 proteins were expressed under the metallothionein gene promoter Drosophila Schneider cells were grown under standard conditions  with antibiotics and up to 12% calf serum (Invitrogen). In a typical transfection 2 ug of plasmid was used with the transfection reagent CellFectin (Invitrogen). In the case of the V5-RBF1, protein expression was induced with 0.7 mM copper sulfate 24 hours after transfection and cells were harvested 48 hours after transfection.Extracts from ovaries and S2 cells were prepared by dounce homogenization of tissue in 1× IP buffer  as previously described S2 cells were transfected as described above with pMT/Rbf1N-V5 and induced for 2 days. The cells were then fixed with 4% formaldehyde in PBS before immunostaining. Mouse anti-V5 (Invitrogen) was used at 1∶200 dilution, and anti-mouse Cy3 secondary antibody was used at 1∶100 (Jackson Immunoresearch). Transgenic Rbf1N-RFP virgins were collected and crossed to males containing salivary gland specific GAL4 drivers Sgs3 or 43B Photobleaching of Rbf1N-RFP was performed with a 543 nm laser in a discrete rectangular area within nuclei. ImageJ Drosophila flies carrying the actin>GAL4/CyO were crossed to UAS>Rbf1N-RFP. Tissues were hand dissected, and flow cytometry of purified follicle cell nuclei and larval neuroblast nuclei was done as previously described http://facs.scripps.edu.software.html). Calculations and ANOVA analysis were performed with Microsoft Excel®.www.flybase.org), Entrez (http://www.ncbi.nlm.nih.gov/Entrez/), and the UCSC Genome Browser (http://genome.ucsc.edu/). Pairwise alignments for comparison of fly and human proteins were generated with the Needleman-Wunsch global alignment algorithm using the PAM250 scoring matrix with a gap extension penalty of 0.5 and an open gap penalty of 10. Multiple protein sequence alignments and phylogenetic trees were generated using ClustalW . Ka/Ks analysis was performed with the Pairwise KaKs Perl script http://www.sbg.bio.ic.ac.uk/phyre/) http://www.cbs.dtu.dk/services/CPHmodels/) Protein sequences were obtained from Flybase Click here for additional data file.Figure S2Drosophila Rbf1 shares the highest percentage of amino acid identity with human p107, most notably in its N-terminal domain. On the other hand, Rbf2 is most identical to Rbf1 throughout the length of the protein. The C-terminal half of the Drosophila Rbf proteins show more overall amino acid similarity to human pRb than p107 or p130. Thus, Rbf1 appears to have a split personality between p107 and pRb. (C and D) Both domains of the Rbf1 and Rbf2 proteins are conserved within Drosophilidae. Multiple sequence alignments of the protein domains of Rbf1 and Rbf2 were used to produce a phylogenetic tree that includes relative distances of divergence. Tree branch lengths indicate that amino acid sequences of both domains of Rbf1 have been more tightly conserved relative to Rbf2. Indeed, Ka/Ks analysis (E) confirms that both domains have been under negative selection and that Rbf1 appears to have been under stronger negative selection than Rbf2. It is also interesting to note that, although Rbf2 protein sequence has experienced greater drift than Rbf1, the Rbf2 N-terminal domain appears to have drifted less than its C-terminal domain, as indicated by the branch lengths of the phylogenetic trees (C and D) and Ka/Ks analysis (E). Rbf2 is not an essential gene, and it has overlapping functions with Rbf1, which might explain the loose conservation of its protein sequence. However, the N-terminal domains of Rbf1 and Rbf2 had similar Ka/Ks values, indicating that they had been under similar selection pressures to retain the amino acid sequence of this domain. (F) A protein structure of Rbf1N was modeled based on the crystal structure of the human RbN. Residues were highlighted based upon conservation determined by pairwise sequence alignments, with red indicating identical amino acids, orange representing conserved substitutions, and yellow being semi-conserved substitutions. The dashed circle encompasses an area of conservation representative of a possible protein interaction surface. (G) Multiple sequence alignment of the conserved surface circled in (F) from widely divergent organisms revealed that this region is highly conserved. Black shading with white letters indicates identical amino acids, and grey shading indicates amino acid similarity.Sequence and Structural Conservation of Rbf1. Pairwise protein sequence alignments were performed to determine the percent amino acid identity between respective N-terminal (A) and C-terminal (B) domains of human and fly retinoblastoma family proteins. Note that percent similarity is in parentheses. The analysis revealed that (5.31 MB TIF)Click here for additional data file.Table S1Drosophila tissues expressing UAS>Rbf1N-RFP driven by actin>GAL4 or CyO control were dissected. The tissues were homogenized and DAPI stained for flow cytometry of purified follicle cell nuclei. Ovaries from a transgenic line carrying a GFP-histone H2Av fusion were used as a control. Follicle cell nuclei undergo several rounds of endoreduplication, resulting in polyploid cells containing 2C, 4C, 8C, 16C, and 32C nuclei. Flow cytometry data was analyzed for DAPI content of follicle cell nuclei in each phase of the cell cycle, which did not reveal any significant differences in ploidy content versus controls.Ploidy of follicle cells not affected by Rbf1N-RFP expression. Ovaries from (0.03 MB XLS)Click here for additional data file.Table S2Drosophila tissues expressing UAS>Rbf1N-RFP driven by actin>GAL4 or CyO control were dissected. The tissues were homogenized and DAPI stained for flow cytometry of purified follicle cell nuclei. Ovaries from a transgenic line carrying a GFP-histone H2Av fusion were used as a control. Follicle cell nuclei undergo several rounds of endoreduplication, resulting in polyploid cells containing 2C, 4C, 8C, 16C, and 32C nuclei. Flow cytometry data was analyzed for number of DAPI-staining follicle cell nuclei in each phase of the cell cycle divided by total number of counted nuclei, which did not reveal any significant differences in cell cycle phases versus controls.Proportion of follicle cells in G or S phases not affected by Rbf1N-RFP expression. Ovaries from (0.03 MB XLS)Click here for additional data file.Table S3Drosophila tissues expressing UAS>Rbf1N-RFP driven by actin>GAL4 or CyO control were dissected. The tissues were homogenized and DAPI stained for flow cytometry of purified follicle cell nuclei. Ovaries from a transgenic line carrying a GFP-histone H2Av fusion were used as a control. Follicle cell nuclei undergo several rounds of endoreduplication, resulting in polyploid cells containing 2C, 4C, 8C, 16C, and 32C nuclei. Flow cytometry data was analyzed for forward light scatter, a measure of nuclear size, for each ploidy level , which did not reveal any significant differences in nuclear size versus controls.Follicle cell nuclear size not affected by Rbf1N-RFP expression. Ovaries from (0.02 MB XLS)Click here for additional data file.


