Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell–bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods . Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-α is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-α receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-α. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx.Infections by  Neisseria meningitidis often leads to asymptomatic colonization (carriage) and rarely results in invasive disease associated with tissue injury. The reasons that make disease-associated isolates (pathogenic isolates) but not asymptomatic carriage isolates able to invade the host to establish disease are not understood. Isolates belonging to the ST-11 clonal complex are most frequently associated with the disease and rarely found in carriers. These hyper-invasive isolates may be able to induce cytopathic effects in target cells. We aimed to investigate the cytopathic effect of meningococcal isolates on epithelial cells using both ST-11 pathogenic isolates and carriage isolates. We showed that cytopathic effects were strongly associated with pathogenic isolates and infected cells exhibited features of apoptosis. This effect is mainly mediated by bacterial endotoxin (lipooligosaccharide) and involved an autocrine signaling mechanism of secreted TNF-α through its receptor TNF-RI. In contrast, carriage isolates down-regulate TNF-RI on the surface of infected cells by increasing TNF-RI shedding into the medium. We suggest that chelating secreted TNF-α protects cells from apoptosis. Our results unravel a differential modulation of TNF-α signaling by meningococcal isolates leading to cell survival or death and would therefore contribute to better understanding of the duality between carriage and invasiveness.Acquisition of  Neisseria meningitidis is the causative agent of meningococcal meningitis and fulminant sepsis. It is a common inhabitant of the human nasopharynx, being asymptomatically carried by approximately 10% of the population, worldwide. The incidence of meningococcal disease varies from 1 to 50 cases per 100,000. The reported fatality rate in meningococcal meningitis is about 10% and up to 20% of survivors suffer from sequelae lead to cytopathic effects that often result in marked tissue and cell damage mainly characterized by the breakdown of cell tight junctions, the deterioration of the cell layers, changes in normal cell morphology, and loss of cells Neisseriae have been shown to induce the expression of apoptosis-related genes and to trigger apoptosis in different cell types Apoptosis plays an important role in the pathogenesis of several infectious agents that induce or block this process Moreover, meningococci-cell interaction is complicated by the high variability of meningococcal isolates. Multilocus sequence typing (MLST) analysis classifies meningococcal isolates according to polymorphisms in seven housekeeping genes. Meningococcal isolates can be clustered into clonal complexes comprising closely related isolates varying by no more than two loci N. meningitidis in comparison to non invasive carriage isolates.The aim of this work was to explore the apoptosis pathway induced by hyper-invasive ST-11 isolates of N-acetyl-Asp-Glu-Val-Asp). In contrast, a basal level of caspase-3 activity similar to uninfected cells was observed in cells infected with the non-cytopathic isolates  that belong to different serogroups . Eight carriage isolates of different serogroups belonging to different clonal complexes were also included . We firsisolates . Furtherisolates . Other eisolates . These rIsolates used in this study (both pathogenic and carriage isolates) are piliated and capsulated as evaluated by specific sera against pili and capsule (data not shown). We therefore explored the impact of these structures on the induction of apoptosis. All tested pathogenic isolates were adherent to Hec-1-B cells . HoweverWe next showed that sonicated extracts of the pathogenic isolate LNP19995 was able to induce apoptosis to a similar level to that observed with live LNP19995 bacteria. In addition, heat-killed bacteria (60°C for 30 min) of the isolate LNP19995 were still able to induce apoptosis in Hec-1-B cells although to a significant lesser extent . These rin vitroMeningococcal LOS is a potent inducer of TNF-α To understand why the non-pathogenic isolate LNP21019 did not induce apoptosis despite the similar release of TNF-α as the pathogenic isolate LNP19995, the bioactivity of secreted TNF-α was evaluated using the cell line L929 as described in We therefore explored the mechanism leading to alteration of TNF-α bioactivity upon infection with the carriage isolate. TNF-α induces apoptosis through signaling with its death domain-containing receptor TNF-RI Pre-incubation of Hec-1-B cells with anti-TNF-RI monoclonal antibody (mAb), but not with anti-TNF-RII, significantly reduced apoptosis induced by the isolate LNP19995 and TNF-α compared to an irrelevant antibody (∼23% decrease). No changes were observed after infection with the carriage isolate LNP21019 . Under aTNFRI. RT-PCR analysis showed that TNFRI was up-regulated upon TNF-α stimulation compared to uninfected cells. Surprisingly, bacterial challenge brought about marked and comparable up-regulation in mRNA levels (TNFRII) nor the level of expression of β-actin encoding gene  showed any alteration under the tested conditions . Levels of sTNF-RII measured by ELISA did not significantly change following infection (data not shown).The reduction of TNF-RI cell surface expression by the carriage isolate did not seem to result from transcriptional down-regulation of its encoding gene A levels . Neithernditions . Moreovenditions . To explnditions . The difN. meningitidis isolates and were not found in uninfected cells. The level of complexes in the supernatant of Hec-1-B cells infected with the carriage isolate LNP21019 was about 4 times higher than that in the supernatant of Hec-1-B cells infected with the pathogenic isolate LNP19995  in meningococcal infected cells. Further analyses confirmed that cells displayed several hallmarks of apoptosis in response to meningococcal infection, namely, phosphatidylserine translocation and activation of caspase-3 and AMP-activated protein kinase.A growing number of studies have shown that apoptosis can be modulated (inhibited or promoted) by bacteria and protozoan parasites  have been reported In the current study, we have shown that both meningococcal LOS and PorB act independently but synergistically to induce apoptosis in several human cell lines. Conflicting results on the role of the major outer membrane porin, PorB, in the induction of apoptosis in pathogenic N. meningitidis induced the secretion of TNF-α and apoptosis in Hec-1-B epithelial cells that could be abrogated by the addition of anti–TNF-α neutralizing antibodies. A relationship between TNF-α secretion and apoptosis in the fallopian tube has also been suggested by studies using a mouse model of infection with a Chlamydia trachomatis mouse-specific pneumonitis strain. Infection with this pathogen leads to a large increase in apoptotic cells in murine oviducts, but treatment with anti–TNF-α antibodies leads to a significant decrease in the level of apoptosis in the upper genital tract The role of endotoxin of Gram negative bacteria  in cytotopathic effects has been documented N. meningitidis (the nasopharynx) could enhance the invasiveness of pathogenic isolates and hence promote the hyper-invasiveness of isolates belonging to the clonal complex ST-11. However, the origin of locally induced TNF-α remains to be determined. It might be produced by resident macrophages and/or airway epithelial cells. It has been shown that TNF-α levels were significantly higher in children with demonstrable H. influenzae growth in nasopharyngeal cultures than in culture-negative children Induction of TNF-α and subsequent apoptosis at the portal of entry of Chlamydia trachomatis and Streptococcus pneumoniae have been shown to reduce the display of TNF-RI at the surface of infected HEp-2 and the airway epithelial cells, respectively through activation of TACE An important mechanism for inducing apoptosis is the activation of the death receptor pathway by extracellular death-inducing ligand TNF-α, which binds to the cognate cell surface receptor TNF-RI N. meningitidis remains poorly understood. Full genome sequencing failed to reveal specific components that could determine transmissibility and colonization by carriage isolates or invasive infection by hyper-invasive isolates. The present study provides further evidence for the ability of pathogenic isolates of the ST-11 complex to exert cytopathic effects on epithelial cells by inducing apoptosis The molecular basis of the pathogenicity of (5) assay kit was purchased from Molecular Probes (France).RPMI 1640, HBSS, and trypsin-EDTA were purchased from Invitrogen . Anti-human TNF-α polyclonal antibody (Anti hTNF-α) was from MBL . Anti-TNF-RI monoclonal  and anti-TNF-RII polyclonal antibodies were from R & D system . FITC-, Rhodamine- and HRP-conjugated secondary antibodies were from Invitrogen. Caspase 3 Assay Kit, Polymixin B and Staurosporine (STRP) were purchased from Sigma Aldrich . FITC-Annexin V kit was from Immunotech . ApoPercentage Apoptosis Kit was purchased from Biocolor Ltd. . DiIC1Neisseria MLST database (http://pubmlst.org/neisseria). All N. meningitidis strains used in this study and their characteristics are listed in pilE::aph3'lpxA::aph3') was obtained by transforming the wild type strain LNP19995 with genomic DNA isolated from the previously described mutant strain Z0204 ctrA or porB genes were PCR-amplified from the pathogenic isolate LNP19995 genomic DNA using the primer sets CtrA-1F/CtrA-100R and PorB0-F/PorB-100R, respectively (aph3' (conferring resistance to kanamycin) and erm (conferring resistance to erythromycin) were inserted into the blunt-ended unique sites pmlI and KpnI which cut within ctrA and porB respectively, resulting into the recombinant vectors pGEM-ctrA::aph3' and pGEM-porB::erm, respectively. These recombinant vectors were separately introduced into LNP19995 and transformants were selected on kanamycin- or erythromycin-supplemented GCB agar plates, respectively. The strain NM0705 inactivated in both porB and lpxA genes was generated by transforming the genomic DNA of Z0305 strain (lpxA::aph3') in the strain NM0401 (porB::erm). Positive clones were selected on GCB agar plates supplemented with 100 µg/ml kanamycin and 15 µg/ml erythromycin. To construct the non-capsulated mutant strain NM0813, ctrA was PCR-amplified from the genomic DNA of LNP21019 using the primers 0801-Fw/0801-Rev  and ELISA were further used to confirm the absence of LOS and PorB expression from corresponding mutants.Meningococcal clinical isolates in France are sent to the National Reference Centre for Meningococci (NRCM) for full determination and typing. Bacteria were grown in GCB medium with Kellogg supplements ectively . PCR fra0801-Rev  and clonBamHI restriction site and reverse primers harbor HindIII restriction site as adapters to facilitate the cloning  and pCV4 (PporB-DsRed), respectively. Plasmids pCV2 and pCV4 were separately transformed into the isolates LNP19995 and LNP21019. Fluorescence of transformants was confirmed using immunofluorescence