Arabidopsis thaliana accessions implicated FLOWERING LOCUS C (FLC) as a circadian-clock regulator. The MADS-box transcription factor FLC is best known as a regulator of flowering time. Its activity is regulated by many regulatory genes in the "autonomous" and vernalization-dependent flowering pathways. We tested whether these same pathways affect the circadian system.The circadian system drives pervasive biological rhythms in plants. Circadian clocks integrate endogenous timing information with environmental signals, in order to match rhythmic outputs to the local day/night cycle. Multiple signaling pathways affect the circadian system, in ways that are likely to be adaptively significant. Our previous studies of natural genetic variation in FLC, were found to regulate circadian period in Arabidopsis. The mechanisms involved are similar, but not identical, to the control of flowering time. By mutant analyses, we demonstrate a graded effect of FLC expression upon circadian period. Related MADS-box genes had less effect on clock function. We also reveal an unexpected vernalization-dependent alteration of periodicity.Genes in the autonomous flowering pathway, including FLC's role in the clock, as it reveals that the network affecting circadian timing is partially overlapping with the floral-regulatory network. We also show a link between vernalization and circadian period. This finding may be of ecological relevance for developmental programing in other plant species.This study has aided in the understanding of  Most eukaryotes and some prokaryotes have evolved a circadian clock to adapt to the 24 h day/night cycle. These clocks drive biological rhythms in many aspects of metabolism, physiology, and behavior, all with a period close to 24 h . CircadiCircadian clocks, including those of Arabidopsis, are reset by light and temperature signals in a characteristic fashion that entrains the clock to the local time in its environment . HoweverA circadian clock maintains accurate timing because it is buffered against many environmental changes, yet several environmental-signaling pathways must affect the circadian oscillator for entrainment to occur. Limiting the input connections to the circadian clock from the rest of the plant-signaling network provides a potential mechanism to balance the opposing requirements of homeostasis and entrainment. In the gene network that regulates flowering time, for example, the circadian clock is an integral part of the photoperiodic sensor, receiving input from light signaling ,4. Curreerecta (Ler) and Columbia (Col) × Ler [FLOWERING LOCUS C (FLC). The Ler allele of FLC is weakly functional due to a transposon insertion within an intron of FLC [er as one parent, therefore FLC function segregates in these recombinant populations [er allele of the QTL shortened the circadian period by 0.8 h, as did independent flc mutant alleles, leading us to conclude that the known allelic variation in FLC could account for the QTL [Substantial natural variation has been detected in clock-affecting genes, based upon assays of rhythmic leaf movement under constant light -11. Thisl) × Ler . In eachn of FLC ,13. The ulations . The Ler the QTL .FLC encodes a MADS-box transcription factor that had no known function in the circadian clock, but was well-characterized as a repressor of flowering. FLC expression is enhanced by FRIGIDA (FRI) and its paralogues, which are active in many late-flowering accessions [FLC transcription is suppressed by genes of the autonomous floral-promotion pathways and by prolonged cold temperatures . As FLC RNA abundance correlates with repression of flowering time and quantitatively mediates the vernalization response of flowering time [FLC expression levels similarly regulated the circadian clock. To assay the plant's endogenous circadian period, rhythmic movement of the primary leaves of Arabidopsis seedlings were measured by video imaging under constant white light. Relatively large numbers of plants were tested in replicate experiments in order to increase the sensitivity of the assays, allowing us to detect small changes in circadian period . FLC RNA abundance was manipulated by two methods. Firstly, we tested a line expressing FLC from the CaMV 35S promoter (35S:FLC). This transgenic construction strongly delays flowering time. The 35S:FLC line had a circadian period lengthened by over 1 hour in multiple experiments  , all uniformly in a Col background. These comprised the functional allelesFRI-Sf2 and FLC-Col (FRI and FLC) and recessive alleles fri-Col and flc-3 (fri and flc) [FLC had a lengthened circadian period compared to flc lines  [FLC lengthened circadian period in both FRI and fri backgrounds. Our