Cross-immunity between seasonal and pandemic A/H1N1 influenza viruses remains uncertain. In particular, the extent that previous infection or vaccination by seasonal A/H1N1 viruses can elicit protective immunity against pandemic A/H1N1 is unclear.Neutralizing titers against seasonal A/H1N1 (A/Brisbane/59/2007) and against pandemic A/H1N1  were measured using an HIV-1-based pseudovirus neutralization assay. Using this highly sensitive assay, we found that a large fraction of subjects who had never been exposed to pandemic A/H1N1 express high levels of pandemic A/H1N1 neutralizing titers. A significant correlation was seen between neutralization of pandemic A/H1N1 and neutralization of a standard seasonal A/H1N1 strain. Significantly higher pandemic A/H1N1 neutralizing titers were measured in subjects who had received vaccination against seasonal influenza in 2008–2009. Higher pandemic neutralizing titers were also measured in subjects over 60 years of age.Our findings reveal that the extent of protective cross-immunity between seasonal and pandemic A/H1N1 influenza viruses may be more important than previously estimated. This cross-immunity could provide a possible explanation of the relatively mild profile of the recent influenza pandemic. The influenza A/H1N1 virus of swine origin, first isolated in April 2009 in Mexico and the United States, has now been spreading worldwide, causing the first influenza pandemic in more than 40 years None of the 107 subjects had reported recent symptoms evocative of influenza infection in the weeks preceding serum sampling, a period during which the pandemic A/H1N1 influenza had barely reached epidemic levels  in France vs baseline), while seasonal vaccinal Brisbane A/H1N1 neutralizing titers only increased by 1.2-fold (95%CI 0.31 to 4.5). As a control, the same pairs of samples were also tested using a standard hemagglutination inhibition (HI) assay measuring antibodies to the pandemic A/H1N1 virus, which showed clear but relatively modest increases in antibody titers, when compared to the increases measured by the PN assay. Using the HI assay, baseline titers were low (titer <40 in 9/10 samples) and increased following vaccination in all subjects, but only 7/10 post-vaccination sera had a titer ≥80 .Reproducibility of the PN assay was evaluated by test-retest analysis on all tested samples, yielding satisfactory intraclass correlation coefficients (ICC) of 0.840 (95%CI 0.774 to 0.888) and 0.846 (95%CI 0.782 to 0.892) for the pandemic and seasonal viruses, respectively. The specificity of the assay was validated by comparing neutralization titers of pre- and post-pandemic A/H1N1 vaccination serum samples from the 10 subjects described above. The results of this analysis are shown on iter ≥80 . A signis = 0.50, 95%CI 0.34 to 0.63, p<0.0001). This finding strongly suggests that previous exposure to seasonal influenza viruses and/or previous seasonal influenza vaccination had conferred some level of protective immunity against the new pandemic A/H1N1 strain in many of the study subjects. We therefore compared the pandemic A/H1N1 and seasonal Brisbane vaccinal A/H1N1 neutralizing titers in sera from subjects having reported inoculation by a seasonal trivalent influenza vaccine during the 2008–2009 epidemic season to titers measured in sera from subjects who did not report such vaccination. As shown on The range of pandemic A/H1N1 neutralizing antibody titers by PN assay in subjects who had no known exposure to this virus was remarkably wide, and often reached surprisingly high levels. Only 9 sera titrated <100 while 43 titrated between 100 and 500, 45 titrated between 500 and 2500, and 10 sera titrated >2500, including two sera with titers >20,000. A comparable range of neutralizing titers was observed against the seasonal Brisbane A/H1N1 strain. Moreover, as illustrated on Based on a highly sensitive pseudovirus neutralization assay, we found that a large proportion of sera from a panel of subjects with no previous exposure to the recently emerged pandemic A/H1N1 influenza strain expressed high titers of neutralizing antibodies against this new virus. Furthermore, our data show that these neutralizing titers correlate significantly with neutralizing titers against a seasonal A/H1N1 virus measured in parallel, and that the pandemic A/H1N1 neutralizing titer is significantly higher in subjects who had been incoculated by a seasonal trivalent influenza vaccine during the previous year. These data are in apparent contradiction with the conclusions of a retrospective study reported by Hancock et al. Our findings are in line with several reports suggesting that some level of A/H1N1 cross-neutralization could be induced by seasonal influenza viruses. First, in some of the studies evaluating the safety and immunogenicity of vaccine preparations against pandemic A/H1N1, high titers of HI antibodies were seen in subjects before vaccination Several factors could explain the high levels of cross-immunity observed in our study. The particularly strong cross-immunity seen in subjects older than 60  is notewThe extent that the cross-immunity observed in this study has had any impact on the relatively mild profile of the current pandemic remains to be determined, and at this stage, our results do not invalidate the clinical and epidemiological utility of mass vaccination against pandemic A/H1N1 influenza virus. In fact, the cross-immunity described here could explain, at least in part, the strong immunological response to a single dose of pandemic A/H1N1 vaccine in adults.Serum samples from 107 subjects were obtained between Aug 18 and Oct 10, 2009 from primary care personnel and patients at Hôpital Saint Louis, Paris, France. Written informed consent was obtained for all patients, and the study was approved by the Paris Saint-Louis ethical committee.tat plasmid, expressing the influenza HA protein either from a vaccinal strain of seasonal A/H1N1 (A/Brisbane/59/2007) or from pandemic A/H1N1 , and grown in the presence of 5 mU/mL of soluble Clostridium perfringens neuraminidase. After 48 hours, the pseudovirus-containing supernatants were harvested and volumes of supernatant expressing an infectious titer of 104 RLU (Relative Light Unit) were incubated with twofold dilutions of decomplemented test serum (starting dilution 1∶20) for 1 hour at 37°C. Subsequently, 200 µL of virus-antibody mixture were transferred to MDCK-SIAT1 target cell cultures in quadruplicate in 96-well plates. The inhibitory effect of serum on pseudovirus infectivity was measured 48 h later by fitting the percent inhibition of RLU values to a sigmoid dose-response curve with variable slope, using the GraphPad Prism 4.0b software. The neutralizing titer was defined as the reciprocal of the serum dilution required to reduce viral infectivity by 50% and expressed as the geometric mean from two independent experiments.The pseudovirus neutralization assay used in this study is based on infectivity of pseudotyped HIV-1 particles expressing influenza HA proteins. Briefly, subconfluent monolayers of human 293-T cells in 6-well plates were co-transfected with 0.7 µg of the HIV-1 pNL4-3-ΔENV-LucR vector and 0.5 µg of the pVITRO2-blasti-mcs-Titration of antibodies to pandemic A/H1N1 by hemagglutination inhibition (HI) was performed by a standard assay using human group O erythrocytes and non-inactivated pandemic A/H1N1 virus.10 values of the titers. Correlations between neutralizing titers were examined by calculating the Spearman correlation coefficient; comparisons of neutralizing titers before and after pandemic A/H1N1 vaccination were done using the Wilcoxon signed rank-sum test; comparison of neutralizing titers between subject groups were performed using the Wilcoxon rank-sum test.Correlations and comparisons between neutralizing titers were done based on the log


Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the intracellular second messengers: cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP). The cAMP-specific PDE family 4 (PDE4) is widely expressed in vertebrates. Each of the four PDE4 gene isoforms (PDE4 A-D) undergo extensive alternative splicing via alternative transcription initiation sites, producing unique amino termini and yielding multiple splice variant forms from each gene isoform termed long, short, super-short and truncated super-short. Many species across the vertebrate lineage contain multiple splice variants of each gene type, which are characterized by length and amino termini.A phylogenetic approach was used to visualize splice variant form genesis and identify conserved splice variants (genome conservation with EST support) across the vertebrate taxa. Bayesian and maximum likelihood phylogenetic inference indicated PDE4 gene duplication occurred at the base of the vertebrate lineage and reveals additional gene duplications specific to the teleost lineage. Phylogenetic inference and PDE4 splice variant presence, or absence as determined by EST screens, were further supported by the genomic analysis of select vertebrate taxa. Two conserved PDE4 long form splice variants were found in each of the PDE4A, PDE4B, and PDE4C genes, and eight conserved long forms from the PDE4 D gene. Conserved short and super-short splice variants were found from each of the PDE4A, PDE4B, and PDE4 D genes, while truncated super-short variants were found from the PDE4C and PDE4 D genes. PDE4 long form splice variants were found in all taxa sampled ; short, super-short, and truncated super-short are detected primarily in tetrapods and mammals, indicating an increasing complexity in both alternative splicing and cAMP metabolism through vertebrate evolution.There was a progressive independent incorporation of multiple PDE4 splice variant forms and amino termini, increasing PDE4 proteome complexity from primitive vertebrates to humans. While PDE4 gene isoform duplicates with limited alternative splicing were found in teleosts, an expansion of both PDE4 splice variant forms, and alternatively spliced amino termini predominantly occurs in mammals. Since amino termini have been linked to intracellular targeting of the PDE4 enzymes, the conservation of amino termini in PDE4 splice variants in evolution highlights the importance of compartmentalization of PDE4-mediated cAMP hydrolysis. Cyclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of cyclic nucleotide second