microscopy and flow cytometry. One isolate of each transformation was selected and designated LNP19995-GFP (/Red) and 21019-GFP (/Red). These transformants were used in indicated experiments.The EGFP and DsRed-N1 encoding genes (ClonTech) were amplified using the primers EGFP-Fw/EGFP-Rev and Red-Fw/Red-Rev, respectively. Both forward primers harbor  cloning . EGFP anB3/PorB4  was inse2 humidified atmosphere in RPMI 1640 and DMEM, respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS). Cells were seeded in 24- or 96-well culture plates, at a density of 5×105 or 5×104 cells/well. Infection was carried out at bacteria to cell ratio of 10: 1 unless otherwise specified. All infection experiments and treatments were carried out in absence of serum to avoid the interference of the serum with PorB The human endometrial carcinoma Hec-1-B, the respiratory A549 and the laryngeal carcinoma HEp-2 epithelial cell lines and the mouse fibroblasts L929 cell lines were from American Type Culture Collection . The human cell lines  and the L929 cells were maintained at 37°C under 5% CO620 nm of infected cells/A620 nm of uninfected cells)×100.Adhesion assay was performed as described previously −, PI−) from early apoptotic cells  and late apoptotic cells . Cells infected in 24-well tissue culture plates as described earlier, were harvested using cold PBS/0.02% EDTA and washed twice in PBS. Double staining with FITC-Annexin V and Propidium iodide (PI) was carried out using the FITC-Annexin V kit, according to the manufacturer's recommendations and then analyzed by FACS. When indicated, apoptosis was also quantified using the ApoPercentage Apoptosis Kit according to the manufacturer's instructions.Annexin V assay. Annexin V specifically binds to phosphatidylserine, a plasma membrane lipid that rapidly relocalizes from the inner leaflet to the outer leaflet in cells that are undergoing programmed cell death (apoptosis). Concomitantly, the extent of overall cytotopathogenicity was measured by the standard PI staining. Co-staining with Annexin V and PI allows differentiation of viable cells (Annexin V(5)) for flow cytometric analysis, as recommended by the manufacturer (Molecular Probes). This dye is reportedly only incorporated into mitochondria with intact membrane potential. After 20 min incubation with the dye at room temperature and darkness, cells were washed and analyzed by flow cytometry. Cells with high fluorescence intensity for DiIC1(5) were considered as living cells. Cells with low fluorescence intensity for DiIC1(5) were considered apoptotic cells.Assay for mitochondrial permeability transition (MPT): To determine variations of mitochondrial permeability transitions (MPT), as indicative of apoptosis, cells were stained with the potential-sensitive dye 1,1′,3,3,3′,3′-hexamethylindodicarbo-cyanine iodide . Standard p-Nitroaniline (pNA) solution was used for calculating caspase activity.Extraction of cellular DNA and gel electrophoresis. Harvested cells were lysed for 1 h at 50°C in lysis buffer  supplemented with proteinase K (5 mg/ml) and digested for 1 h with RNase A (0.5 mg/ml). After two phenol/chloroform (1∶1) extractions, the DNA was precipitated with ethanol, dried, resolved in TE , separated on a 1.5% agarose gel and visualized under ultra-violet light after ethidium bromide staining.3) and 1% FBS (staining buffer). Cells were labeled with anti TNF-RI mAb, anti-TNF-RII or irrelevant isotype-matched IgGs for 20 min. Cells were then washed twice with staining buffer and labeled with FITC-conjugated secondary antibody for 20 min. Cells were then fixed in 2% paraformaldehyde in staining buffer. Samples were analyzed using a FACSCalibur flow cytometer  equipped with FITC signal detector FL1-H . Relative fluorescence intensities were recorded from a total of 10,000 events. Results were analyzed using WinMDI 2.8 software.To measure cell surface expression of TNF-RI, cells were collected in HBSS containing 0.1% sodium azide (NaNThe immunofluorescence staining was performed as described previously g and kept frozen at −80°C until use. The amounts of TNF-α secreted in the supernatants were determined using the TNF-α enzyme-linked immunosorbent assay (ELISA) kit  in accordance with the manufacturer's instructions. TNF-α bioactivity was assessed by measuring cytopathic effect on L929 murine fibroblasts cells. In brief, L929 cell monolayers cultured for 24 h in 96-well plates (5×105 cells/well), were overlaid with twofold serial dilutions of culture supernatants from Hec-1-B infected cells in RPMI 1640 supplemented with polymixin B to a final concentration of 1 µM to avoid the effect of free circulating LOS. After incubation at 37°C for 24 h the cells were carefully washed with PBS, and processed for NBB staining as described above. Estimates of the concentrations of bioactive TNF-α in the supernatants were obtained by comparison with calibration curves established with an rhTNF-α standard. TNF-α bioactivity in Hec-1-B supernatant samples was inhibited by anti–TNF-α Ab, but not by control rabbit IgG, indicating that the cytopathic activity in Hec-1-B supernatants represents TNF-α. TNF-α bound to TNF-RI was detected in supernatants of N. meningitidis-infected Hec-1-B cells by a mixed Ab ELISA. The supernatants were added to 96-well microtiter plates coated with an anti TNF-RI mAb, and, after 2 h at room temperature, the plates were washed and incubated for 1 h at room temperature with a rabbit anti-human TNF-α Ab (0.2 µg/ml). After washing, HRPO-conjugated goat anti rabbit IgG  was determined. Peroxidase activity was determined by addition of citrate buffer and A450 was performed using a microplate reader. Calibrated dilutions of rhTNF-α captured by immobilized anti–TNF-α Ab were used as an internal standard for the comparative measurements of TNF-α complexed with TNF-RI.Supernatants of infected cells were collected, cleared of bacteria and cells by centrifugation at 14,000×al., 2002). To assess the expression analysis of TNFRI and TNFRII the primer pairs TNFR1-Fw/TNFR1-Rev, TNFR2-Fw/TNFR2-Rev were used . Fifty micrograms of extracted protein was run on SDS-PAGE and transferred to a nitrocellulose membrane. The blots were then probed overnight at 4°C with the relevant antibodies, washed and probed again with species-specific secondary antibodies coupled to HRPO and signal was detected by chemiluminescent reagents.Total RNA was prepared using Total RNA isolation kit  according to manufacturer's recommendations. cDNA synthesis and PCR conditions were previously described (Deghmane et ere used . β-actinere used  were uset test (two-way Annova) test, and considered significant if P<0.05.Data are expressed as the mean±SD of triplicate samples, and the reproducibility was confirmed at least in three separate experiments. Statistical analysis were performed using student Figure S1N. meningitidis. At 9 h after infection, epithelial cell DNA was extracted, separated in a 1.5% agarose gel, and stained with ethidium bromide. The 100-bp ladder is shown. Uninfected or STRP treated cells were used as controls.DNA fragmentation in Hec-1-B cells following infection with pathogenic ST-11 isolates or carriage isolates of (0.75 MB TIF)Click here for additional data file.Figure S2LNP19995 but not LNP21019 induced apoptosis in A549 and Hep-2 epithelial cell lines. Cells were treated with TNF-α alone or in combination with CHX, or infected with either isolate. After 9 h of incubation in presence of anti-TNF-α neutralizing antibody (closed bars) or irrelevant antibody , cells were stained with FITC-Annexin V and PI and apoptotic cells were analyzed by FACS. Data represent the mean±SD of three independent experiments. *** represent P<0.001 regarding untreated cells.(0.16 MB TIF)Click here for additional data file.Figure S3Level of TNF-RI increased in infected cells. (A) FACS analysis revealed up-regulation of intracellular level of TNF-RI at 9 h post-infection. The solid open histograms: unstained cells, the dotted histograms: isotype control stained cells, the gray-filled histograms anti-TNF-RI stained cells. (B) Consistently, immunoblot analysis showed a strong increase in the amount of TNF-RI protein, but not TNF-RII in infected or TNF-α-treated cells compared with uninfected cells.(0.36 MB TIF)Click here for additional data file.Table S1Summary of cytopathic and apoptotic features of meningococcal isolates used in this study.(0.05 MB PDF)Click here for additional data file.


P450 reductase (NADPH: cytochrome c (P450) reductase, EC 1.6.2.4) plays an important role in the reductive activation of the bioreductive drug tirapazamine (SR4233). Thus, in a panel of human breast cancer cell lines, expression of P450 reductase correlated with both the hypoxic toxicity and the metabolism of tirapazamine [Patterson et al (1995) Br J Cancer 72: 1144-1150]. To examine this dependence in more detail, the MDA231 cell line, which has the lowest activity of P450 reductase in our breast cell line panel, was transfected with the human P450 reductase cDNA. Isolated clones expressed a 78-kDa protein, which was detected with anti-P450 reductase antibody, and were shown to have up to a 53-fold increase in activity of the enzyme. Using six stable transfected clones covering the 53-fold range of activity of P450 reductase, it was shown that the enzyme activity correlated directly with both hypoxic and aerobic toxicity of tirapazamine, and metabolism of the drug under hypoxic conditions. No metabolism was detected under aerobic conditions. For RSU1069, toxicity was also correlated with P450 reductase activity, but only under hypoxic conditions. Measurable activity of P450 reductase was found in a selection of 14 primary human breast tumours. Activity covered an 18-fold range, which was generally higher than that seen in cell lines but within the range of activity measured in the transfected clones. These results suggest that if breast tumours have significant areas of low oxygen tension, then they are likely to be highly sensitive to the cytotoxic action of tirapazamine and RSU 1069.


The impact of recombinant human tumour necrosis factor-alpha  U mg protein-1) on blood flow, oxygen consumption and growth of a moderately TNF-sensitive rat tumour (DS-carcinosarcoma) was studied. Tumour growth was stimulated at low TNF doses (1 and 10 micrograms kg-1) and significantly retarded at higher TNF dose levels (0.1 and 1 mg kg-1). Growth changes were concomitant with variations in oxygen consumption, lactate release and acidification of the metabolic micromilieu. Both single and repeated application of low TNF doses (1-10 micrograms kg-1 i.v.) increased tumour perfusion whereas single administration of high TNF dose levels (0.1-1 mg kg-1 i.v.) reduced tumour blood flow. After repeated application of high TNF doses tumours shrank to such small sizes that perfusion measurements could not be performed within the observation period of two weeks. It is concluded that TNF effects on solid tumours are at least partially mediated by changes in tumour perfusion. Thus, an altered tumour sensitivity towards other treatment modalities, e.g. irradiation, chemotherapy or hyperthermia, can be expected after TNF therapy. A beneficial TNF effect would critically depend on the dose level employed and on the sequence and timing of various combination regimes.