findings are also consistent with the function of FLC in flowering time. fri;FLC plants flower slightly later than fri;flc mutants under non-inductive photoperiods, indicating FLC can function independently of FRI in the floral pathway [FLC transcript is therefore sufficient to increase the circadian period of the plant. The greater effect of 35S:FLC compared to endogenous FLC suggests that increasing FLC RNA levels increase circadian period in a graded manner, similar to FLC's dose-dependent delay of flowering [FLC expression in 35S:FLC plants contributed to this result, though such effects on the location of FLC expression have not previously been implicated in flowering-time control.ing time . We sougand flc) . Lines h. (1999) , who sholowering ,18. It mFRI caused an additional increase in period in the presence of functional FLC . This indicates that FRI can increase circadian period weakly and less consistently than FLC. In contrast, functional FRI strongly enhances FLC RNA levels and severely delays flowering [FRI does not enhance FLC expression in the cells that regulate leaf movement as much as 35S:FLC.We suggest that functional C Figure . Althouglowering ,18, highFLC expression levels, including the genes of the autonomous pathway, would affect circadian period. In order to compare the network of circadian regulators to the pathways that regulates flowering time, we analyzed the circadian period of plants carrying mutations in several genes of the autonomous flowering-time pathway .FCA is one of the genes that defines the autonomous flowering-time pathway. FCA encodes a nuclear-localized protein with RNA recognition motifs. It is involved in 3' RNA processing of FCA transcripts through physical interaction with the FY protein [FCA promotes flowering by repression of FLC RNA levels [fca lesion have high levels of FLC RNA and flower late. Assays on fca mutants revealed an increase in circadian period of nearly 1 hour compared to the Col-0 wild-type , in line with the hypothesis that elevated FLC levels increase circadian period    . To test whether this was due to an increase in FLC activity in the mutant, we assayed the fve; flc double mutant. We found a statistically insignificant decrease in circadian period (0.24 h) in the double mutant compared to the fve single mutant , so the period of the fve; flc double mutant retained a significantly lengthened period  compared to the wild type, and this modest effect was not statistically significant   Figure , Table 2e dosage , FPA int pathway , and has pathway . These dSOC1  is a MADS-box gene that activates flowering, in response to signals from the autonomous and photoperiodic flowering-time pathways [SOC1 is described as a target of FLC-mediated transcriptional repression in the flowering-time pathway [soc1 mutant has a small increase in circadian period relative to wild type , indicating that SOC1 normally shortens circadian period . The resulting period of soc1; flc was identical to that of Col  , is the member of this family with the highest level of identity with FLC [FLM to have a similar function in the repression of flowering as FLC. However, unlike FLC, FLM expression is not increased by FRI, and FLM does not contribute to the vernalization requirement [flm mutant revealed that it had no circadian defect (P = 0.69)  in repressing flowering time [svp mutant showed a significant lengthening in period of 0.7 h compared to wild type  . We found in duplicate experiments that vernalization consistently decreased circadian period (P < 0.001) but that none of the single-gene or gene interaction effects was significant .e Spring . flc mutresponse , which mresponse . Based ot Figure  and 4. TFLC itself, modulate the period of a circadian clock in Arabidopsis. The effects on period are modest, but our data measurements are accurate, as detected in plots of relative amplitude error (RAE) versus period length for individual cotyledon traces of a genotype in an experiment  [fve-4, fve-4;flc-3 [ld-1, ld-1;flc-3, fpa-7 [flm [ld-3 [fca-9 [svp-41 [ld-1 from the Nottingham Arabidopsis Stock Centre. 