messengers; cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) ,2. The PPDE4A, PDE4B, PDE4C, and PDE4 D which arose via a gene duplication event in a common eukaryotic ancestor before the separation of sponges and eumetazoans [The PDE4 gene family is composed of four gene isoforms; etazoans . Althougetazoans . Mammalietazoans ,10 . In this study we first established the timing of PDE4 gene and splice variant form evolution in vertebrates using NCBI EST and ENSEMBL genome databases. We then identified evolutionary conserved splice variants (based on amino terminus conservation) within the vertebrate lineage that may likely form the functional foundation of PDE4 mediated cAMP degradation.We identified 162 PDE4 ESTs from 15 species encompassing the vertebrate phyla and one invertebrate  for all PDE4 splice variants used in this study , PDE4D1 (ENSTRUT00000034687), and Zebrafish 4D2 (XM_688538) and 4D3 (XM_001332019), all group into the "PDE4C" clade , E. caballus PDE4A1 (cDNA acc. # XR_044570), R. norvegicus 4A (protein acc. # EDL78317), M. musculus 4A (protein acc. # EDL25164) and 4 D (protein acc. # CAQ51655), H. sapiens PDE4C (ENSP00000336624). Splice variants grouped according to PDE4 gene isoform type and not splice variant form . All teleost long form transcripts were incorporated, as the catalytic domain phylogeny   suggest more than one PDE4 gene type is present in this species. Ciona intestinalis (outgroup) root placement established two main clades composed of PDE4A and PDE4B, and PDEs 4C and 4 D genes present in vertebrates studied  contain two PDE4B and PDE4C genes, however only O. lapites, G. aculeatus, and T. rubripes contain two PDE4A genes  hypothesis of Ohno [The PDE4 phylogeny was constructed from one representative long form splice variant sequence for each of our 17 vertebrate species  Figure . PreENSEd Figure   are located in tandem within intronic regions between the same domain junctions for all species examined  tested using Ensembl and NCBI EST databases  through humans (NP_001032416), but not in teleosts. PDE4B super-short form transcripts were detectable in EST libraries from teleosts (ENSTRUP00000002631) through humans (EF595686). PDE4B truncated super-short forms were not detectable in any EST libraries. PDE4C short and super-short transcripts were not detectable in any EST libraries screened. A PDE4C truncated super-short form splice variant transcript and genomic amino terminus exon were identified in both C. jacchus (ENSCJAP00000025959) and H. sapiens (ENSP00000336624), but absent in non-primates analyzed in this study  and paralogous gene conservation increased the number of long forms for PDE4A,4B, and 4C gene isoforms in D. rerio, and G. aculeatus (O. latipes PDE4A and 4C) through an increase in gene dosage, in lieu of long form alternative splicing. Extensive PDE4 alternative splicing is limited in non-mammalian ESTs and does not appear to be a common property of all vertebrate PDE4 gene isoforms.The intracellular targeting and microdomain diversity of the PDE4 family suggests a functional expansion through the incorporation of different splice variant forms , as well as the alternative splicing of multiple splice variant 5' exons from each PDE4 gene isoform . There is very limited evidence of alternative splicing in teleost PDE4 genes. Only one alternative splicing event (PDE4B super-short)  and duplicated (in teleosts), both of which can increase PDE4 proteomic diversity. In a recent study, a correlation was detected between the size of the gene family and frequency of alternative splicing events, as duplicated gene families had significantly fewer alternative splicing events than single copy or less duplicated gene families . This saThe mammalian evolution of each of the PDE4 splice variant forms  appears to have occurred at the same domain junctions independently within each gene isoform Figure . The losPDE4A are conserved across multiple species. The amino terminus of the PDE4A long form PDE4A8 is composed of two exons, both of which are conserved in the genomes of S. scrofa, B. taurus, C. familiaris, and C. jacchus, while these exons are not well conserved in M. musculus  to 98% (C. jacchus) sequence homology. The 5' exon corresponding to this amino terminus (PDE4A1) is conserved in the genomes of species from T. rubripes to H. sapiens . The PDE4A2 splice variant has EST support in both M. musculus and C. jacchus. The human PDE4A3/10 ortholog is detectable in the transcriptomes of B. Taurus, R. norvegicus, M. musculus, and C. jacchus  shares little sequence identity in these species (not shown).Four long forms and one super-short form splice variant from s Figure . EST seas Figure . A PDE4Aa Figure  , to 25% in teleosts (O. latipes). Two exons encode this amino terminus (PDE4B1), each of which were found in the genomes of these same species. Transcripts for the second PDE4B long form splice variant, orthologous to H. sapiens PDE4B3, were detectable in transcriptomes of X. laevis, S. scrofa, B. taurus, M. domestica, E. caballus, R. norvegicus, M. musculus, and C. jacchus . The 5' exon comprising this amino terminus was found in the genomes of all species ranging from teleosts to humans . A PDE4B super-short form orthologous to human PDE4B5 has both EST and genomic conservation from T. rubripes to H. sapiens,  from the s Figure . Long fonus PDE4B, each ofnus PDE4B, each ofnus PDE4B, each ofPDE4C is the smallest PDE4 gene isoform, and also contains the fewest total (two) conserved splice variants . Genomic sequence conservation of the PDE4C1 splice variant 5' exon was found in M. domestica, B. Taurus, C. familiaris, C. jacchus, and H. sapiens. The 5' exon sequence encoding this amino terminus (PDE4C1) is not intact in T. rubripes, and M. musculus . Genomic conservation for the 5' exon of PDE4C3 was found in B. taurus, C. familiaris, M. musculus, C. jacchus, and H. sapiens. T. rubripes and M. domestica lack an ATG start site (M. domestica and T. rubripes) and 5' sequence identity (T. rubripes) for this amino terminus.s Figure , . The PDE4 D gene is the only PDE4 gene isoform with detectable transcripts for short (orthologs to H. sapiens PDE4D1), super-short (orthologs to H. sapiens PDE4D6), and truncated super-short (orthologs to H. sapiens PDE4D2) splice variant forms. Both the short form PDE4D1 5' exon and truncated super-short form PDE4D2 have EST evidence and genomic conservation in R. norvegicus and H. sapiens  was found in T. rubripes, X. tropicalis, A. carolinensis, M. domestica, S. scrofa, B. taurus, C. familiaris, M. musculus, and C. jacchus. Splice variants orthologous to human PDE4D6 and PDE4B5 have homologous amino termini to each other .The 9 Figure ,  during heart failure [2 adrenoreceptor activity [Our multi-species PDE4 gene isoform alignments performed using PipMaker and transcript presence determined by NCBI EST database screens showed the evolutionary conservation of select PDE4 splice variant amino termini across species. Conservation across multiple species has been recognized as a key indicator of functional importance ,28, alth failure . The lonactivity ,35,36. TC. familiaris to H. sapiens, though genome conservation for the 5' exon of PDE4D6 extends to teleosts  4D6 upstream promoter regions appear to not have "TATA" and "GC" motifs, necessary for transcriptional initiation  share high nucleotide sequence identity (77-100% between vertebrates sampled)  early in vertebrate evolution  as compared to mammals   in different areas of the brain.The splice variants PDE4B5 and PDE4D6 appear to represent an exception to the rule among PDE4 splice variant amino termini; they have homologous amino termini  within gene isoforms , coding regions found in the LR1, LR2, and terminal catalytic domain exons are highly conserved across species Figures , 4, 5, 6PDE4A splice variants has not been shown to date. The observed conservation at this site across all isoforms strongly suggests ERK regulation within PDE4A splice variants as well  have been identified and described in PDE4 s ,26,39. Wn humans . This hiC. intestinalis) and primitive vertebrates (teleosts), multiple gene duplications produced the present day PDE4 gene isoforms (A-D). The production of multiple protein products from these genes through alternative splicing is limited in primitive vertebrates. Integration of distinctive long form amino termini and expansion of splice variant type  is largely introduced in tetrapods and greatly expanded in mammals. This growth in splice variant identity (unique amino termini) and different form types appears at different times throughout vertebrate evolution and occurs independently within each gene isoform. Sequence drift appears to be responsible for the development of PDE4 amino termini-coding exons, as well as promoter development.In summary, this phylogenetic analysis of PDE4 gene isoforms and alternative splicing has addressed several gaps in our understanding of the evolutionary basis for the functional diversity seen in the multitude of PDE4 splice variants. Following the divergence of invertebrates  have remained essentially completely conserved. The conservation of these regulatory domains, which provide an additional level of functional regulation, underpins the importance of these domains in an evolutionary functional context.http://www.ncbi.nlm.nih.gov and ENSEMBL http://www.ensembl.org genomic databases. The UCR-1, UCR-2, and catalytic domain nucleotide and amino acid sequences from human, rat and mouse PDE4A-D splice variants were used to identify annotated and unannoted PDE4 transcript and protein sequences in vertebrates and invertebrates  using NCBI BLASTP, TBLASTN, EST database searches and Ensembl (release 41). Select species, spanning the vertebrate lineage, used for this study include: mammals-Homo sapiens (H. sap), Callithrix jacchus (C. jac), Mus musculus (M. mus), Rattus norvegicus (Rnor), Canis familiaris (C. fam), Equus caballus (E. cab), Bos Taurus (B. tau), Sus scrofa (S. scr), Monodelphis domestica (M. dom), reptiles- Anolis carolinensis (A. car), amphibians- Xenopus tropicalis (X. tro), teleosts-Danio rerio (D. rer), Takifugu rubripes (T. rub), Oryzias latipes (O. lat), Gasterosteus aculeatus (G. acu); chordates- Sea lamprey Petromyzon marinus (P. mar).PDE4 nucleotide and protein sequences were obtained from a search of NCBI PDE4 translated amino acid sequences were aligned