Brucella spp., which are adapted to intracellular life within cells of a large variety of mammals. Brucella can be considered a furtive pathogen that infects professional and non-professional phagocytes. In these cells Brucella survives in a replicative niche, which is characterized for having a very low oxygen tension and being deprived from nutrients such as amino acids and vitamins. Among these vitamins, we have focused on riboflavin (vitamin B2). Flavin metabolism has been barely implicated in bacterial virulence. We have recently described that Brucella and other Rhizobiales bear an atypical riboflavin metabolic pathway. In the present work we analyze the role of the flavin metabolism on Brucella virulence. Mutants on the two lumazine synthases (LS) isoenzymes RibH1 and RibH2 and a double RibH mutant were generated. These mutants and different complemented strains were tested for viability and virulence in cells and in mice. In this fashion we have established that at least one LS must be present for B. abortus survival and that RibH2 and not RibH1 is essential for intracellular survival due to its LS activity in vivo. In summary, we show that riboflavin biosynthesis is essential for Brucella survival inside cells or in mice. These results highlight the potential use of flavin biosynthetic pathway enzymes as targets for the chemotherapy of brucellosis.Brucellosis is a worldwide zoonosis that affects livestock and humans and is caused by closely related  Brucella spp. which are adapted to intracellular life within cells of a large variety of mammals. The most pathogenic species for humans are B. melitensis, B. suis and B. abortus whose preferred host are goats, pigs and cattle respectively. Transmission to humans occurs mainly through the consumption of contaminated unpasteurized dairy products and through direct contact with infected animals. An estimated of 500,000 human infections per year still occur worldwide Brucellosis is a worldwide zoonosis that affects livestock and humans and is caused by closely related Brucella can be considered a furtive pathogen that infects professional and non-professional phagocytes. No classical virulence factors, such as exotoxins, cytolysins, capsules, fimbria, plasmids, lysogenic phages, resistant forms, antigenic variation, endotoxic lipopolysaccharide or apoptotic inducers have been described in Brucella spp. so far Brucella resides within a membrane-bound compartment, the Brucella-containing vacuole (BCV), which sequentially interacts and fuses with early endosomes, lysosomes and further traffics to the endoplasmic reticulum (ER) to generate an ER-derived replicative niche Brucella extensively replicates without restricting basic cellular functions or inducing obvious damage to cells Brucella are a complex array of molecular determinants that confer the pathogen the metabolic ability to thrive in the harsh intracellular conditions allowing it to invade, resist intracellular killing, build the replicative niche and replicate.Once internalized, BrucellaBrucella needs to survive to oxidative stress, microaerobic conditions and nutrient starvation and flavoenzymes or flavins themselves may be playing a key role in those processes.Although riboflavin (vitamin B2) is the precursor of the essential flavin coenzymes FMN and FAD which participate in a myriad of biochemical reactions, flavin metabolism has been barely implicated in bacterial virulence. Flavoenzymes are involved in dozens of crucial cellular processes such as energy metabolism, RedOx reactions, detoxification and biosynthesis The metabolic pathway of riboflavin in bacteria, plants and fungi has been described in some detail Brucella and other Rhizobiales bear an atypical riboflavin metabolic pathway Brucella codes both a Type-I and a Type-II LS called RibH1 and RibH2 respectively. Both enzymes show low catalytic activity in vitro, suggesting that natural selection operating over the riboflavin pathway favored the evolution of catalysts with low reaction rates. The rationale behind this is that the excess of flavins in the intracellular pool in Brucella could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress We have recently described that B. abortus intracellular survival, focusing at its two LS isoenzymes. Mutants on the ribH1, ribH2 genes and a double ribH mutant were generated. These mutants and different complemented strains were tested for viability on cells and mice. In this fashion we have established that at least one LS must be present for B. abortus viability and that RibH2 and not RibH1 is neccesary for Brucella intracellular replication due to its LS activity in vivo. In summary, we show that flavin biosynthesis is essential for Brucella survival inside cells and mice.In the present work we analyze the role of flavin metabolism on ribH genes were generated. The ribH1 (BAB1_0791) mutant was made by clean non-polar deletion because this gene is believed to be located in an operon (rib operon). On the other hand, ribH2 (BAB2_0545) is an isolated ORF, thus the ribH2 mutant was generated by disrupting the ribH2 gene with an antibiotic resistance cassette  was obtained but at the expense of keeping a rescue copy of ribH1 in an expression plasmid  (ribH1Km plasmid could be cured in the presence of another plasmid from the same incompatibility group harboring different inserts but with an ampicillin resistance plasmid (pBBR4)  in vivo  are added to the media. Under these conditions, both the empty plasmid and the ribH2_W22A construct were able to replace RibH1 in the ribH1-ribH2 mutant strain (ribH genes of B. abortus code for LS isoenzymes.Given the above described mutagenesis studies and the fact that both RibH1 and RibH2 exhibit poor oflavin) . Thus we in vivo . As expebH2_W22A . RibH2_Wt strain . The samt strain . Overallin vivo, we decided to evaluate whether they play a role in virulence. For this purpose, we analyzed the intracellular replication of single and double RibH mutants in a macrophagic cell line and the persistence of these mutants in mice.Once established that both RibH1 and RibH2 can function as active LS ribH1 strain showed a similar behavior to that of the wild-type strain. In contrast, ribH2 showed a marked decrease in CFU counts at all time-points, suggesting that RibH2 is an important factor for intracellular life style. Complementation of ribH2 by ectopic expression of RibH2 in a plasmid recovers this attenuated phenotype: this strain exhibited an increased survival at early times (1 and 4 h p.i.) and a similar curve to that of the wild-type strain at later times. These results are indicative that RibH2 is more important in the intracellular phase than RibH1. In spite of the fact that the double ribH mutant is unable to synthesize flavins, its intracellular flavin pool content is enriched when it is grown in the presence of 500 µM riboflavin . In clear agreement, the double ribH mutant exhibited an increased survival at early time points, but is unable to replicate at later times p.i.  . This remes p.i. . This isribH2 BCVs in HeLa cells by scoring the recruitment kinetics of the late endosome/lysosome glycoprotein LAMP-1 and the lysosomal luminal hydrolase cathepsin D. As previously described, LAMP-1 is rapidly acquired and then gradually excluded from wild-type BCVs ribH2 BCVs acquired this glycoprotein with similar kinetics but 40,3%±5,0 of BCVs retained LAMP-1 at 24 h, suggesting that this mutant fails to control vacuole maturation in HeLa cells  of 1.50 Å when 128 Cα are aligned from a total of 157–158 Cα. We have previously speculated that one plausible explanation for the important role of RibH2 in Brucella virulence is that this new type of decameric LS could harbor a novel yet unknown function ribH2 attenuated strain with different constructs (ribH2 is complemented with an active LS (endogenous RibH2 or the heterologous LS from yeast), the bacteria not only recover their virulence but also acquire an increased capacity to survive inside macrophages. In clear contrast, the complementation with the point-mutated RibH2_W22A does not recover the attenuated phenotype. Thus, it is unlikely that RibH2 is exerting a function different than LS.Although nstructs . When riBrucella is an intriguing observation, our working hypothesis is that the two ribH genes may be differentially expressed during the Brucella infection cycle. Brucella would use RibH1 for flavin biosynthesis during the extracellular phase and RibH2 during intracellular growth. In any case, the role of RibH2 points out to the importance of the biosynthetic pathway of flavins in virulence. We speculate that the LS activity could be a key point for the regulation of this pathway. In order to corroborate this hypothesis we are currently studying the regulation of ribH genes under different conditions.Although the expression of two very similar LSs with the same function in ribH1-ribH2 mutant is only viable at very high concentrations of exogenous flavins. This result supports previous bioinformatic analyses that indicate that Brucella lacks riboflavin transport activities. We infected cells with the ribH1-ribH2 strain previously grown at high levels of riboflavin  appears to function as a photoreceptor that is directly related to Brucella survival and replication within macrophages Brucella pathogenesis is a question of biological relevance and appears not to be limited to the genus Brucella. We have performed bioinformatic and biochemical analyses in other bacteria Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium spp. and some pathogens like Ochrobactrum anthropi, Pseudomonas syringae pv. tomato and Roseobacter denitrificans also present two ribH genes in their genomes exhibiting one Type-I and one Type-II LS. They are located in the respective genomes in the same fashion as in Brucella, meaning that ribH1 is coded within the rib operon while ribH2 is located as an isolated ORF with the regulatory RFN element upstream. A Type-I LS-ribH gene duplication might have occurred in a bacterial ancestor where one of these genes evolved to a Type-II LS, providing positive fitness for symbiosis or pathogenesis. Vertical and horizontal gene transfer could be responsible for the distribution of the ribH genes through the different taxa where two different types of LS are found.Two recent discoveries involving flavins in virulence and symbiosis in Rhizobiales have raised particular interest: Taga Rhodococcus equi and Actinobacillus pleuropneumoniae, become avirulent when their riboflavin operons are disrupted ribH1-ribH2 strain generated in this work could also be a very good candidate for developing a Brucella vaccine because it can not synthesize riboflavin and it is completely attenuated in cells and in mice. This strain also lacks the serological marker RibH2 which could eventually be used to discriminate between vaccinated and infected animals Brucella survival inside cells and that the LSs constitute attractive targets for chemotherapy and vaccine design.Two mammal pathogens, Brucella strains have been manipulated in a BSL-3 facility at UNSAM University. Bacterial strains, plasmids and oligonucleotides used in this study are listed in B. abortus strains were grown in Tryptic Soy Broth , Tryptic Soy Agar  or Gerhardt-Wilson minimal medium at 37°C on a rotary shaker (250 rpm) or stove for plates Escherichia coli strains were grown at 37°C on a rotary shaker (250 rpm) in Luria-Bertani