35S:FLC ; soc1-2,0;flc-3) ;fve-4, f-4;flc-3 ;ld-1, ld3, fpa-7 . Mutantsa-7 [flm , ld-3 [2lm [ld-3 . We are 3 [fca-9  from Car [svp-41  from Petet al. 1995 [-2sec-1 continuous cool white fluorescent light for 7 days, at 21–22°C, followed by entrainment to three 12 h-light, 12 h-dark cycles. Growth of the first pair of primary leaves was recorded under 30–40 μmolm-2sec-1 continuous cool white fluorescent light, at 21–22°C for 7 days. Seedlings were arranged randomly with respect to genotype within each experiment, to avoid positional bias in the imaging arrays. For vernalization, stratified seed were germinated for 4 days, as described above (at which point cotyledons were expanding), then incubated at 2°C for 8 weeks under low-intensity white light (0.5–1.0 μmol m-2sec-1). After the vernalization treatment, seedlings were transferred to continuous light for 4 days, then entrained and imaged as above. In order to confirm the effectiveness of vernalization, imaged seedlings were transferred from agar to soil immediately after the leaf-movement assay. Flowering time of these lines was measured as the number of rosette leaves when the floral bolt was 1 cm high. These studies confirmed the expected effects of FLC and FRI upon vernalization-responsiveness (data not shown).Seedlings were grown and followed by assays of rhythmic leaf movements by video imaging, as described by Dowson-Day and Millar, 1999; Millar al. 1995 ,49. Brieal. 1995  that incFRI-FLC interactions and vernalization effects on period were assessed using the Wald test with variances derived from REML. The significance of the differences between the mean period of pairs of genotypes was assessed using the standard error of each difference, derived from REML. Figures Leaf movement data were analyzed by Fast Fourier Transform non-linear least squares program FFT-NLLS , essentiNS, AJM, and SJD conceived the experiments and wrote the paper, with critical experimental and intellectual revisions from SDM and RMA. SDM and RMA generated reagents. NS carried out the experiments. JRL conducted statistical analysis.Robustness of rhythmicity of all lines examined MS EXCEL file containing 3 worksheets. These include RAE plots for all genotypes examined (sorted with respect to Figure Click here for file


The first Japanese case of fibrolamellar carcinoma (FLC) of the liver is described. The 17-year-oldJapanese male affected died of the disease 25 months after palliative operation. The diagnosis of FLC hadnot been confirmed before autopsy. Pre-operative diagnosis of FLC is important to surgeons. Acccordingto the literature, FLC is rare in Asia. The endemic discrepancy may suggest that the carcinogenic factorsare different from those of general hepatocellular carcinoma.


Escherichia coli and Salmonella lipopolysaccharide are modified by membrane enzymes including ArnT, EptA and LpxT. ArnT and EptA catalyse the periplasmic addition of the positively charged substituents 4-amino-4-deoxy-L-arabinose and phosphoethanolamine respectively. These modifications are controlled by the PmrA transcriptional regulator and confer resistance to cationic antimicrobial peptides, including polymyxin. LpxT, however, catalyses the phosphorylation of lipid A at the 1-position forming 1-diphosphate lipid A increasing the negative charge of the bacterial surface. Here, we report that PmrA is involved in the regulation of LpxT. Interestingly, this regulation does not occur at the level of transcription, but rather following the assembly of LpxT into the inner membrane. PmrA-dependent inhibition of LpxT is required for phosphoethanolamine decoration of lipid A, which is shown here to be critical for E. coli to resist the bactericidal activity of polymyxin. Furthermore, although Salmonella lipid A is more prevalently modified with l-4-aminoarabinose, we demonstrate that loss of Salmonella lpxT greatly increases EptA modification. The current work is an example of the complexities associated with the structural remodelling of Gram-negative lipopolysaccharides promoting bacterial survival.During its transport to the bacterial surface, the phosphate groups of the lipid A anchor of  Lipopolysaccharide (LPS) is a complex bacterial surface structure that is composed of lipid A, a core oligosaccharide, and the O-antigen polysaccharide. LPS is characterized by its endotoxicity, which is provided by the lipid A domain, a hydrophobic molecule that anchors LPS to the outer membrane . Given tSalmonella and Escherichia coli consists of a β-1′-6-linked glucosamine disaccharide backbone bearing six fatty acyl chains, that is phosphorylated at the 1 and 4′ positions. This molecule is further glycosylated at the 6′-position with two 3-deoxy-d-manno-octusolonic acid (Kdo) moieties that link the lipid A domain to the remaining polysaccharide of LPS  and phosphoethanolamine (pEtN) respectively   species is mediated by the undecaprenyl phosphotransferase, LpxT (3+) (3+) . Under these conditions PmrA activation is mediated by a second two-component system, PhoP–PhoQ. According to the proposed mechanism, activation of PhoP in Salmonella induces the synthesis of PmrD, which regulates PmrA activity post-transcriptionally by preventing dephosphorylation of PmrA  . Other s of PmrA . It was  as previously described (2+ (0.01–0.1 mM) produced lipid A species modified with L-Ara4N and pEtN, but lacking 1-PP (2+ (1–10 mM) concentrations  medium supplemented with increasing concentrations of FeSO4. Fe2+ is quickly oxidized to Fe3+ serving as a specific signal for activation of the PmrA–PmrB system  harbouring a PmrA constitutive (PmrAc) phenotype , CH040 (ΔarnT) and the double mutant CH034 (ΔarnTΔeptA) in the W3110 background. 