using the Clustal function of Mega v4, according to the default settings. PDE4 splice variant relationships based on the catalytic domain were determined using the Neighbor Joining algorithm of Mega v4. The tree shown represents three independent runs, each with 1000 bootstrap replicates using the PAM matrix of amino acid evolution. One long form splice variant nucleotide sequence from each clade was selected for each species of interest -upstream conserved region 1 (UCR1) which contains a PKA phosphorylation motif from residues 36-39, linker region 1 (LR1), upstream conserved region 2 (UCR2) which contains the amino terminus for truncated super-short forms, linker region 2 (LR2), catalytic domain, which contains the metal-dependent phosphohydrolase motif from residues 356-609 and a Kinase Interaction Motif (KIM) from residues 484-497, and carboxy termini which contains an ERK phosphorylation site from residues 621-623 and "FQF" motif from 660-662.Click here for fileSupplemental Figure S2. Phylogenetic classification of PDE4 Splice Variants Used in This Study. Supplemental Figure S2 is a Bayesian phylogenetic tree constructed using PDE4 catalytic domain amino acid sequences from each PDE4 gene isoform, from each taxa used in this study.Click here for fileSupplemental Table S2. Supplemental Table S2 lists orthologous PDE4 splice variants with different designations in nomenclature. Three PDE4A and three PDE4 D splice variants have homologous amino termini but have different names between humans and rodents.Click here for fileSupplemental Figure S3. PDE4 Exon Formation Through Sequence Drift. Supplemental Figure S3 is a multiple sequence alignment of flanking and coding nucleotide sequences orthologous to the H. sapiens splice variant PDE4A3/10, showing the formation of this exon occurred through sequence drift.Click here for fileSupplemental Figure S4. PDE4 Splice Variant Promoter Formation Through Sequence Drift. Supplemental Figure S4 is a multiple sequence alignments of the PDE4D6 upstream flanking region, PDE4B5 and PDE4D6 5'exons, and corresponding amino acid sequences.Click here for file


Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX.Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h) resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300.Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets. Chromatin-associated histone H2AX is a key regulator of the cellular response to genotoxic stress and DNA double-strand breaks -6. H2AX Previous studies in yeast have shown that histone levels need to be tightly regulated and that high levels of non-chromatin bound histones are cytotoxic . SolubleThe present study was designed to determine the mechanisms that lead to increased soluble H2AX. We found that even short-term stalling of replication forks is sufficient to cause an increase of soluble H2AX. Furthermore, we show that transient overexpression of H2AX, but not H2A, promotes apoptotic cell death. In addition, we demonstrate that overexpression of H2AX leads to a nuclear redistribution and/or loss of the active form of RNA polymerase II as well as the transcriptional co-activators CBP and p300. Taken together, our results suggest a model in which replication stress leads to increased soluble H2AX, which sensitize cells to undergo apoptosis in a process that involves a shut-down of gene transcription.bona fide normal DNA damage response pathways. Cells were transfected with constructs encoding GFP-tagged H2AX, mutant GFP-H2AX-S139A or empty GFP vector [To test whether DNA replication stress can induce soluble H2AX, we used the human osteosarcoma cell line U-2 OS that, unlike GIST cells, does not depend on a single oncogenically activated tyrosine kinase such as KIT and has P vector . ExpressP vector -19. TranTo further corroborate our results, U-2 OS cell populations stably expressing GFP-tagged H2AX, H2AX-S139A or empty vector were generated. In stable cell populations, H2AX was undetectable in S3 fractions from untreated cells Fig. . HoweverTaken together, these results show that transient stalling of DNA replication forks is associated with an increase of soluble histone H2AX.We next tested whether an increase of soluble H2AX can lead to enhanced apoptosis. As shown in Fig. To determine whether replication stress sensitizes H2AX-overexpressing cells to undergo apoptosis, we used U-2 OS cell populations stably expressing either empty GFP vector, GFP-tagged H2AX or GFP-H2AX-S139A. Cells were treated with aphidicolin for 24 h and the proportion of apoptotic cells was assessed Fig. . A signiWe tested next whether overexpression of core histone H2A was able to stimulate apoptosis. Transiently transfected cell populations were stained with an anti-myc-tag antibody and assessed for apoptotic nuclear morphology. The proportion of transfected cells was similar to H2AX-transfected populations. No enhanced cell death was detected when myc-tagged H2A was overexpressed under transient conditions in comparison to empty myc-tag vector Fig. .Taken together, these results indicate that overexpression of histone H2AX, but not histone H2A, can sensitize cells to undergo apoptosis.In order to determine the mechanisms that underlie H2AX induced cell death, we performed a morphological analysis of U-2 OS cells transiently transfected with empty GFP vector, GFP-H2AX or GFP-H2AX-S139A Fig. . Excessi1S2P3T4S5P6S7 that become hyperphosphorylated and hypophosphorylated during the transcription cycle. Phosphorylation at serine 5 is associated with transcription initiation whereas serine 2 phosphorylation is predominantly found during transcription elongation [We lastly asked whether chromatin aggregation induced by overexpression of histone H2AX can lead to a downregulation of active gene transcription . The majongation . Active ongation ,24. The ongation . These pongation ,27. LikeWe detected a significant increase of cells with redistribution of pRNA pol II S2 into nuclear speckles or loss of pRNA pol II S2 expression below detection level by immunofluorescence microscopy in cells ectopically expressing H2AX  or H2AX-S139A  when compared to controls  in comparison to GIST cells (approximately 40%) . One posIt is conceivable that replication stress is a frequent event in tumor cells ,33. GiveThe fact that soluble H2AX induces apoptosis in tumor cells opens new avenues for cancer therapy. Identification of drugs that induce soluble H2AX, e.g. by inhibiting a potential suppressor of H2AX, would be promising. Given the role of the ubiquitin-proteasome system in the regulation of H2AX levels in GISTs , it is aIn summary, we report here that DNA replication stress rapidly leads to an increase of soluble H2AX. Overexpression of H2AX was found to sensitize cells to undergo apoptosis, which very likely involves the disruption of ongoing gene transcription. Our findings encourage further studies to exploit H2AX and the cellular mechanisms that regulate its expression levels as novel anti-cancer drug targets.This report shows that stalling of DNA replication leads to an increase of soluble H2AX in human tumor cells. Soluble H2AX was found to sensitize cells to undergo apoptosis and this activity was associated with a transcriptional shut-down. These results encourage further studies to exploit histone H2AX and the mechanisms that regulate its expression as anti-cancer drug targets.U-2 OS osteosarcoma cells and H1299 lung cancer cells were obtained from ATCC  and maintained in Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum , 50 U/ml penicillin and 50 μg/ml streptomycin . Cells were transfected with an empty GFP vector or N-terminal GFP fusion plasmids encoding wild-type H2AX and H2AX in which serine 139 has been substituted by alanine (H2AX-S139A) using FuWhole cell lysates were prepared by scraping cells into RIPA buffer  containing protease inhibitors . Suspensions were incubated for one hour at 4°C and cleared by centrifugation for 30 min at 14,000 rpm at 4°C. Protein concentrations were determined by the Bradford assay . 30 μg of protein were loaded on a 4–12% Bis-Tris gel  and blotted onto a nitrocellulose membrane. Primary antibodies used were directed against GFP , H2AX , ORC2 (BD Biosciences Pharmingen), phosphorylated RNA pol II S2  and actin .For immunofluorescence micoscopic analyses, cells were grown on coverslips, washed in PBS, fixed in 4% paraformaldehyde/PBS (15 min), permeabilized with 1% Triton-X 100/PBS (15 min) and blocked in 10% normal donkey serum  for 15 min at room temperature. After incubation with primary antibodies overnight at 4°C in a humid chamber, cells were incubated with FITC-conjugated or Rhodamine Red-conjugated secondary antibodies  and counterstained with DAPI . For double-immunofluorescence analysis, cells were stained for the first antigen followed by a second primary antibody and an AMCA-labeled secondary antibody (Jackson Immunoresearch). Primary antibodies used for immunofluorescence stainings were directed against γ-H2AX , myc-tag , phosphorylated RNA pol II S2 , CBP and p300 . Cells were analyzed using an Olympus AX70 epifluorescence microscope equipped with a SpotRT digital camera.2, 10 mM NaF, 340 mM sucrose, 10% glycerol, 1 mM DTT, 0.1 mM PMSF, 1 mM sodium orthovanadate, 0.1% Triton-X 100, 5 μg/ml leupeptin 5 μg/ml aprotinin) and incubated on ice for 5 minutes. The nuclei (P1) were collected by low speed centrifugation at 1,300 g and lysed in Buffer B  for 10 minutes on ice. The insoluble chromatin fraction (P3) was collected by centrifugation at 1,700 g. The supernatant contained the soluble nuclear proteins (S3). The chromatin pellet (P3) was resuspended in SDS sample buffer and the DNA was sheared by sonication in a 550 Sonic Dismembrator (Fisher) at 15% amplitude.Subcellular fractionation was performed according to Mendez and Stillman . Cells wApoptotic cells were visualized using the In Situ Cell Death Detection Kit  according to manufacturer's instructions.Statistical significance was assessed using Student's t test for independent samples. P values ≤ 0.05 were considered significant.The authors declare that they have no competing interests.YL, JAP and AC carried out the immunoblot and immunofluorescence experiments and participated in the interpretation of results. SD analyzed the immunofluorescence experiments and participated in the design and interpretation of experiments. AD designed this study, participated in the analysis of experiments and drafted the manuscript.DNA replication stress induces soluble H2AX in H1299 lung carcinoma cells. Immunoblot analysis of soluble nuclear (S3) and chromatin bound (P3) protein fractions obtained from H1299 cells transiently transfected with empty GFP vector, GFP-H2AX or GFP-H2AX-S139A and treated with 10 μM aphidicolin (APH) for 1 h to block DNA replication. Staining for GFP detects expression of tagged proteins, whereas staining for H2AX detects endogenous protein. Immunoblot for ORC2 is shown to rule out carry-over between fractions. Ponceau S staining demonstrates protein loading.Click here for file