broth. For E. coli strains antibiotics were used at the following concentrations: Km, 50 µg/ml; Gm, 20 µg/ml; Amp, 100 µg/ml and streptomycin (Str), 25 µg/ml.All live ribH1) unmarked chromosomal mutant two PCR fragments were generated from regions flanking this ORF. Oligonucleotides ribH1_F1 and ribH1_R1 were used to amplify a 0.3 Kbp fragment including codons 1–7 and oligonucleotides ribH1_F2 and ribH1_R2 were used to amplify a 0.3 Kbp fragment including 150–157 codons of ribH1. Both fragments (containing complementary regions) were ligated by overlapping PCR using oligonucleotides ribH1_F1 and ribH1_R2. The resulting fragment containing the ribH1 deletion allele was cloned into pBluescript-KSII (Stratagene) in which the Bacillus subtilis sacB gene encoding levansucrase  sucrose in the growth medium) had been cloned in the PstI site to generate pBlueKSIIsacBΔribH1. This plasmid was introduced in B. abortus 2308 by electroporation. The first recombination event (integration of the suicide vector in the chromosome) was selected by Cb resistance and 10% (w/v) sucrose sensitivity in TSA plates. Cells were grown overnight in TSB in the absence of antibiotic selection to promote recombination. The second recombination event  was selected by Cb sensitivity and 10% (w/v) sucrose resistance in TSA plates. Colonies were screened by PCR using oligonucleotides ribH1_F1 and ribH1_R2 that amplify a fragment of 0.6 Kbp in the mutant strain and a fragment of 1.2 Kbp in the wild-type strain. The mutant strain carrying an unmarked deletion of ribH1 was called ribH1.For the construction of the ORF BAB1_0791 (ribH2) was amplified using oligonucleotides ribH2_F1 and ribH2_R1 and ligated to pGem-T-Easy vector (Promega) to generate pGem-TribH2. The plasmid was linearized with EcoRV. Linearized pGem-TribH2 was ligated to a 0.7 Kbp SmaI fragment containing a acc(3)-I cassette to generate pGem-TribH2::acc(3)-I. This plasmid was electroporated into B. abortus 2308 where it is incapable of autonomous replication. Homologous recombination events were selected using Gm resistance (3 µg/ml) and Cb sensitivity (50 µg/ml) in TSA plates. PCR and sequencing analyses showed that the ribH2 wild-type gene was replaced by the disrupted one. The mutant strain obtained was called ribH2.A PCR product of 0.9 Kbp containing ORF BAB2_0545 -I plasmid was electroporated into this strain and homologous recombination events were selected as previously described. PCR and sequencing analyses showed that the ribH2 wild-type gene was replaced by the disrupted one. The mutant strain obtained was called ribH1-ribH2+pribH1Km.A 1 Kbp PCR product containing E. coli S17-1 B. abortus strains were cultured separately, cells were harvested during the exponential phase and 1 ml of each culture was pelleted and washed twice with TSB. Bacteria were then centrifuged, resuspended in residual liquid, mixed and plated together in a single spot in a TSA plate without antibiotics. Plates were incubated at 37°C overnight, afterwards the mix was plated in TSA-Nal plates to select Brucella and exconjugants were selected for the appropriate plasmidic antibiotic resistance.ribH1 gene was first cloned into pGem-T-Easy and then excised and ligated to the EcoRI site of pBBR4 ribH1 where ribH1 is under the lac promoter. This plasmid was conjugated into the ribH2 strain by biparental mating to generate ribH1 + pribH1Amp.A 1 Kbp PCR product containing the ribH2 gene was excised from pGem-TribH2 and ligated to the EcoRI site of pBBR4. The resulting plasmid, pribH2, was conjugated into the ribH2 strain by biparental mating to generate ribH2 + pribH2.A 0.9 Kbp EcoRI fragment containing S. cerevisiae genomic DNA. The resulting 0.6 Kbp PCR fragment was purified and digested with XhoI and XbaI and ligated into pBBR4 digested with the same restriction enzymes. The resulting plasmid, pLS_sce, was confirmed by sequencing and conjugated into ribH2 strain by biparental mating to generate ribH2 + pLS_sce.ORF YOL143C was amplified using the oligonucleotides LS_sce_F and LS_sce_R from ribH2_W22A mutation was generated using the oligonucleotides W22A_F and ribH2_R2 to amplify the first part of the ribH2 ORF, then the PCR product was purified and used as a “megaprimer” for a second amplification round using the oligonucleotide ribH2_F2. The resultant PCR product was purified, digested with BamHI and ligated to pBBR4 digested with the same restriction enzyme. The resulting plasmid, pribH2_W22A, was confirmed by sequencing and conjugated into ribH2 strain by biparental mating to generate ribH2 + pLS_sce.The in vivo, we developed a plasmid swap experiment based the instability of two vectors from the same incompatibility group harbored in the same bacterial population. To this end, a pBBR4 plasmid harboring different inserts  was introduced by biparental mating conjugation into the ribH1-ribH2 + pribH1Km strain. We selected the presence of both vectors with Amp and Km. Three independent Ampr/Kmr clones from each conjugation were cultured for at least 15 generations in TSB added with Amp only, then dilutions were plated in TSA-Amp plates. One hundred colonies from these plates were replica plated in TSA with Amp or Km. When necessary, riboflavin (500 µM) was added to the TSB or TSA. The percentage of Ampr/Kms clones was calculated for each case (n = 100).To demonstrate that RibH1 and RibH2 are LSs 5 cells per well) and inoculated with RPMI 1640 supplemented with 5% fetal calf serum (FCS) containing 5×105 bacteria (MOI = 5∶1). In order to ensure close contact between cells and bacteria, multiwell plates were centrifuged at 400 g for 10 min After 30 min of incubation at 37°C in a 5% CO2 atmosphere, cells were gently washed with phosphate buffered saline  and then incubated for 1 h in medium supplemented with Str (100 µg/ml) and Gm (50 µg/ml) to kill remaining extracellular bacteria. Thereafter, the antibiotic concentration was decreased to 20 µg/ml of Str and 10 µg/ml of Gm. At the indicated times p.i., the number of intracellular viable bacteria was determined as follows: cells were washed three times with PBS and treated for 5 min with 0.5 ml of 0.1% Triton X-100. Lysates were serially diluted and plated on TSA plates with the appropriate antibiotic to determine colony forming units per ml (CFU/ml). Each determination is the average of two independent experiments. Values are expressed as mean ± standard error of the mean.Monolayers of J774.A1 cells were seeded in 24-well plates (105 cells per coverslip) and inoculated with minimal essential medium  supplemented with 5% FCS and 2 mM glutamine containing 5×107 CFU of bacteria (MOI = 500∶1). Multiwell plates were centrifuged at 400 g for 10 min After 1 h of incubation at 37°C in a 5% CO2 atmosphere, cells were gently washed with PBS (pH 7.4) and then incubated for 1 h in medium supplemented with 100 µg/ml of Str and 50 µg/ml of Gm to kill remaining extracellular bacteria. Thereafter, the antibiotic concentration was decreased to 20 µg/ml of Str and 10 µg/ml of Gm. At the indicated times p.i. cells were washed and fixed for immunofluorescence.HeLa cells were seeded in coverslips . Cells were observed on the microscope using a 100× oil immersion objective. Projections were saved in TIFF format and imported to ADOBE PHOTOSHOP CS where images were merged using RGB format. To determine the percentages of bacteria that co-localized with the studied intracellular markers a minimum of 100 intracellular bacteria  were counted. The assays were performed in duplicate.HeLa cells were washed five times with PBS to remove non-adherent bacteria and fixed for 15 min in 3% paraformaldehyde (pH 7.4) at 37°C and then processed for immunofluorescence labeling. Briefly, coverslips were washed three times with PBS, incubated for 10 min with PBS added with 250 mM NHFor immunofluorescence, the primary antibodies used were mouse anti-human LAMP-1 H4A3  or rabbit anti-human cathepsin D (Dako). The secondary antibodies used were goat anti-mouse or goat anti-rabbit Alexa Fluor 568 . For DNA staining, Hoechst dye at 2 µg/ml  was used. For Western Blots, the primary antibodies used were monoclonal anti-RibH2 and polyclonal anti-RibH1 mice serum (both generated in our laboratory). The secondary antibody used was goat anti-mouse IgG (Fc Specific) Peroxidase Conjugate (Sigma).Bacteria were grown overnight with the proper antibiotics and extra added riboflavin when necessary. The same amount of protein was loaded into each lane in the gels. Chemiluminiscent ECL Plus Western Blotting Detection System (Amersham Biosciences), Storm Image and Detection system (Molecular Dynamics) and Immobilon-NC Transfer Membranes (Millipore) were used for the process.5 CFU of the indicated strains. At 14, 30, 40 or 60 days p.i. mice were sacrificed and spleens were removed and homogenized in 2 ml of PBS. Tissue homogenates were serially diluted and plated on TSA plates with the appropriate antibiotics to determine colony forming units per spleen (CFU/spleen). Values are expressed as mean ± standard errors of the mean (N = 4).60-day-old female BALB/c mice were inoculated intraperitoneally with 5×10All research involving animals has been conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all experimental protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) of Leloir Institute.T-test . p-values<0.05 were considered statistically relevant.Data are presented as mean ± standard error of the mean. Significance was reported using Student's unpaired two-tailed Figure S1Plants, fungi, and microorganisms share a common flavin biosynthetic pathway. Animals take riboflavin as a vitamin in their diets to synthesize FMN and FAD cofactors (by RK and FS respectively).(2.12 MB EPS)Click here for additional data file.Figure S2r/Kms clones carrying empty pBBR4 or pribH2_W22A derived from the plasmid swap experiment were plated in TSB-Amp plates without the addition of riboflavin. Control replica plates in TSB-Km plates with riboflavin added are also shown.Amp(1.32 MB TIF)Click here for additional data file.Figure S3B. abortus (black) and ribH2 mutant (red) were lysed and intracellular CFUs per ml quantified at different times after inoculation. Data shown (mean ± standard error of the mean) are representative of three independent experiments performed.HeLa cells infected with wild-type (2.02 MB TIF)Click here for additional data file.


Tumour necrosis factor (TNF) is directly cytolytic to certain tumour cell lines in vitro, although TNF-resistant variants can be selected from these susceptible lines by exposure to TNF. While studying TNF-susceptible L929 cells and their resistant variant, L929/R, we noted that within L929 colonies the cells were widely spaced whereas they were closely packed in L929/R colonies. L929/R cells also adhered more strongly to plastic and differed from L929 in cell shape. Similar observations were made with TNF susceptible and resistant variants of two other cell lines (RK13 and a plastic adherent U937 subline). The tendency of resistant cells to grow closely together suggests the possibility of inter-cell communication for the TNF resistant state. However, like L929 and U937, L929/R and U937/R did not communicate by gap junctions and we could find no evidence of extracellular mediators of TNF resistance. Rather the differences in colonial morphology, cell shape and plastic adherence may be secondary to an underlying mechanism which defines TNF susceptibility/resistance.