32P-lipid A profiles were evaluated in cells grown in LB medium supplemented with 200 µM FeSO4 (eptA), strong induction of single L-Ara4N modification was observed (arnT), both single and double pEtN-lipid A substitutions were evident (eptA) grown in Fe3+ produced a peak at m/z 1928.8 atomic mass units (amu) indicative of L-Ara4N modification (arnT) produced major peaks at m/z 1920.8 and 2043.5 amu corresponding to the predicted pEtN modified species (3+ induction (data not shown). Similar lipid A profiles were obtained in N-minimal medium containing 10 mM Mg2+, at pH 5.8 (eptA or arnT mutants. Only in the double mutant CH034 (ΔarnTΔeptA) could any 1-PP lipid A be detected , CH040 (ΔeptA) and the double mutant CH034 (ΔarnTΔeptA) were similar to that of wild-type (data not shown).We examined whether inactivation of ArnT or EptA, or both, would restore LpxT-dependent phosphorylation of lipid A. For this purpose, we created the isogenic strains CH030  background (pmrAc background even in strain WDarnTeptA (lane 5). Moreover, single and double pEtN-lipid A species were highly induced compared with L-Ara4N-lipid A substitutions  gene is downstream of the yeiR gene separated by only 38 bp. To elucidate whether both genes are transcribed into the same mRNA, yeiR and lpxT transcripts were evaluated by reverse transcription-PCR  was reported to be under PmrA-regulation  harbouring these plasmids were grown in LB medium supplemented with or without 200 µM FeSO4 (lpxTp (lpxT promoter) was not upregulated in the presence of Fe3+. Furthermore, W3110 and CH020 (ΔpmrA) did not exhibit differences in lpxTp activation. Contrary, eptAp (eptA promoter) was induced sevenfold in the presence of Fe3+, and this induction was lost in strain CH020 (ΔpmrA). These data confirmed that eptAp was activated in a PmrA-dependent manner. Background activity from plasmid pRS415 was limited to 50–80 units or lower in the negative controls, which is consistent with the reported plasmid background  strains when grown in LB medium (data not shown). These results confirm that PmrA does not regulate lpxT transcription.To perform β-galactosidase assays, the 250 bp upstream region of µM FeSO4 . Our res8 or 7.5 . Again, lpxT at the transcriptional level, growth conditions promoting PmrA activation result in inhibition of LpxT-dependent phosphorylation of lipid A in whole cells. To determine if LpxT is successfully assembled into the membrane, we monitored the expression of a chromosomal lpxT–gfp fusion protein under the control of its native promoter in strain CH01. The fusion protein was also expressed in strain CH021 . LpxT–GFP was fully functional in whole cells as indicated by the production of 1-PP lipid A  and CH020 (ΔpmrA) strains were prepared from cultures grown in LB medium with or without 200 µM FeSO4. Phosphorylation of lipid A by LpxT was evaluated using [4′-32P]Kdo2-lipid A  catalysed phosphate addition to [4′-32P]Kdo2-lipid A even under Fe3+ inducing conditions (3+) PmrA is necessary for the loss of LpxT activity in the bacterial membrane. A more hydrophobic reaction product resulting from the PagP catalysed transfer of palmitate  was grown under Fe3+ inducing conditions there was an unexpected increase in pEtN modification of lipid A with a concomitant loss of L-Ara4N modification compared with wild-type  expressing pWSK29 plasmid-born lpxT were evaluated  . On the other hand, pEtN addition is lost upon introduction of plasmid born LpxT even under Fe3+ inducing conditions. Together, these results suggest that even though LpxT activity is inhibited during PmrA activation it is possible to overcome this inhibition by overexpression of the protein.As we reported previously  , lane 3.6RP-(X12–54)-PSGH-(X31–54)-SRX5HX3D (190) within the predicted active site of LpxT (WT) and the H190A (LpxTH190A) mutant were expressed in plasmid pWSK29. EptA was expressed in plasmid pACYC184 (pACeptA) avoiding plasmid incompatibility. Expression of LpxTH190Ain trans was unable to complement the loss of 1-PP in strain MST01 (ΔlpxT) (WT (lane 4). Coexpression of LpxTWT and EptA produced both 1-PP and pEtN-lipid A species, with LpxTWT clearly the predominate activity (lane 6). In contrast, coexpression of LpxTH190A and EptA led to extensive double pEtN modification (lane 7) identical to that seen when EptA was