Helicobacter pylori, the major cause of human gastric diseases, affects histone modification. We therefore investigated the effects of H. pylori infection on histone modifications in a global and promoter-specific manner in gastric epithelial cells. Infection of gastric epithelial cells by wild-type H. pylori induced time- and dose-dependent dephosphorylation of histone H3 at serine 10 (H3 Ser10) and decreased acetylation of H3 lysine 23, but had no effects on seven other specific modifications. Different cag pathogenicity island (PAI)-containing-clinical isolates showed similar abilities to induce H3 Ser10 dephosphorylation. Mutation of cagA, vacA, nonphosphorylateable CagA mutant EPISAcagA, or disruption of the flagella showed no effects, while deletion of the entire cagPAI restored the H3 Ser10 phosphorylation to control levels. Analysis of 27 cagPAI mutants indicated that the genes that caused H3 Ser10 dephosphorylation were similar to those that were previously found to induce interleukin-8, irrespective of CagA translocation. This effect was independent of ERK or p38 pathways and type I interferon signaling. Additionally, c-Jun and hsp70 gene expression was associated with this histone modification. These results demonstrate that H. pylori alters histone modification and host response via a cagA-, vacA-independent, but cagPAI-dependent mechanisms, which contribute to its persistent infection and pathogenesis.Histone modifications are critical in regulating gene expression, cell cycle, cell proliferation, and development. Relatively few studies have investigated whether  Helicobacter pylori, a Gram-negative bacterium, is a major cause of chronic gastritis, peptic ulcers, and gastric malignancies, including gastric non-cardia adenocarcinoma and mucosal-associated lymphoid tissue (MALT) lymphoma H. pylori infection induces both acute and chronic gastritis, which is present as superficial mucosal inflammation in the gastric mucosa. In vitro experiments demonstrate that H. pylori activates multiple intracellular pathways including mitogen-activated protein kinases (MAPK), NF-κB and activator protein-1 as well as the Wnt/β-catenin pathway, which affect various cellular functions. These include increased inflammatory cytokine production, increased apoptosis, and epithelial cell turnover cag pathogenicity island (PAI), and vacuolating cytotoxin (VacA) are responsible for these effects H. pylori VacA has also been shown to inhibit T-cell proliferation and the cell cycle, and therefore suppress the immune response H. pylori induces the transcription of thousands of host genes, while at the same time represses another set of genes Chronic infection of the human stomach by Listeria monocytogenes secretes listeriolysin O (LLO), which induces a dramatic dephosphorylation of histone H3 at serine 10 (H3 Ser10) and deacetylation of histone H4, and this correlates with changes in host gene expression during the early infection Clostridium perfringens toxin perfringolysin (PFO) and Streptococcus pneumoniae toxin pneumolysin (PLY) also induce the same dephosphorylation of histone H3 Ser10; this decreased phosphorylation of H3 Ser10 is associated with the previously reported decreased inflammatory cell responses during bacteria infection Shigella flexneri toxin OspF blocks phosphorylation of MAPK ERK2 in the nucleus; this subsequently prevents histone H3 Ser10 phosphorylaton, which is a prerequisite of NF-κB activation and downstream gene transcription, and leads to a compromised inflammation in mouse tissue Immune subversion by histone modification is an important mechanism used by multiple bacteria and viruses during infection H. pylori infection is chronic and persistently enhanced inflammation with increased inflammatory cell infiltration in the local gastric mucosa and increased inflammatory cytokine production. A small percentage of infected individuals manifest the clinical presentation of gastritis, peptic ulcer, or gastric malignancy H. pylori infection effects histone modifications has not been as thoroughly evaluated. In the present study, we investigated if H. pylori infection modulates host gastric epithelial cell histone modification. In addition, we correlated H. pylori-induced histone modification with the changes in gastric epithelial cell functions and detected bacterial genes that are responsible for these effects, and explore their impact on pathogenesis.One prominent feature of H. pylori cagPAI-dependent dephosphorylation of histone H3 Ser10 and deacetylation of H3 lysine (K) 23. The effects of H3 Ser10 dephosphorylation are independent of major H. pylori virulence factors including CagA, VacA, and flagella. In addition, this cagPAI-dependent effect is common to multiple H. pylori strains, and the histone H3 Ser10 dephosphorylation is independent of ERK and p38 pathways, and type I interferon (IFN) signaling. Additionally, H3 Ser10 dephosphorylation is associated with changes in host gene expression. These results indicate a novel mechanism of H. pylori pathogenesis through histone modifications that has potential implications on H. pylori-induced chronic and persistent infections.We demonstrate a H. pylori in gastric epithelial cells, we first monitored the histone H3 Ser10 phosphorylation status in whole cell extracts by Western blot  did not alter the dephosphorylation of H3 Ser10 in AGS cells, compared to the wild-type 60190 strain  are the cause of H. pylori-induced H3 Ser10 dephosphorylation.The major virulence factors of rylation . In addi0 strain . These rH. pylori genes products within the cagPAI are the cause of H3 Ser10 dephosphorylation in AGS cells, G27-MA (and its derivatives) did not induce H3 Ser10 dephosphorylation in MKN45 cells (data not shown), likely due to the fact that G27-MA is a MDCK cell adherent strain H. pylori Sydney strain 1 which has a handicapped cagPAI cagPAI, induced H3 Ser10 dephosphorylation (data not shown). The results suggest both pathogen and host factors may affect histone dephosphorylation.Although we showed that H. pylori can cause H3 Ser10 dephosphorylation, we next investigated if H. pylori 26695 also induced other histone modifications. We monitored several common histone protein modifications, including the acetylation of H3K9, H3K14, H3K18, H3K23, H3K9K14, H3S10P/K9Ac, H3S10P/K14Ac, dimethylation of H3K9, hyperacetylation of H4, and acetylation of H4K8 in gastric epithelial cells. The results indicated that H. pylori 26695 cagPAI induced dephosphorylation of H3S10P/K14Ac, in a similar manner to the H3 Ser10 dephosphorylation caused by H. pylori in both AGS (cagPAI-dependent (data not shown). We also detected decreased H3K23 acetylation during H. pylori infection in a cagPAI-dependent manner in AGS cells, while other modifications tested in this study revealed no difference as compared with uninfected control cells , a histone deacetylase inhibitor which non-specifically increases the chromatin acetylation status, resulted in increased multiple histone H3 acetylations (data not shown). Interestingly, this effect is associated with some commonly reported gene transcription pattern alterations in nd c-fos . We notecagA is not responsible for the H3 Ser10 dephosphorylation in gastric epithelial cells as shown above, we investigated which gene or gene product within the cagPAI was responsible for this effect. To determine this, we used a series of H. pylori cag mutant strains cag genes that are necessary for IL-8 induction  Since H. pylori or L. monocytogenes infection cxcl2, prkdc, dusp4, cox2, c-Jun, hsp70, cyclin D1, and il-8, using chromatin immunoprecipitation (ChIP). We infected AGS cells with wild-type H. pylori G27-MA, which showed dephosphorylation of H3 Ser 10, and performed RT-PCR and ChIP assays. Interestingly, we noted that c-Jun and hsp70 gene expression was correlated with the H3 Ser10 phosphorylation in the promoter region of these genes . c-Jun and hsp70 mRNA expression levels in H. pylori-stimulated AGS cells represented up- or down- regulated genes, respectively. The results are in line with previously reported gene array H. pylori selectively regulates gene expression in host cells associated with chromatin modification, which subsequently regulates cellular functions.To determine if the changes in histone modification are associated with alterations in host gene expression, we tested a group of genes that has previously reported to be changed during se genes . On the H. pylori infection status  hours post-infection  has been shown to induce H3 Ser10 phosphorylation at the IL-6 promoter and this is also associated with increased IL-6 mRNA and protein expression H. pylori has been shown to regulate p21(WAF1) expression associated with histone H4 acetylation in gastric epithelial cells c-fos, expression L. monocytogenes has been shown to regulate il-8 gene expression through histone modification, including increased H4K8 acetylation and phosphorylation of histone H3 Ser10 in endothelial cells il-8 and c-fos genes, including the up-regulation of c-fos and down-regulation of il-8 in MKN45 cells upon H. pylori infection. These results suggest that altered chromatin structure can function to “mask” or “unmask” gene transcription start sites and therefore affects their transcription. The opposite effects between il-8 and c-fos gene transcription provide additional information on the bacterial-induced gene regulation, and may suggest different roles of histone deacetylation/acetylation and transcription factors play on each of their gene promoter in gastric epithelial cells.The effects of specific histone modifications in host cells appears to be cell type and promoter specific: in the mouse macrophage, H. pylori has been shown to affect the cell cycle