Tumour necrosis factor (TNF)-alpha induces apoptosis in a human acute myeloid leukaemia cell line, Kasumi-1. To examine the role of protein phosphorylation in signal transduction of TNF-alpha-induced apoptosis, a variant cell line resistant to TNF-alpha was established by an intermittent challenge of Kasumi-1 cells with increasing concentrations of TNF-alpha for 6 months. The mechanism of resistance to TNF-alpha appears to be in the post-receptor pathway because expression of p55 TNF receptor in the variant cells is increased compared with that of the parental Kasumi-1 cells. In renaturation assays, TNF-alpha induced a rapid activation of different protein kinases of different molecular weights, including the 50 kDa protein kinase (PK50) followed by the 35 kDa protein kinase (PK35), in the parental Kasumi-1 cells. The dose-response of TNF-alpha required to activate PK50 and PK35 was closely related to concentrations of TNF-alpha that induced apoptosis. Treatment of Kasumi-1 cells with ceramide also activated PK35. In TNF-resistant variant cells, activation of PK35 in response to TNF-alpha or ceramide was practically nil. These findings suggest that activation of PK35 through the ceramide pathway may play an important role in signal transduction of TNF-alpha in the Kasumi-1 cell line, while the decreased activation of PK35 may explain the insensitivity of the variant cells towards TNF-alpha.


Epithelioid sarcoma is a highly malignant soft tissue tumour that is refractory to conventional chemotherapy and irradiation. Since permanent cell lines of this tumour are extremely rare, in vitro data on compounds with significant antiproliferative effects are still lacking. Therefore, we investigated the effects of retinoic acid (RA) and tumour necrosis factor alpha  on tumour cell proliferation of three different clonal subpopulations  derived from the same human epithelioid sarcoma cell line, GRU-1. In GRU-1A both RA (P=0.01) and TNF-alpha (P=0.002) exhibited highly significant and dose-dependent growth inhibitory effects, which could further be increased by a combined application of both compounds (P<0.006). GRU-1B proved to be sensitive to RA (P=0.006), whereas no response to TNF-alpha was observed. GRU-1C was resistant to both RA and TNF-alpha. The antiproliferative effect of TNF-alpha was mediated by TNF receptor 1(TNF-R1) and correlated positively with both the number of TNF-R1 per cell and receptor affinity. No correlation was detected between RA-induced growth inhibition and the expression pattern of the RA receptors (RARs) RAR-alpha, RAR-beta, and RAR-gamma. Plating efficiency, however, could exclusively be reduced by RA in GRU-1B, the only cell line expressing RAR-alpha. Taken together, these data are the first showing significant antiproliferative effects in human epithelioid sarcoma by RA and TNF-alpha. Whereas the TNF-alpha response seems to depend on the expression of TNF-R1, no simple correlation could be found between RA sensitivity and the expression pattern of RARs.


Increasing lines of evidence show that activation of Kupffer cells plays a pivotal role in initiation and progression of alcoholic liver disease. Kupffer cell activation is evoked by endotoxin , which leads to rapid elevation of intracellular calcium accompanied by release of a variety of mediators including cytokines, eicosanoids and reactive radical species. Among them, TNF-alpha is a critical factor in the pathogenesis of alcoholic liver disease.Thalidomide  was initially used as a sedative and antiemetic during pregnancy but was withdrawn from the market due to its teratogenic effects. Before it was banned, thalidomide was recognized to reduce dramatically symptoms associated with erythema nodosum leprosum, a complication of Hansen's disease. Subsequently, it was shown that thalidomide suppresses TNF-alpha production by macrophages and other cell types such as activated T cells ,2, whichAccordingly, the purpose of this study was to determine whether thalidomide could prevent alcohol-induced liver injury. Here we report that thalidomide prevents liver damage caused by chronic ethanol exposure through not only suppression of TNF-alpha production but abrogation of Kupffer cell sensitization to LPS.In this study, a model of alcoholic liver injury based on the sensitization of Kupffer cells, in which rats are given ethanol (5 g/kg body wt) once every 24 hours . Gut per2+]i was measured using a microspectrofluorometer with fura-2, and TNF-alpha in the culture media was measured using an enzyme-linked immunosorbent assay (ELISA) kit .Kupffer cells were isolated by collagenase digestion and [Cat test as appropriate. p < 0.05 was selected prior to the study to reflect significance.All results were expressed as mean – S.E.M. Statistical differences between means were determined using analysis of variance (ANOVA) or ANOVA on ranks or Bonferroni's post-hoc test or Student's Ethanol administration once every 24 h for 8 weeks caused pronounced pathological changes in the liver lobule, including steatosis, necrosis and inflammation in the liver, confirming our earlier results. In marked contrast, these pathological changes were prevented almost completely by coadministration of thalidomide (5 mg/kg). Likewise, ALT was increased to 91 – 7 IU/L in the 8-week ethanol group, which was three times higher than the values in the control, non-treated rats  was administered i.v. and liver histology were evaluated 24 hours later (data not shown). LPS caused focal necrosis with neutrophil infiltration in the control liver. In the group treated with thalidomide for 4 weeks, liver histology revealed only slight infiltration of neutrophils, but lacking evidence of necrosis. This result agrees with serum transaminase levels, which were slightly lower in the thalidomide-only group. In the 4-week ethanol group, LPS injection resulted in marked aggravation of these pathological parameters. Moreover, steatosis was prominent in this group. Treatment with thalidomide dramatically improved liver injury, which exhibited near normal histology. The increased ALT levels observed 24 hours after LPS injection in the 4-week ethanol group was blocked completely by thalidomide. Similar results were obtained with AST values . Liver from control rats expressed the 55 kD CD14. Thalidomide did not change the amount of CD14 protein; however, the band was far more intense in liver from rats treated with ethanol chronically. Furthermore, the ethanol-induced increase in CD14 protein levels was abrogated completely with co-administration of thalidomide for 4 weeks.As reported previously, Kupffer cell sensitization to LPS is caused by LPS. Therefore, we determined the effect of thalidomide on gut-permeability Table . ThalidoAfter addition of LPS (100 ng/ml) to Kupffer cells isolated from the untreated, control rats, the amount of TNF-alpha mRNA increased and reached a maximal value after 1 hour, followed by a gradual decline over 2 hours (data not shown). Inclusion of thalidomide (5 micromolar) in the culture media did not alter the peak level of TNF-alpha mRNA occurring at 1 hour after LPS. However, 3 hours after LPS addition, the amount of TNF-alpha mRNA in Kupffer cells treated with thalidomide was 30% lower than that of control Kupffer cells, indicating that thalidomide accelerated decrease in TNF-alpha mRNA Table . Thalidoin vitro (data not shown). Treatment with TNF-alpha (10 ng/ml) for 24 hours resulted in enhancement of CD14 expression in Kupffer cells. Inclusion of thalidomide (5 micromolar) in the culture media reduced CD14 expression to a level comparable to the control.To explore the mechanism by which thalidomide abrogated Kupffer cell sensitization to LPS caused by chronic ethanol, we determined the effect of TNF-alpha on CD14 expression in Kupffer cells obtained from normal rats LPS-induced TNF-alpha production by isolated Kupffer cells was compared between groups cultured for 24 hours in the presence or absence of TNF-alpha (10 ng/ml) prior to LPS challenge. Kupffer cells pretreated with TNF-alpha produced about 30% more TNF-alpha in response to LPS (100 ng/ml) than the control cells cultured in the absence of TNF-alpha Table . ThalidoThe results of this study showed that thalidomide prevented liver injury caused by chronic ethanol treatment Table . To inveTNF-alpha has a pleiotropic cytokine and regulatory mechanisms of TNF-alpha production in macrophages and its intracellular signaling have been studied extensively . It was in vitro.This scenario is supported by the facts that pretreatment with TNF-alpha led to an increased TNF-alpha synthesis in response to LPS and enhancement of CD14 expression in Kupffer cells, and that inclusion of thalidomide in the culture media inhibited this TNF-alpha-induced autocrine activation of TNF-alpha synthesis Table . FurtherThe precise mechanisms of the thalidomide actions remain to be pursued, however, the results of this study suggest that thalidomide prevents liver injury caused by chronic ethanol consumption through destabilization of TNF-alpha mRNA and abolishing Kupffer cell sensitization to LPS. Though currently its side effects preclude the use of thalidomide in clinical settings and require rigors of the drug distribution program and prescription decision, efforts to synthesize thalidomide analogues lacking teratogenic effects but having more potent efficacy are underway . Given t


Xenopus laevis tadpole visual system, a model frequently used to examine the anatomical and functional development of neural circuits.Imbalances in the regulation of pro-inflammatory cytokines have been increasingly correlated with a number of severe and prevalent neurodevelopmental disorders, including autism spectrum disorder, schizophrenia and Down syndrome. Although several studies have shown that cytokines have potent effects on neural function, their role in neural development is still poorly understood. In this study, we investigated the link between abnormal cytokine levels and neural development using the Using a test for a visually guided behavior that requires normal visual system development, we examined the long-term effects of prolonged developmental exposure to three pro-inflammatory cytokines with known neural functions: interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. We found that all cytokines affected the development of normal visually guided behavior. Neuroanatomical imaging of the visual projection showed that none of the cytokines caused any gross abnormalities in the anatomical organization of this projection, suggesting that they may be acting at the level of neuronal microcircuits. We further tested the effects of TNF-α on the electrophysiological properties of the retinotectal circuit and found that long-term developmental exposure to TNF-α resulted in enhanced spontaneous excitatory synaptic transmission in tectal neurons, increased AMPA/NMDA ratios of retinotectal synapses, and a decrease in the number of immature synapses containing only NMDA receptors, consistent with premature maturation and stabilization of these synapses. Local interconnectivity within the tectum also appeared to remain widespread, as shown by increased recurrent polysynaptic activity, and was similar to what is seen in more immature, less refined tectal circuits. TNF-α treatment also enhanced the overall growth of tectal cell dendrites. Finally, we found that TNF-α-reared tadpoles had increased susceptibility to pentylenetetrazol-induced seizures.Taken