expressed alone (lane 8). The latter result was confirmed by mass spectrometry (data not shown) yielding similar results to those shown in EptA and LpxT are both inner membrane proteins with their active sites located in the periplasmic compartment  , lane 5 E. coli plays a role in polymyxin resistance in a cpmrA background using strain WD101. The minimal inhibitory concentrations (MICs) were determined on LB agar using ETest polymyxin B strips (pmrAc) was 40-fold more resistant to polymyxin compared with its isogenic parent, W3110. Loss of arnT, which still allows for the synthesis of single and double pEtN modifications  unable to modify its lipid A with either L-Ara4N or pEtN was slightly more resistant  than wild-type E. coli   . This slµg ml−1) . Finallyfication  and compfication . MICs wefication .E. coli, we performed a comparative analysis of the lipid A profiles of Salmonella typhimurium LT2 (wild-typeST) and its isogenic mutant CH05 (ΔSTlpxT)  . In SalmST, both 1-PP and L-Ara4N modified lipid A were easily detected, whereas pEtN modification was less evident (STlpxT (strain CH05) there was a drastic increase in lipid A modifications even in the absence of iron induction with pEtN modification predominating (3+ induction promoted L-Ara4N modification and inhibited LpxT modification in wild-type LT2 (lane 7), whereas in strain CH05 (ΔSTlpxT) pEtN modification was again increased (lane 10). The identification of Salmonella lipid A species was based upon previous reports (STarnT), CH07 (ΔSTeptA), and CH057 . The doubly modified lipid A species prevalent in the ΔSTlpxT background during Fe3+ induction were lost upon introduction of ΔSTeptA (lane 11). On the other hand, deletion of eptA in the wild-type background had little effect on the lipid A profile (lane 8). Here again, these data support a role for LpxT in the reduction of EptA activity. Furthermore, loss of functional Salmonella LpxT results in a shift from lipid A species primarily modified with L-Ara4N to modification with pEtN.In wild-type evident , lane 1.minating , lane 4. reports ; 2001; aE. coli and Salmonella, the PmrA–PmrB and PhoP–PhoQ two-component systems are primarily responsible for regulating the expression of the molecular machinery required for the structural modification of LPS. PmrA controls synthesis of both ArnT and EptA, two inner membrane enzymes that modify the phosphate groups with amine-containing substituents by the addition of L-Ara4N  and increasing the overall negative charge of the bacterial surface  were the same in both wild-type (W3110) and PmrAc (WD101) strains. These data are supported further by microarray analysis in pmrAc and pmrA null mutants where levels of lpxT transcripts did not change in cells grown in 400 µM Fe3+-containing LB medium  L-Ara4N addition drastically decreases and pEtN modification predominates, indicating the ability of LpxT to inhibit EptA modification in Salmonella as well. Previously, it was shown that EptA  did not play any significant role in providing resistance to CAMPs in S. typhimurium . Other membrane phosphatases are dedicated to the hydrolysis of Und-PP ;, and somotility . A proteE. coli K-12 strains were grown at 37°C on LB broth or agar (DIFCO), or N-minimal medium containing 0.1% casamino acids and 38 mM glycerol , kanamycin (30 µg ml−1), chloramphenicol (30 µg ml−1) and polymyxin B were used where appropriate.All relevant strains are listed in RPfuTurbo DNA polymerase (Stratagene) or Takara Ex Taq DNA polymerase (Takara). PCR products were isolated from agarose gels using QIAquick PCR gel extraction kit. Primers were purchased from Invitrogen. All restriction endonucleases and T4 DNA ligase were purchased from New England Biolab. Total RNA was extracted with Trizol Max bacterial RNA isolation kit (Invitrogen) from cells grown in LB up to an OD600 of 1.0. RNA was treated with RQ1 RNAse-free DNAse (Promega) to eliminate DNA contamination. RNA quality was evaluated by gel electrophoresis (Genomic DNA was extracted with Easy DNA Kit (Invitrogen). Plasmid DNA was purified with QIAGEN Spin Prep Kit (Stratagene). DNA fragments were amplified using phoresis . cDNA syvir phage transduction was used to move selectable deleted genes from the donor DY330 strain to the recipient W3110  according to the manufacturer's instructions with plasmid pWSlpxT as the template. Primers LpxTA190-S and LpxTA190-A were used to mutate amino acid H190 to A190 resulting in plasmid pWSlpxTH190A.Site-directed mutagenesis of 4. Membrane pellets from cells lysed by French Press were homogenized and washed twice as previously described (Cells expressing the YeiU-GFP fusion protein were grown in LB with or without 200 µM FeSOescribed  and was 