H. pylori infection are not clear. Interestingly, during the submission of this work, a paper by Fehri et al. H. pylori-induced cagPAI dependent H3 Ser10 dephosphorylation. H3 Ser10 dephosphorylation and cyclin B1 correlated with transient pre-mitotic arrest. Further they showed vaccinia-related kinase 1 (VRK1) activity but not Aurora B activity are responsible for this transient cell cycle delay. In addition, they pointed out that this effect was IkB kinase alpha (IKKα) dependent H. pylori-induced cell cycle arrest is independent of cagPAI H. pylori-induce a cagPAI dependent H3 Ser 10 dephosphorylation, and the current work further suggest this modification is also associated with gene transcription control. Since H3 Ser10 phosphorylation has been linked with both cell cycle and transcription control H. pylori pathogenesis remain to be established in the future.H3 Ser 10 is cell cycle mitotic marker in proliferating cells, and H. pylori-induced H3 Ser10 phosphorylation levels. SP600125, the JNK inhibitor, which has been reported to reduce global H3 Ser10 phosphorylation H. pylori-induced a fast inhibition on H3 Ser10 phosphorylation within one hour, a time we reason most likely linked to transcriptional control through chromatin remodeling. Therefore, the current results provide a signaling link between bacterial infection and H3 Ser10 dephosphorylation.MAPK pathways including ERK and p38 have been reported to mediate the increased H3 Ser 10 phosphorylation, but not decreased H3 Ser10 phosphorylation il-8, cox2, cxcl2, prkdc, dusp4, and cyclin D1 were not associated with, or were independent of, histone modification in the current infection model. However, altered c-jun and hsp70 gene expression was associated with the H3 Ser10 dephosphorylation. Increased c-Jun protein phosphorylation and mRNA and reduced hsp70 mRNA has been observed during H. pylori infection H. pylori infection is not clear, but several studies have suggested it has cytoprotective effects, by reducing stress-induced denaturation and aggregation of intracellular proteins, and protecting the mitochondria, and interfering with the stress-induced apoptotic program H. pylori infection by inhibiting the expression of iNOS from gastric epithelial cells The global gene expression profile regulated by phosphorylated histone H3 Ser10 has yet to be identified. In this work, we noted, several commonly expressed genes, including H. pylori-induced pathophysiology in human stomach related to histone modifications. The delicate interplay between the host and pathogen may potentially lead to the different outcomes of H. pylori infection. Identification of the mechanisms by which H. pylori affects chromatin structure at gene promoters will allow us a better understanding of the gene transcription control, and subsequent alteration of cellular functions.In conclusion, these observations provide novel evidence in microbial pathogenesis as well as 2 incubator.Tissue culture reagents were purchased from GIBCO . The human gastric epithelial cell line, AGS, and the mouse fibroblast cell line, L929, were purchased from American Type Culture Collection . The human gastric epithelial cell line MKN45 was purchased from JCRB Cell Bank . Cells were grown in Ham's F-12 (AGS) or RPMI 1640 (MKN45 and L929) medium supplemented with 10% fetal bovine serum (FBS) without antibiotics at 37°C in a humidified 10% COH. pylori stimulation.MAPK inhibitors, including MEK1/2 inhibitor U0126, which inhibits extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation, p38 inhibitor SB203580, and c-Jun/SAPK N-terminal kinases (JNK) inhibitor SP600125, were purchased from Calbiochem . Cells were treated with the above inhibitors 30 minutes before H. pylori infection. Controls without inhibitors were treated with medium alone and an equal concentration of DMSO.Rabbit polyclonal anti-histone antibodies including histone H3 phosphorylated at serine 10 (p-H3 Ser10); histone H3 acetylated at lysine 9 (H3K9Ac); histone H3 acetylated at lysine 18 (H3K18Ac); histone H3 acetylated at lysine 23 (H3K23Ac); histone H3 phosphorylated at serine 10 and acetylated at lysine 9 (H3S10P/K9Ac); histone H3 phosphorylated serine 10 and acetylated lysine 14 (H3S10P/K14Ac); histone H3 dimethylated at lysine 9 (dimeH3K9); total H3; and histone H4 acetylated at lysine 8 (H4K8Ac) were purchased from Cell Signaling Technology . Histone H3 acetylated at lysine 14 (H3K14Ac); histone H3 acetylated at lysine 9 and lysine 14 (H3(K9K14)Ac); and hyperacetylated H4  were purchased from Upstate Biotechnology . Specific histone deacetylase inhibitor trichostatin A (TSA) was purchased from Sigma . Stock solutions were prepared in DMSO solution at 100 mM. In some experiments, cells were treated with the above inhibitors 30 minutes before H. pylori strains were used in the current study including 26695 and its isogenic cagPAI mutant (entire cag island deletion) strain 8-1  H. pylori strain 60190 and its vacA mutant strain (60190 VacA/KO), which contains a kanamycin cassette insertion  cag+ strains   cagA, vacA, cagPAI, cagA-vacA, and the nonphosphorylateable CagA mutant EPISAcagA and EPISAvacA-cagAcagPAI genes , and strain P12, its ΔcagA and ΔcagPAI mutants 2 at 37°C, and then harvested with a sterile cotton swab and resuspended in phosphate buffered-saline (PBS) solution. The bacteria were pelleted at 1,400×g for 10 minutes and resuspended in 5 ml of culture medium and added to the cell culture media at different bacteria to cell ratios , as indicated. Infections lasted from 0.5 to 6 hours in 10% CO2 at 37°C. Under these conditions, H. pylori remained alive and motile . We also noted that wild-type 26695, P12, and G27-MA strains induced hummingbird phenotype.A total of 52 different 5) or MKN45 cells (1×106) per well in 6-well culture plates were infected with bacteria at designated doses for designated times, washed three times with PBS, and lysed with cell lysis buffer directly on the dish (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue). Prior to loading, samples were boiled at 100°C for 3 minutes, cooled down on ice, and then separated on 15% SDS polyacrylamide gels. Proteins were subsequently transferred from gels onto nitrocellulose membranes (Bio-Rad). Membranes were blocked for 1 hour at room temperature in Tris-buffered saline plus 0.025% Tween-20 (TBS-T) with 5% nonfat dry milk (pH 7.4). Various histone antibodies were diluted at 1∶1000 in TBS-T with 5% nonfat dry milk solution. Membranes were then incubated with antibodies at 4°C overnight and washed three times with TBS-T (pH 7.4). The secondary antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies, were used at a 1∶1000 dilution in TBS-T with 5% nonfat dry milk and incubated at room temperature for 3 hours (pH 7.4). Bands were detected with an enhanced chemiluminescence detection kit . In some experiments, proteins were separated on 8% polyacrylamide gel, blotted on a polyvinylidene difluoride membrane, and examined for p-H3 Ser10 with protein A coupled to alkaline phosphatase. The original membrane was stripped with stripping buffer (50 mM Tris-HCl (pH 6.7), 2% SDS, and 200 mM β-mercaptoethanol) at 60°C for 30 minutes, followed by three washes with TBS-T and blocked for 1 hour with TBS-T in 5% nonfat dry milk solution (pH 7.4) and re-probed with total histone H3 antibodies to assess protein loading.AGS cells (5×10Rt–Et), where Rt is the mean threshold cycle for the reference gene HPRT and Et is the mean threshold cycle for the experimental gene. Data were expressed as arbitrary units and fold changes were adjusted to the non-stimulated control cells. Primer sequences are provided in Total RNA from AGS and MKN45 cells was purified using the RNeasy Mini Kit (Qiagen), as described previously Cells were seeded and treated essentially the same way as described for Western blot. The procedures to detect IL-8 production from AGS cell was described previously 6) per well in 150 mm dish were infected with bacteria at MOI of 150:1 for 6 hours, washed three times with PBS, and cross-linked with 1% formaldehyde (Sigma) for 10 minutes. Cells were harvested in cell lysis buffer, and sonicated with 6×10 second pulses to generate 0.4–1.2 kb size. The sonicated chromatin was then split equally into four parts, one part without any process served as input control, the other 3 parts were immunoprecipitated with: rabbit polyclonal histone H3 Ser10 antibody  H. pylori treated control. Total H3 and non-immune isogenic IgG was used as monitoring control. PCR primer for both ChIP and RT-PCR are listed in Chromatin immunoprecipitation (ChIP) assays were performed as previously described “t” tests, and differences were considered significant if P values were <0.05.All quantitative data were expressed as mean ± SEM. Data were compared by using paired or unpaired Student Figure S1cagPAI mutants on H3 Ser 10 dephosphorylation during H. pylori infection in AGS cells. AGS cells (5×105) were treated in the presence or absence of wild-type H. pylori 26695 and its various mutants strains at MOI of 150:1. The cell lysate was then subjected to immunoblot analysis with rabbit anti-phospho-histone H3 Ser10 antibodies, anti-total H3 antibodies were used to re-probe the membrane and monitor protein loading. A representative blot from each strain is presented.Effects of (0.18 MB TIF)Click here for additional data file.Figure S2cagPAI mutants on H3 Ser 10 dephosphorylation during H. pylori infection in AGS cells. Data are mean±SEM from 3–6 densitometry scans, adjusted with total histone H3, and expressed as fold changes over the appropriate control. **P<0.05, *P<0.01 when compared with controls.Comparison of the effects of (0.25 MB TIF)Click here for additional data file.Figure S3H. pylori-induced H3 Ser10 dephosphorylation is independent of IFN α/β signaling or bacterial DNA transfer into host cells. Mouse fibroblast cell line L929 (2×105) and human gastric epithelial cell line AGS (5×105) were treated with H. pylori G27-MA (G27) or its isogenic mutant strains in antibiotic-free medium for 10 hours at an MOI of 100:1, control cells were treated with medium alone. Supernatant from L929 cells were collected and used to measure IFN α/β production (panels A and B). RNA was extracted from AGS cells with the same treatment and cDNA was made for quantitative RT-PCR assay as described in (0.21 MB TIF)Click here for additional data file.