together our data are consistent with a model in which TNF-α causes premature stabilization of developing synapses within the tectum, therefore preventing normal refinement and synapse elimination that occurs during development, leading to increased local connectivity and epilepsy. This experimental model also provides an integrative approach to understanding the effects of cytokines on the development of neural circuits and may provide novel insights into the etiology underlying some neurodevelopmental disorders. Pro-inflammatory cytokines are immune signaling peptides, proteins, and glycoproteins that are secreted largely in response to immunological stimuli, such as infection or inflammation. Many of these cytokines are also found within the central nervous system, and are thought to play important signaling roles, independent of their immunological functions ,2. They Xenopus laevis tadpoles. The visual system of Xenopus tadpoles is frequently used as a model for the development of neuronal microcircuitry [Xenopus tadpole visual system is therefore one of the few model systems in which we can link molecular, cellular and circuit level events in a behaving animal as it goes through development.One common feature in many of these disorders is that they are thought to result from abnormal development of brain connectivity, rather than an overt pathophysiology or neurodegeneration -18. One ircuitry ,20. It iircuitry -25. In tircuitry ,27. Locaircuitry . Proper ircuitry , and thiircuitry . The Xen+ and Ca2+ channels [2+ release from internal stores [+ and voltage-gated Ca2+ channels in some neurons [In this study, we focused on a set of three pro-inflammatory cytokines - IL-1β, IL-6 and TNF-α - that are known to be elevated in neurodevelopmental disorders and have known effects on neuronal function. IL-1β is known to reduce current through voltage-gated Nachannels ,30 as wechannels , and curchannels . It alsochannels ,34. IL-6l stores . TNF-α cl stores ,37. TNF-l stores ,39. It a neurons ,41.Our general experimental approach consisted of rearing tadpoles in media containing elevated concentrations of the different cytokines between developmental stages 44 and 49. During this time period, several well-characterized developmental changes at the level of synaptic transmission, intrinsic excitability, retinotectal circuit refinement and visually guided behavior are known to occur -24. AfteP < 0.0001; Figure P = 0.789; IL-1β: n = 20 trials, 56 ± 5%, P = 0.1342; IL-6: n = 20 trials, 49 ± 6%, P = 0.82; Figure P = 0.0001; TNF-α: n = 20 trials, 71 ± 1%, P = 0.001; IL-1β: n = 20 trials, 68 ± 3%, P = 0.0001; Figure P = 0.04; Figure In order to test whether rearing tadpoles in elevated cytokine levels results in visual processing deficits, we screened cytokine-reared tadpoles using a visual avoidance test . This teWe next tested whether cytokine treatments resulted in abnormally targeted visual projections, which could account for the behavioral deficits. To do this we used a line of transgenic tadpoles expressing green fluorescent protein (GFP) driven by the Isl2b promoter, which primarily restricts expression to retinal ganglion cells. This allowed us to directly visualize the retinotectal projection using fluorescence microscopy. The pattern of innervation by the retinal ganglion cell axons enables the identification of four major targets that have been previously described  Figure . HoweverTo further examine the effects of elevated cytokine levels during development, we chose to focus on the electrophysiological effects of TNF-α. Due to the possibility that IL-6 may have had more widespread effects on the developing tadpole, we did not examine the effects of this cytokine further. Between IL-1β and TNF-α, the effects of TNF-α on excitatory synaptic transmission are better understood; thus, we focused our attention on TNF-α, leaving IL-1β and IL-6 as the subjects of future studies. Prior work has shown that acute application of TNF-α enhances excitatory synaptic transmission in hippocampal neurons by increasing surface expression of AMPA-type glutamate receptors ,37. This+ and K+ currents, as well as the amplitude and frequency of spontaneous AMPAR-mediated EPSCs (sEPSCs) in control brains and brains that had been exposed to TNF-α (0.5 mg/ml) for at least 1 hour. Tectal neurons exhibit voltage-gated Na+ and K+ currents , but no detectable difference in sEPSC amplitude . In our hands we have found that both the amplitude and frequency distribution of sEPSCs are not significantly different from those measured from mEPSCs recorded in tetrodotoxin (TTX) [To test whether long-term elevations of TNF-α affect intrinsic excitability and synaptic properties of tectal neurons, we performed electrophysiological recordings on tectal neurons from tadpoles reared in TNF-α for 10 days, between developmental stages 44 and 49. We first looked for changes in intrinsic properties. We found no significant differences in the maximal amplitudes and current-voltage (I-V) relationships of both voltage gated NaP = 0.0425). Because no increase in sEPSC amplitude was detected, one possibility that would account for the change in AMPA/NMDA ratio is that TNF-α is causing a premature 'unsilencing' of immature NMDAR-only synapses, rather than a strengthening of pre-existing AMPAR-containing synapses. If this possibility were true, then we would expect to see a decrease in the number of so-called silent synapses after chronic treatment with TNF-α.As retinotectal synapses mature, they increase the relative amount of AMPAR-mediated currents relative to NMDAR-mediated currents, as a result of 'unsilencing' of immature synapses containing only NMDAR ,25. ThisP = 0.0005). One interpretation of these data is that long-term treatment of TNF-α during development results in changes consistent with enhanced maturation of excitatory synapses, including those of the retinotectal projection. Other mechanisms, such as enhanced stabilization of mature synapses or decreased synapse elimination, are also consistent with these data.Silent synapses were measured by stimulating single retinotectal fibers, using minimal stimulation ,47,48. SDuring development, the tectal circuitry undergoes a significant amount of refinement, both in the retinotectal projection as well as in the local circuitry within the tectum. Much of this refinement involves strengthening and stabilization of some synapses, and weakening and elimination of others. In the prevailing model, synaptic maturation and AMPAR insertion is believed to stabilize existing synapses and dendritic branches . SynapseP = 0.007). These data are consistent with a lack of refinement of the retinotectal projection.To assess the effects of TNF-α on refinement of the retinotectal projection, we estimated the number of retinotectal fibers innervating a single tectal cell. Over development, the number of retinotectal fibers innervating a single tectal cell is known to decrease. This calculation was done by first measuring the amplitude of the synaptic response evoked by single-fiber stimulation. The stimulus intensity was then increased until the retinotectal synaptic response leveled-off at a maximal value. We then divided the maximal amplitude by the minimal amplitude to get an estimate of the number of retinotectal fibers innervating a single tectal cell Figure . TypicalXenopus tectum, a large proportion of excitatory synapses originate from local tectal circuits. Activation of these local circuits by afferent activity results in persistent recurrent activity within the tectum [P = 0.05, F-test). One alternative possibility is that the prolonged recurrent synaptic activity could be due to enhanced intrinsic excitability of tectal neurons. However, as shown in Figure In the e tectum . This ree tectum . We prede tectum . In conte tectum . We founP = 0.006 for length, P = 0.039 for growth). This observation is consistent with enhanced synapse stabilization.One further test of this hypothesis would be that if TNF-α treatment results in premature stabilization of synapses onto tectal neurons, then tectal neuron dendrites should reflect this effect. Tectal dendrites grow through a balance of branch addition and retraction . New denP < 0.0001; Figure One possible consequence resulting from increased recurrent circuitry within the tectum is that this circuitry may facilitate the generation of seizures. Interestingly, some neurodevelopmental disorders are often associated with an increased tendency toward epilepsy ,52-55. TXenopus visual system result in impairment of visually guided avoidance behavior. This behavior is known to require normal maturation of retinotectal circuits. Both IL-1β and TNF-α appear to selectively disrupt tectally-mediated behavior but not the OMR, a visual behavior largely independent of tectal function. All three cytokines tested did not appear to have noticeable effects on the gross anatomical projection pattern of visual inputs. TNF-α was tested for long-term effects on synaptic transmission in the tectum, and it was found to result in increased frequency of sEPSC, enhanced AMPA/NMDA ratios and decreased number of immature, NMDA-only synapses when compared to developmentally matched controls. The TNF-α treated group also lacked normal refinement of the retinotectal projection as well as refinement of intratectal circuitry, as assayed electrophysiologically. This lack of refinement resulted in abnormal patterns of recurrent synaptic activity. Anatomically, individual tectal neurons were observed to have enhanced dendritic growth rates after treatment with TNF-α Finally, the TNF-α treated group had an increased susceptibility to PTZ-induced seizures. Taken together, our data are consistent with a working model in which TNF-α results in premature maturation of excitatory synapses within the tectum. This enhances the stabilization of tectal synapses, and impairs normal synapse elimination that occurs during refinement of the retinotectal projection and local tectal circuits. As a result, tectal cells will have increased local interconnectivity, which would cause deficits in visual processing as well as an increased tendency to seizures.Our data indicate that elevated levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α during early development of the Our data indicate that elevated cytokine levels during development are sufficient to induce abnormalities in tectal microcircuitry as shown by behavioral studies. Furthermore, our electrophysiological data suggest that the direct effect of TNF-α on excitatory synaptic transmission may be responsible for preventing the normal maturation of tectal microcircuitry. Accumulating evidence from the developing tectum and other brain regions has suggested that functional and anatomical maturation of neural circuits are concurrent, and that functional synaptic maturation is a critical step in stabilizing developing synapses. This idea is known as the 'synaptotropic hypothesis' ,58. In oCould the other cytokines tested be acting in a similar manner? Because IL-6 may have also had an effect on the OMR, we cannot rule out that its behavioral deficit is due to a more general visual or developmental deficit. However, IL-1β did have a specific effect on visual avoidance. IL-1β is known to affect long-term potentiation, reduce AMPAR-mediated transmission and alter NMDAR function ,33,34. TXenopus tadpoles and zebrafish embryos show distinct seizure-like activity when presented with