600 of 0.05 in 5 ml of fresh medium and labelled with 2.5 µCi ml−132Pi (Amersham) in LB broth or N-minimal media as indicated. Cells were harvested at an OD600 of 1.0 by centrifugation and the isolation of 32P-labelled lipid A carried out by mild acid hydrolysis as previously described (32P-lipid A species (∼10 000 cpm·per lane) were analysed by TLC in a solvent system of chloroform, pyridine, 88% formic acid and water  and visualized by phosphorimaging analysis (Bio-Rad PMI).Cultures were diluted to an OD600 1.0 served as the enzyme source. Membrane pellets were homogenized and washed twice as previously described (2-[4′-32P]lipid A was prepared as previously described and served as the lipid acceptor (2-[4′-32P]lipid A (∼5000 cpm nmol−1). Endogenous undecaprenyl-pyrophosphate within the membrane fraction served as the phosphate donor. The reaction mixture was incubated at 30°C for 1 h. Reaction products were analysed by TLC in a solvent system of chloroform, pyridine, 88% formic acid, water  and visualized by phosphorimaging analysis (Bio-Rad PMI).Double-washed membrane fractions from cultures harvested at OD600 of 0.05. FeSO4 was added to starting dilutions for inducing conditions when necessary. Cells were harvested at mid-exponential phase (OD600 of 0.5–0.6) and β-galactosidase activity of bacterial lysates was determined as described by Bacteria were grown overnight in LB medium, washed in the same medium to be cultured, and diluted to an OD600 of 0.05 and applied to LB agar. Quantitative MIC values were determined using ETest gradient Polymyxin strips (AB Biodisk) after 24 h.Cells were grown overnight followed by a 1:100 dilution in LB. Cells were cultured up to exponential phase, diluted to an OD600 of ∼1.0. Lipid A was released from cells and purified as previously described (Typically, 200 ml cultures of each strain were grown at 37°C until cultures reached an ODescribed  by the U


Aspergillus and the ciliate Tetrahymena. In the former, a single Nek plays an essential role in cell cycle regulation; in the latter, which has more than 30 Neks in its genome, multiple Neks regulate ciliary length. Mammalian genomes encode an intermediate number of Neks, several of which are reported to play roles in cell cycle regulation and/or localize to centrosomes. Previously, we reported that organisms with cilia typically have more Neks than organisms without cilia, but were unable to establish the evolutionary history of the gene family.NIMA-related kinases (Neks) have been studied in diverse eukaryotes, including the fungus We have performed a large-scale analysis of the Nek family using Bayesian techniques, including tests of alternate topologies. We find that the Nek family had already expanded in the last common ancestor of eukaryotes, a ciliated cell which likely expressed at least five Neks. We suggest that Neks played an important role in the common ancestor in regulating cilia, centrioles, and centrosomes with respect to mitotic entry, and that this role continues today in organisms with cilia. Organisms that lack cilia generally show a reduction in the number of Nek clades represented, sometimes associated with lineage specific expansion of a single clade, as has occurred in the plants.This is the first rigorous phylogenetic analysis of a kinase family across a broad array of phyla. Our findings provide a coherent framework for the study of Neks and their roles in coordinating cilia and cell cycle progression. Never In Mitosis A from the fungus AspergillusAspergillus genome, plays multiple roles in cell cycle progression and localizes to the fungal equivalent of the centrosome, the spindle pole body The NIMA-related family of serine/threonine kinases (Neks) are widespread among eukaryotes. They are defined by similarity in their N-terminal catalytic domains to the founding member, Centrosomes are typically located near the nucleus in the centre of the cell, where they serve as organizers of the microtubular cytoskeleton during both interphase and mitosis Chlamydomonas and Tetrahymena regulate ciliary length and/or disassembly In mammalian cells, the elder centriole of the centrosomal pair often directly nucleates a cilium. Cilia have functions in both motility and sensory signaling Aspergillus as an ancestor to mammals. The assumption by these authors is that the last common ancestor of Fungi and Metazoa had one Nek, similar to NIMA, and this gene underwent an expansion in Metazoa and especially in mammals. This conflicts with previously published phylogenies Chlamydomonas and Tetrahymena. However, these earlier Nek phylogenies had few taxa and poor support at many nodes. We set out to establish a more complete Nek family tree, by