SRD5A2) encodes a critical enzyme involved in the conversion of testosterone to dihydrotestosterone. A germline mis-sense substitution (A49T) leads to a variant SRD5A2 protein, which has a 5-fold higher in vitro V max than the wild-type protein . The A49T variant was recently associated with 2.5 to 3.28-fold increased risk of prostate cancer (PC) in African-American and Hispanic men . Also, Jaffe et al (2000) reported an association between A49T and more aggressive disease among Caucasian patients. Here, we report that the prevalence of the A49T variant in 449 Finnish PC patients was 6.0%, not significantly different from 6.3% observed in 223 patients with benign prostatic hyperplasia or 5.8% in 588 population-based controls . There was no association between A49T and the family history of the patients nor with tumour stage or grade. Our results argue against a prominent role of the A49T variant as a genetic risk factor for prostate cancer development and progression in the Finnish population. © 2001 Cancer Research Campaign www.bjcancer.comProstatic steroid 5-alpha-reductase gene (


To the Editor:Streptococcus pneumoniae is a major human pathogen. In 2007, Park et al. identified a novel serotype, 6C  (reference 06/Q1704/105). A total of 697 nasopharyngeal swab specimens were collected from unselected (not selected by a method) children In the first year of this study, we identified 3 (3.1%) serotype 6C pneumococci belonging to 3 sequence types (STs): ST65, ST1714, and ST1692 . ST1714 <0.01), which was explained mostly by a large increase in ST1692 (p<0.03) (No serotype 6C ST65 was observed in the second year. We isolated more serotype 6C pneumococci in year 2 than in year 1 (p(p<0.03) . A recenOur study showed a large increase in ST1692 in serotype 6C pneumococci during the implementation of PCV7 and an increase in serotype 6C. Depending on the extent of cross-protection between vaccine-related serotypes, introduction of conjugate vaccines could induce clearance or emergence of vaccine-related serotypes. This introduction could also contribute to their substitution with novel or existing serotypes that are better adapted to the ecologic niche. However, our data may only be relevant to carried pneumococci and not reflected in pneumococcal disease epidemiology. Nevertheless, the increase in serotype 6C pneumococci in the United Kingdom, which is supported by a similar observation in the United States (Genotypes of serotype 6C pneumococci isolated from children in 2006-2007 (year 1) and 2007-2008 (year 2), United Kingdom. ST, sequence type.


T15+ foci-forming B cells were firstdetected 6 days after birth and expanded rapidly to constitute greater than 80% of the totalPC-specific foci by day 10. Injection of heat-killed S. pneumoniae (R36A) into neonatal miceresulted in priming of the antibody response to PC, with an idiotype profile reflecting thatof precursors of foci-forming B cells at the time of antigen administration. Priming of 2-dayoldmice with 2 X106 and 2 X107 R36A induced a five- and ten-fold increase in the antibodyresponse to phosphorylcholine 6 to 8 weeks later. However, only 10 to 15% of the serumantibodies expressed the normally dominant T15 idiotype. Doses below 2 x105 R36A showedno detectable priming activity. PC-specific hybridomas derived from mice injected with2 X107 R36A 2 days after birth lacked the idiotypic and molecular characteristics typical ofT15+ antibodies. Antibodies to phosphorylcholine, raised by immunization of 6-week-oldmice are normally protective against pneumococcal infection. However, serum antibodiesfrom mice treated with R36A 2 days after birth and responding to phosphorylcholinefollowing challenge with R36A at 6 weeks of age failed to protect against deliberate infectionwith virulent S. pneumoniae. These observations imply that the antigen phosphorylcholinedoes not play a role in the selective expansion and dominant expression of the T15 idiotype.Precursors of B cells capable of responding to a T-independent form of phosphorylcholine(PC) in splenic focus assays were detected in the spleens of neonatal mice as early as 4 daysafter birth. The earliest anti-PC B cells were T15


Escherichia coli ribosomal protein S5, in which glycine is changed to aspartate at position 28 [S5(G28D)], results in cold sensitivity and defects in ribosome biogenesis and translational fidelity. In an attempt to understand the roles of S5 in these essential cellular functions, we selected extragenic suppressors and identified rimJ as a high-copy suppressor of the cold-sensitive phenotype associated with the S5(G28D) mutation. Our studies indicate that RimJ overexpression suppresses the growth defects, anomalous ribosome profiles and mRNA misreading exhibited by the S5(G28D) mutant strain. Although previously characterized as the N-acetyltransferase of S5, our data indicate that RimJ, when devoid of acetyltransferase activity, can suppress S5(G28D) defects thus indicating that the suppression activity of RimJ is not dependent on its acetyltransferase activity. Additionally, RimJ appears to associate with pre-30S subunits indicating that it acts on the ribonucleoprotein particle. These findings suggest that RimJ has evolved dual functionality; it functions in r-protein acetylation and as a ribosome assembly factor in E. coli.A specific mutation of  Escherichia coli (E. coli) is a highly complex and co-ordinated process that remains largely uncharacterized despite its importance to cell physiology. The ribosomal RNAs (rRNAs) are synthesized as a primary transcript, which must be processed, folded and bound by ribosomal proteins (r-proteins) to form functional ribosomes  and large (50S) subunits. The small subunit contains a 16S rRNA and 21 r-proteins (S1-S21), while the large subunit contains two rRNAs, 5S and 23S, as well as 34 r-proteins. Thus, the process of ribosome biogenesis minimally involves the appropriate folding and interaction of over 50 r-proteins and three rRNAs (composed of more than 4000 nucleotides).Ribosome biogenesis in in vitro  mutations  strain. Additionally, we have shown that the suppressor function is independent of its acetyltransferase activity. We also demonstrate that RimJ associates with pre-30S subunits and can immunoprecipitate some 30S subunit components, including 16S rRNA, indicating that it exerts its function at the level of the RNP. Thus, an additional in vivo role has been identified for RimJ and leads to the proposal that RimJ acts as both a ribosome assembly factor and an r-protein modification enzyme.Here, we describe the identification and characterization of a role of RimJ (E. coli genomic library was transformed into the S5(G28D) strain and colonies that could grow at the non-permissive temperature (20°C) were identified. A total of 24 independent isolates were subsequently re-screened for growth at the non-permissive temperature (data not shown). A third round of screening, where plasmid DNA isolated from each of the cold-tolerant colonies was transformed again into S5(G28D) cells, resulted in the identification of five independent suppressors.In an earlier study, the specific mutation of glycine to aspartate at position 28 in r-protein S5 was shown to result in spectinomycin resistance and cold sensitivity, whereby, at a reduced temperature (20°C), growth was undetectable for the mutant strain  that allows regulated expression of the gene. Induced expression of rimJ from p-rimJ alleviates the cold-sensitive phenotype of S5(G28D) thereby confirming that rimJ can act as an extragenic suppressor.To identify the gene(s) responsible for the suppression of the cold sensitivity of the S5(G28D) strain, plasmid DNA from each of the five isolates was sequenced from both ends of the insert. Sequence analysis revealed that all of the isolates contained overlapping DNA fragments (data not shown). Although DNA sequences on either side of the overlapping elements were unique, all suppressor DNA fragments contained the entire Overexpression of RimJ decreases the doubling time of S5(G28D) cells at all temperatures, although, the effect is more pronounced at the non-permissive temperature. At low temperature, the S5(G28D) strain has a doubling time at least five times longer than its parental strain and is virtually immeasurable . OverprorimJ should alter the growth of the S5(G28D) strain. To study this genetic link, a strain bearing a deletion of rimJ (JW1053) and the S5(G28D) mutation was constructed and the phenotype of the double mutant was analysed. Examination of doubling times revealed a growth defect of the [S5(G28D)ΔrimJ] double mutant strain relative to the parental strains, S5(G28D) or ΔrimJ. At reduced temperature (20°C), growth is virtually undetectable for both [S5(G28D)ΔrimJ] and S5(G28D) strains (doubling time of > 300 min). At the permissive temperature (37°C), the double