a convulsant agent [Another interesting finding in our study is that TNF-α reared tadpoles have a significantly higher seizure susceptibility than controls Figure . It is knt agent ,57, and nt agent . Alternant agent . Prior snt agent . It remaSeveral neurodevelopmental disorders, including autism, Down syndrome and schizophrenia have been associated with abnormal maturation of neuronal microcircuitry ,17 and tWe find that cytokine-reared tadpoles exhibit some of the features associated with a number of neurodevelopmental diseases and disease models: abnormal development of neuronal microcircuits, lack of developmental pruning, and increased tendency to seizures. However, many of these disorders have been primarily associated with a wide variety of genetic causes, and animal models interfering with these different genes appear to replicate many of the phenotypic aspects of the various disorders ,62-65, aTaken together our studies present a novel integrative model to study neuro-immune interactions. Our assays test multiple parameters of development ranging from behavior, neuroanatomy and whole-brain electrophysiology. We show that a select group of pro-inflammatory cytokines can lead to developmental abnormalities consistent with inappropriate formation of neuronal microcircuits. While we tested only three cytokines, our assays could be scaled up to study a much broader range of immune molecules and combinations of molecules, providing for a powerful large-scale assay for abnormal neural development. We believe our findings might provide valuable insight into novel therapeutic approaches for the treatment and understanding of neurodevelopmental disorders.Xenopus laevis tadpoles were raised on a 12 hour light/dark cycle at 20 to 22°C.All of our experiments were carried out in accordance with Institutional Animal Care and Use Committee standards. The tadpoles were staged according to Nieuwkoop and Faber developmental stages (as described in ) and genTadpoles were reared in 15-ml wells of a 6-well plate containing 5 to 6 ml of IL-6 (0.065 μg/ml), IL-1β (0.10 μg/ml), or TNF-α (0.50 μg/ml). Treatment began at stage 44 (approximately 7 dpf) and media was exchanged every 2 to 3 days. Tadpoles were treated until they reached stages 48 to 49, about 18 to 20 dpf. A maximum of four tadpoles were reared in each well, and control tadpoles, which were derived from the same clutch as the treated groups, were also reared in the same wells and rearing media volume. TNF-α was purchased from Sigma . IL-1β and IL-6 were purchased from R&D Systems .et al. [Behavioral testing was performed as described in Dong et al. . In brieet al. .t-test was used to test whether the percentage of tadpoles on the stationary side was different from a random distribution in which tadpoles are evenly split between the stationary and non-stationary sides . This dX. laevis. After fertilization, the embryos were placed in dishes of Steinberg's solution and kept at 19 to 20°C. The tadpoles were screened for GFP expression and fed on day 5 post-fertilization. On day 6, at stage 44 the tadpoles were treated with cytokines as described earlier. On the completion of the tenth day of cytokine treatment, the tadpoles were euthanized and fixed in paraformaldehyde. The brains were then dissected from the tadpoles and placed in phosphate-buffered saline in preparation for whole mount immunostaining. The tissue was permeabilized using 1% Triton X-100 and 0.1% bovine serum albumin in phosphate-buffered saline (PBT), and blocking was done with 10% normal goat serum in PBT. The primary antibody incubations were done at 4°C O/N using 1:1,000 rabbit αGFP  to label retinal ganglion cell axons. The secondary antibody incubation was done at room temperature for 4 hours using 1:200 α rabbit Alexa-488. The brains were then mounted on coverslips and pictures were taken on the fluorescent microscope. For high resolution images an Olympus confocal microscope was used.Isl2b:GFP tadpoles are transgenic tadpoles expressing GFP driven by the Isl2b promoter , which primarily drives expression in retinal ganglion cells . Isl2b:Get al. [et al. [2, 3 MgCl2, 5 HEPES, 10 glucose, and 0.1 picrotoxin, pH 7.2; osmolarity, 255 mOsm). The brains were opened along the dorsal midline and pinned to a submerged block of Sylgard in a recording chamber maintained at room temperature (approximately 20 to 22°C) with continuous perfusion of external saline. Using a broken glass recording pipette, the ventricular membrane surrounding the tectum was scraped, exposing tectal neuron somata for recording. A bipolar stimulating electrode  was placed on the optic chiasm to stimulate retinal ganglion cell axons. Visualized whole-cell voltage-clamp recordings were made using glass micropipettes (8 to 12 MΩ) filled with K+ gluconate intracellular saline .For whole-brain recordings, tadpole brains were prepared as described by Wu et al.  and Aize [et al. . TadpoleBecause developmental variability exists along the rostrocaudal axis , we consI-V plots were generated by measuring either the peak inward or the peak outward current in response to a series of depolarizing pulses, ranging from -60 to +20 mV . Spontant-test, unless otherwise specified, and were done using InStat or Prism software . Error bars are standard error of the mean.To calculate AMPA/NMDA ratios, we collected evoked synaptic responses at -60 mV to measure AMPAR-mediated EPSCs and at +60 mV to measure NMDAR-mediated EPSCs. The peak AMPA response was used to calculate the AMPAR component and the amplitude between 15 and 17.5 ms post response onset was used to calculate the slower NMDA component. Although this time window may not be an accurate absolute measure of NMDA peak response, the method still allows us to calculate relative changes in AMPA/NMDA ratio. This method was successfully used to detect developmental changes in the AMPA/NMDA ratio in this and other preparations ,49,50. TMicropipettes were filled with a solution containing a plasmid encoding farnesylated enhancedGFP (EGFP-F) at a concentration of 5 μg/μl. Tadpoles  were anesthetized by immersion in 0.02% MS-222. The micropipette was positioned in the superficial tectum under microscope guidance using a Narashige micromanipulator, a 0.5-s 200 Hz burst of 1-ms square wave pulses was then applied using a grass SD9 stimulator as described in Ruthazer et al., 2005 E.S. Ruthazer, K. Haas, A. Javaherian, K.R. Jensen, W.C. Sin and H. Cline, Vivo Time-lapse Imaging of Neuronal Development. In: R. Yuste and A. Konnerth, Editors, Imaging in Neuroscience and Development: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2005), pp. 191-204 . AnimalsFor imaging, tadpoles were anesthetized by immersion in 0.02% MS-222 and immobilized in Sylgard chambers  carved to fit their body shapes with a cover glass on top. Single cells were then visualized and imaged on a custom-built two-photon microscope in 1-μm z-steps. After the first image animals were placed into rearing medium containing 0.5 μg/ml recombinant human TNF-α . This solution was then replaced 2 days later. On the fourth day cells were imaged again. Three-dimensional tracings of imaged neurons were made with Imaris software .Seizures were induced in the tadpoles by the bath application of 10 mM PTZ, a known convulsant, for 20 minutes . AnimalsAMPAR: AMPA receptor; dpf: days post-fertilization; EPSC: excitatory post-synaptic current; GFP: green fluorescent protein; IL: interleukin; NMDAR: NMDA receptor; OMR: optomotor response; pA: pico amperes; sEPSC: spontaneous EPSC; TNF: tumor necrosis factor.The authors declare that they have no competing interests.RHL performed the behavioral screen and the majority of the electrophysiology. He also performed data analysis and participated in producing the final manuscript. EAM performed the neuroanatomy experiments and interpreted the results. NS performed and analyzed the functional imaging experiments. MRB performed and analyzed the seizure experiments. KED performed additional electrophysiological experiments. ESR oversaw the functional anatomy. NM-A oversaw the gross neuroanatomical experiments and provided the Isl2b:GFP tadpoles. CDA conceived of the study, participated in its design and coordination, performed some electrophysiology experiments and helped draft the manuscript and prepare the figures.Progression of PTZ-induced seizures in control and TNF-α reared tadpoles. Movie shows a control well and a well containing TNF-α reared tadpoles. Tadpoles are stage 48 and are clutchmates. PTZ is introduced at t = 0. At t = 20 s normal swimming behavior is observed in both groups. At t = 5:30 minutes the TNF-α group is showing periods of increased locomotor activity. At t = 7:30 minutes the TNF-α group is undergoing clear seizure behavior characterized by extreme tail bends and fast turning behavior followed by periods of immobility. At t = 10 minutes the TNF-α group continues to seize while the control group begins to exhibit some increased locomotor activity. At t = 18 minutes both groups exhibit regular seizures interspersed with periods of immobility.Click here for file


FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive.  FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. The FWA promoter is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is necessary. Here we show that the direct repeat structure is highly diverse in species within the genus Arabidopsis. Unexpectedly, we found that the direct repeat structure is dispensable for the epigenetic silencing and methylation of the FWA promoter. Rather, the SINE-related promoter sequence is sufficient for these features. Frequent independent formation of the tandem repeats suggests that they may be a consequence of the epigenetically controlled system.Genomic imprinting, mono-allelic gene expression depending on the parent-of-origin, is an epigenetic process known in mammals and flowering plants. A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive.  Parental imprinting, mono-allelic gene expression depending on its parent-of-origin, is an epigenetic process known in mammals and flowering plants. In both mammals and plants, many imprinted genes are under control of DNA methylation, as their imprinting is abolished by mutations in DNA methyltransferase genes FWA gene in the flowering plant Arabidopsis thaliana is one of the most extensively studied systems linking the control of DNA methylation and imprinting. The FWA gene was originally identified through characterization of epigenetic mutants causing a heritable late-flowering phenotype. The phenotype was due to ectopic expression of the FWA gene in vegetative tissue FWA gene is silent in vegetative tissues and expressed specifically in the endosperm in an imprinted manner FWA silencing depends on cytosine methylation, as it is derepressed by mutations in the maintenance methylase gene MET1The trans-acting components, a cis-requirement for the epigenetic FWA silencing has also been identified. Promoter of the FWA gene has two pairs of tandem repeats that are heavily methylated de novo methylation induced by double-stranded RNA, we previously showed that the critical methylated element corresponds to the SINE-related tandem repeats per se can direct DNA methylation, or whether the tandem repeat structure is necessary for control of methylation and imprinted expression.In addition to these FWA promoters in A. thaliana and five other Arabidopsis species. The results demonstrate that the tandem repeat structure is necessary neither for imprinted FWA expression, nor for vegetative FWA silencing. Instead, the SINE-related sequence itself appears to be the primary target of the epigenetic control, which might have subsequently induced frequent tandem repeat formation during evolution. In addition, considerable intra-specific variation was found in vegetative FWA expression, which might present sources for epigenetic variation in reproductive strategy.Here we investigate the evolution and natural variation of FWA silencing in A. thaliana is a SINE-related direct repeat FWA promoter and FWA expression, we identified FWA orthologues in three related Arabidopsis species, A. halleri, A. lyrata, and A. arenosa. We found that the gene structure, including coding regions as well as introns and promoter sequences, was conserved among these species and A. thaliana  lyrata (three copies) and ssp. petraea (four copies)  . In A. hpromoter .A. thaliana, the FWA gene is silent in vegetative tissues and expressed in the endosperm in an imprinted manner . Both the silencing of the paternally-derived copy and silencing in vegetative tissues depend on DNA methylation in the FWA promoter.In A. thaliana, FWA transcripts were detected in immature seeds in A. lyrata and A. halleri. To determine whether the FWA gene shows imprinted expression in these species, RNA was isolated from immature seeds after inter-strain crosses, and the origin of FWA transcripts was identified using sequence polymorphisms in the transcript. In both A. lyrata and A. halleri, the transcript detected was derived from the allele of maternal origin .The vegetative re seeds  and in pre seeds , which cFWA expression in two allotetraploid species, A. kamchatica (ssp. kamchatica and kawasakiana) and A. suecica. The former is an allotetraploid between A. lyrata and A. halleriA. arenosa and A. thalianaWe further examined FWA expression in A. kamchatica (ssp. kamchatica and kawasakiana) .We were able to detect vegetative sakiana) . As expes silent . Interestophytes , leavingFWA expression in A. suecica , which shows vegetative silencing of the FWA.The methylation level is high throughout the repeat region ilencing . Interes. lyrata . The metA. lyrata, the number of repeats in the FWA promoter correlated with the level of vegetative silencing. In A. lyrata ssp. petraea strain Mue-1, which has four copies of the tandem repeat, the vegetative expression was much lower than that in the two strains of A. lyrata ssp. lyrata that have three copies of the tandem repeat  is a kind gift from Maarten Koornneef. A. lyrata ssp. lyrata pn3 was collected in Pores Knob, Wilkes County, NC. The A. lyrata ssp. lyrata MN47 was developed at Cornell University from material originally isolated by Charles Langley in Michigan, USA. The A. lyrata ssp. petraea strain Mue-1, was collected in Muehlberg, Germany. A. halleri ssp. gemmifera Tada, OID06-6, and Mino were isolated in Inagawa-cho, Osaka, Japan, Taka-cho, Hyogo, Japan, and Mino-shi, Osaka, Japan. A. halleri ssp. halleri H-RB was isolated in Bistrita, Romania, ssp. tatrica T-PLDH1 in Vysoke Tatry, Poland, ssp. ovirensis O-AUO26 in Karinthia, Austria. A. kamchatica ssp. kamchatica FJSB1 was isolated in Subashiri, Shizuoka, Japan, and ssp. kawasakiana Shirahama was isolated in Ohmi-shirahama, Shiga, Japan. A. suecica was from Sendai Arabidopsis Seed Stock Center (JS7). Synthetic suecica (CS22665 and CS22666) and A. arenosa (CS3901) are from Arabidopsis Biological Resource Center at Ohio State University.The sources of the 96 natural accessions (cs22660) of FWA gene in related species were amplified by PCR and cloned into pGEM-TEasy or pCR2.1-TOPO vector using the primers lyrata-7f and lyrata-10r for lyrata and arenosa alleles, lyrata-7f+lyrata-9r for halleri alleles, FWApro1+As-3 for thaliana alleles. In each strain, multiple clones were sequenced and regions of inconsistent sequences were directly sequenced from the genomic DNA again. Sequences of both alleles were determined in out-crossing species, such as A. halleri and A. lyrata. 96 ecotypes of the tandem repeat region was amplified using primer pair Ateco96F+Ateco96R, and their nucleotide sequences were determined directly from the genomic DNA. Data were analyzed using Sequencher . Harr plot analysis was performed with GENETYX-MAC software . Unit size to compare was 10 bp, and dot plot matching number was 9 bp http://www.ddbj.nig.ac.jp/search/clustalw-j.html). A phylogenetic tree was constructed by the neighbor joining method with K2P distance [43], and bootstrap probabilities of 1,000 trials were calculated.Sequences of all primers used are shown in Supplementary FWA transcript was detected by RT-PCR of 40 cycles using the first strand cDNA as a template with primer pair FWA-RT-F2+FWA-RT-R1. GAPC was used as control and amplified by RT-PCR of 25 cycles using primer pair GAP3+GAP5. In A. suecica, allele-specific RT-PCR was performed using primer pair specific for FWA in A. thaliana (AtFWA-RT-F1+AtFWA-RT-R1) and specific for FWA in A. arenosa (FWA-RT-F5+AtFWA-RT-R4). In hybrid between A. lyrata/A. halleri and A. thaliana, allele specific primer pair was used for RT-PCR to amplify FWA in A. lyrata/A. halleri (FWA-RT-F5+AtFWA-RT-R5) and that of A. thaliana (AtFWA-RT-F1+AtFWA-RT-R1). The PCR condition was 95°C for 10 sec followed by 25 or 40 cycles of 95°C for 30 sec, 57°C for 30 sec, and 72°C for 30 sec.Total RNA was isolated from leaves and pistils from open flowers using SV Total RNA Isolation system . From 2 µg total RNAs, first strand cDNA was synthesized using random primers by a First-strand cDNA synthesis Kit . A. lyrata and A. halleri were isolated by RNAqueous-Micro . Using all of isolated total RNAs, first strand cDNA was synthesized using FWA-specific primer AlFWAcDNA-R by SuperScript III Reverse Transcriptase (Invitrogen). FWA transcripts in seed of A. lyrata and A. halleri were amplified by RT-PCR of 35 cycles using the first strand cDNA as a template with primer pairs (AhAlFWAcDNA-F2+lyrata2r) and (FWA-RT-F3+FWA-RT-R3), respectively. Products from genomic DNA and mRNA could be distinguished by size.Total RNAs from one premature fertilized seed after 15DAP in FWA transcript level was compared between two strains of A. kamchatica by real-time PCR  on a LightCycler . With the primer pairs FWAreal1+FWA-RT-R1 (FWA) and GAPreal2+GAPreal3 (GAP). The PCR condition was 95 for 10sec followed by 25 (GAP) or 40 (FWA) cycles of 95°C for 5 sec, 55°C for 10 sec, and 72°C for 7 sec. Data were analyzed with LightCycler Software version 3.5 (Roche).time PCR  with theFWA were amplified using primer pairs as follow. AtFWA; (AtFWA-Bis-F1+AtFWA-Bis-R1), AaFWA; (AhFWA-Bis-F1+arenosaRTbisR), AlFWA; (arenosaRTbisF+lyrataRTbisR), and AhFWA; (AhFWA-Bis-F1+AhFWA-Bis-R2). The amplified PCR fragments were gel purified by GENECLEAN III Kit  and cloned into pGEM-T easy vector , and 10 independent clones were sequenced.Bisulfite sequencing was performed according to Paulin et al. Sequence data from this article have been deposited with DDBJ/GenBank/EMBL libraries under accession numbers AB363659–AB363674, AB367805–AB367817, and AB367818–AB367913.Text S1Supplementary materials and methods.(0.03 MB PDF)Click here for additional data file.Figure S1FWA genes in Arabidopsis species. Harr plot analysis shows that not only exons (black boxes) but also promoter and introns are conserved. A big gap found in each panel reflects thaliana-specific big tandem duplication. Gray boxes indicate the regions related to the SINE.(0.05 MB PDF)Click here for additional data file.Figure S2FWA gene to detect tandem repeats. Black boxes and gray bar indicate exons and the SINE-related region, respectively. In the SINE-related regions, two, three, and four tandem repeats were found in A. arenosa, A. lyrata ssp. lyrata, and A. lyrata ssp. petraea, respectively. Two pairs of tandem repeats were found in A. thaliana. No tandem repeat was found A. halleri.Harr plot analysis of 5′ region of the (0.08 MB PDF)Click here for additional data file.Figure S3FWA expression in eleven plants of A. halleri ssp. gemmifera isolated around Kyoto area in Japan. (A) The vegetative FWA expression was detectable in two isolates, while it was undetectable in the other nine isolates. (B) Same expression pattern was detected in different individuals isolated from the same area. (C) Map of the locations these plants were isolated. Abbreviation of the isolates: AK (Akebe), MH (Mikohata), IK (Ikuno), OM , RK (Rokko), TD (Tada), MK (Myouken), MN (Mino), OH (Ohara), IB (Mt. Ibuki), OE (Mt. Ooe). The plants were isolated from the field and were grown in the laboratory. Three of them, Mino, Omoidegawa, and Tada, were further characterized after isolation of the seeds and growth in the laboratory (Variation in vegetative boratory .(0.12 MB PDF)Click here for additional data file.Figure S4FWA genes in allotetraploids and parental species. (A) Two FWA copies in A. kamchatica. Each of them was similar to that in A. halleri (left panel) or A. lyrata (right panel). The similarity was found not only in exons (black boxes) but also promoter and introns. Gray boxes indicate the SINE-related regions. Copy number of tandem repeats in the lyrata-like copy was three as is the case in ssp. lyrata. (B) Two FWA copies in A. suecica. Each of them was similar to that in A. thaliana (left panel) or A. arenosa (right panel). (C) A phylogenetic tree of nucleotide sequences of full length cDNA of FWA in the Arabidopsis species constructed by the neighbor-joining method. Arabis glabra FWA was used as the outgroup. Bootstrap values with 1,000 repeats are indicated at the nodes of the neighbor-joining tree.Structure of (0.08 MB PDF)Click here for additional data file.Figure S5FWA gene in two strains of in A. kamchatica, SHR1 (ssp. kawasakiana), and FJSB1 (ssp. kamchatica). (A) Promoter alignment revealed insertion of a possible transposable element in the SHR1 allele. Blue and red letters indicate the predicted target site duplication and terminal inverted repeat, respectively. This insertion was found in majority of A. kamchatica, ssp. kawasakiana strains isolated in Japan (not shown). Gray boxes: the SINE-related sequences. Green letter shows the target site duplication of the SINE insertion. The circle shows the transcription start site. Black arrows show regions duplicated in A. thaliana. (B) Methylation status of the halleri-type FWA gene in the two strains. The predicted transposon (white box) was heavily methylated in SHR1. (C) Expression analysis of the FWA gene. Related FWA expression level measured by real-time PCR are shown below the gel. The transposon insertion and increase in the DNA methylation correlates with reduction in the FWA transcript level in SHR1.Differences in promoter structure and expression pattern of the halleri-type (0.04 MB PDF)Click here for additional data file.Figure S6FWA gene. The analyses are based on the 12 strains of A. thaliana. K and p in 200 bp silent sites are plotted in 1 bp intervals. Under the sliding window plot, exons (from third to last) are shown by black boxes and repeated regions by arrows.Sliding window analyses of silent divergence and diversity in the (0.02 MB PDF)Click here for additional data file.Table S1FWA promoter.Proportion of methylated cytosine around the (0.04 MB PDF)Click here for additional data file.Table S2FWA gene within A. thaliana.Patterns of nucleotide variation in the (0.05 MB PDF)Click here for additional data file.