mutant strain had an exaggerated doubling time (69 min) as compared with S5(G28D) (44 min) and ΔrimJ (36 min). This significant alteration in growth properties can be also observed on solid media (rimJ (JW1053) results in a slight growth defect compared with the parental BW25113 strain at all temperatures (rimJ is tolerated in a S5(G28D) background, this double mutation is deleterious and that the suppression observed with RimJ overexpresson is not merely non-specific.If there is a functional link between RimJ overexpression and the S5(G28D) protein, one prediction would be that deletion of id media . As expeeratures . This su-rimJ strain as compared with what is found in the S5(G28D) + pBAD strain , further indicating that RimJ plays a role in ribosome biogenesis.One likely cause of the growth defects observed in the S5(G28D) strain is the abnormal ribosomal subunit/functional ribosome ratio observed in this strain. Ribosomes from strains carrying the S5(G28D) mutation are characterized by an increase in free 30S and 50S subunits compared with functional 70S particles and an accumulation of precursor 30S particles, especially at the non-permissive temperature . To begilacZ gene or the lacZ gene with mutations in the 5′ end enable miscoding to be assessed by monitoring β-galactosidase activity in vivo , the known function of RimJ was evaluated. RimJ is the N-acetyltransferase of S5 (N-acetyltransferase) domain (rimJ strain (CWZ387) (in vivo.In an attempt to dissect the manner by which extra copies of ) domain . Mass an) domain  are acet(CWZ387) . These f-rimJ to change the two cysteines at positions 54 and 105 in E. coli RimJ to alanine or phenylalanine residues to create four different singly and doubly substituted proteins . To determine if these mutations were sufficient to obliterate acetyltransferase function, the mutant RimJ proteins were overexpressed in the rimJ deletion strain  constructs revealed that the majority of S5 was unacetylated when either the empty vector or RimJ was induced. However, S5 was clearly acetylated when wild-type RimJ was induced (rimJ suppression.To explore the requirement of the acetyltransferase function of RimJ for suppression of phenotypes associated with the S5(G28D) mutation, RimJ mutants that lack acetyltransferase activity were constructed. Cysteine residues have been implicated in the enzymatic activity of many acetyltransferases . Site-dime, rimI , lane 1. induced . These rrimJ, RimJ, was used for cold sensitivity suppression analysis. Overexpression of RimJ in S5(G28D) at the non-permissive temperature results in similar growth as seen with wild-type RimJ . Subunit dissociation and polysome analysis of ribosomes from S5(G28D) strain with RimJ overexpression further suggest that there is no stable interaction of RimJ with polysomes (data not shown). Thus it appears that these fast sedimenting molecules are aggregates of RimJ that can be observed upon extensive overexpression.To test the association of RimJ with ribosomal particles as has been shown for many assembly factors, fractions were collected across the ribosome profiles of S5(G28D) with RimJ overexpressed at the non-permissive temperature. Immunochemical analysis revealed RimJ in fractions containing free pre-30S subunits, but apparently not in mature 30S subunits or in 50S subunits or 70S particle containing fractions . These r4Cl tested, even at 0.5 M NH4Cl (data not shown), suggesting that the co-sedimentation of RimJ with pre-30S subunits is not fortuitous but likely due to a valid interaction. To augment our hypothesis that RimJ binds to 16S rRNA containing RNPs in vivo, we performed immunoprecipitations using RimJ. Analysis of the precipitated complexes for RNA revealed that RimJ associates with 16S rRNA (The stability of interaction of RimJ with pre-30S particles was further studied by isolating pre-30S particles from S5(G28D) strain with RimJ overexpressed and re-sedimenting these particles through gradients containing increasing concentrations of ammonium chloride. RimJ was found to be associated with pre-30S subunits at different concentrations of NH16S rRNA  but not 16S rRNA  but not 16S rRNA , indicat16S rRNA . These fin vivo.In our previous work, we identified a unique point mutation within S5 which results in a change of a glycine residue to an aspartate at a highly conserved position  was created by transducing JW1053 to kanamycin and spectinomycin resistance using a phage P1 lysate of S5(G28D). The deletion of rimJ in this strain was confirmed by PCR amplification and the presence of the S5(G28D) mutation was ascertained by S5 gene (rpsE) amplification and subsequent sequencing using rpsE-specific primers.Strains S5(G28D), ΔE. coli pUC-based genomic library  was transformed into the S5(G28D) strain and colonies that could grow at the non-permissive temperature (20°C) on 2XYT plates containing ampicillin (100 μg ml−1) and spectinomycin (60 μg ml−1) were identified.A high-copy Cells were grown at 37°C (permissive temperature) to lag phase or grown at 37°C for 60 min prior to being shifted to 20°C (non-permissive temperature). Cell growth was continued for another 30 min at the non-permissive temperature before adding 0.2% arabinose, when appropriate. Optical density at 600 nm was measured every 30 min during the course of the growth  to obtain growth curves and determine the doubling times.For dilution plating, an equal number of cells from overnight cultures grown in LB plus appropriate antibiotics at 37°C were subcultured into fresh LB and grown at 37°C to an OD of ∼0.3. Ten-fold serial dilutions were made using fresh LB and plated on LB plates plus appropriate antibiotics and 0.2% arabinose, when required. Plates were incubated at 37°C overnight or at the non-permissive temperature for at least 48 h.For analysis of ribosome profiles, cell growth was carried out as done for estimation of doubling times (mentioned above). Ribosome analysis was essentially carried out as described  was performed with amounts of RNA (OD measured at 260 nm) equalized. 16S rRNA and 23S rRNA were used as markers for 30S and 50S subunits, respectively, and analysed by agarose gel electrophoresis.To localize RimJ in the ribosome profiles, fractions of 1 ml were collected across the profiles of S5(G28D) with p-rimJ gene was PCR amplified from E. coli (CSH142) genomic DNA using the primers: 5′-CAGGCTATTCATATGTTTGGCTATCGCAGTAACGTG-3′ and 5′-TCCGCGGCTGAATTCTTAGCGGCCGGGCGTCCA-3′.The rimJ was induced by a final concentration of 0.2% arabinose. The construct carried a V5-epitope for indirect detection of the recombinant RimJ and a polyhistidine tag that was utilized for immunoprecipitation.It was ultimately inserted into the pBAD-DEST49 vector by GATEWAY technology (Invitrogen). Expression of rimJ was conducted using QuickChange kit (Stratagene). The primers used for site-directed mutagenesis were:DNA sequencing of the suppressor element of S5(G28D) was performed using pUC/M13 sequencing primer (5′-TGTAAAACGACGGCCAGT) and reverse (M13) primer (5′-CAGGAAACAGCTATGACC-3′) at the Iowa State University sequencing facility. Site-directed mutagenesis to create substitutions of cysteine residues in p-(C54F) sense primer: 5′-GACGAAAGCCACTTTTATCCATCAGGC-3′ and the corresponding antisense primer;(C54A) sense primer: 5′-GACGAAAGCCACGCCTATCCATCAGGC-3′ and the corresponding antisense primer; and(C105A) sense primer: 5′-CTTTTCATGCCGCCTATCTCGG-3′ and the corresponding antisense primer.The underlined codons correspond to the substituted amino acids.rimJ or p-rimJ, growth was carried out at 37°C. MALDI and ESI samples were prepared by adding two volumes of 1 M magnesium acetate and glacial acetic acid mixture  to the 30S subunits and the extracted r-proteins (30S subunits were collected by sucrose gradient sedimentation of cell lysates from wild-type CSH142 or S5(G28D) cells grown at both 37°C and 20°C. For the CWZ387 strain in the presence of pBAD, p-proteins  were deslacZ S5(G28D) strain used here was available from our previous work  strain  strain with various constructs and p-rimJ were reported as ratios to those observed in the isogenic MC252 strain.Cells to be assayed for the activity of β-galactosidase, as described previously  was isolated from the 30S subunits  of CWZ38rimJ ( non-induced) or overexpressed RimJ (p-rimJ induced) were prepared in B-150 buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.2% Triton-X, 1X Roche-Complete Protease Inhibitor). Cleared extracts containing ∼3 mg ml−1 total protein were incubated with Ni-NTA Agarose beads (Qiagen) for 3 h at 4°C. After washing six or more times with B-150 or twice with 50 mM imidazole, bound complexes were analysed for 16S rRNA or 23S rRNA by primer extension or by Western blotting using either S3, S4 or V5 (against recombinant RimJ) antibodies.Cell extracts from S5(G28D) cells with pBAD, p-