sampling distantly-related phyla, by using Bayesian inference methods, and by testing specific hypotheses in a Bayesian framework. To our knowledge, no similar analyses have been published for a eukaryotic kinase family. Our results suggest that the last common ancestor of the eukaryotes, which must have been a ciliated cell able to divide The breadth of organisms in which Neks are found and the lack of sequence conservation outside the kinase domains has meant no large-scale phylogenetic analysis has been possible via traditional methods. Many authors refer to NIMA as “ancestral” to the mammalian Neks, yet no eukaryotic phylogeny would ever place Species from a broad range of eukaryotic lineages, with publicly-available sequenced genomes, were selected for analysis . We genebona fide Neks, as they are outgroup to a clade containing multiple eukaryotic kinase families (not shown).In the course of our BLAST searching, we found some prokaryotic kinases which were reciprocal best hits with Neks. However, these sequences do not represent 6 generations, sampled every 100 generations, and run until the average standard deviation of split frequencies between the two runs remained below the 0.01 convergence threshold for at least 1×105 generations. Ninety percent of each run was burned in before the parameters were analyzed. Human Cdk2 was used to root all trees in either Treeview We used the Metropolis-coupled Markov chain Monte Carlo program MrBayes v. 3.1.2 Chlamydomonas dataset we first analyzed the unconstrained dataset and obtained a harmonic mean of the log likelihood values of the MCMC samples (a priori imposed constraint (i.e. the hypothesis). To test hypotheses on the unikonts plus  samples . We then samples , and recThalassiosira or the green alga Ostreococcus.We use the terminology of Cavalier-Smith Leishmania and Trypanosoma, as well in the genome of Giardia  all sequences recognized as having a Nek-like kinase domain had that domain at the N-terminus of the predicted protein; when C-terminal domains were predicted by SMART, these were always protein-protein interaction domains. These results are consistent with the view that Neks have rapidly-evolving C-terminal regulatory/interaction domains, allowing differential localization, regulation, or function in various lineages.Large numbers of Neks were found in the genomes of the kinetoplastids  Giardia . InclusiChlamydomonas Neks was the largest dataset we could analyze in a reasonable time (∼1 week on a desktop computer), while inclusion of the bikont Neks to produce the tree in A dataset consisting of the unikont Neks plus the a posteriori-defined clades, each of which contains representatives from most species tested and most of which have well-supported nodes  branch near the well-supported Nek2 clade .Addition of Nek sequences from other bikonts, as well as the (unikont) chytrid fungus rahymena –7. Thek2 clade . The Nekchytrium . Satisfyeria Nek . Similareria Nek , and nowChlamydomonas Cnk10p, Tetrahymena Nrk29 and Cryptosporidium cgd7 3760 are members of a well-supported Nek1/3/5 clade (posterior probability of 0.94). This suggests that some of the clades arising from the polytomy could be orthologous to members of the unikont Nek1/3/5 clade. Above, we provided additional evidence that Cnk10p is a Nek1/3/5 family member  of unikonts and bikonts most likely had at least five Neks and variations in the numbers of Neks in different lineages was due to expansion or loss of multiple Neks; this is a novel finding and changes our view of this kinase family and the relationships of its members. Second, this work confirms and extends our previous suggestion that Neks are more abundant in the genomes of organisms which must co-ordinate ciliary assembly and disassembly with respect to the cell cycle.Currently, there is a paucity of functional data relevant to the cell biology of the Nek family. Consequently, there is insufficient data available to discern clear roles for particular Nek subfamilies from comparison of the Nek repertoires. However, the availability of the relationships between the various Neks will make future research on any particular Nek more relevant to the field at large. While most Neks that have been studied are associated with cilia/centrosomes/cell cycle, we do not propose that all Neks subserve this triumvirate. Indeed, expansion of the subfamilies was inevitably associated with both loss of function and acquisition of new roles de novo when differentiating into gametes Giardia genome has >70 Neks; Giardia flagella undergo a complex maturation process which takes four cell cycles Although we cannot discern cellular roles for individual Nek subfamilies, it is clear that Neks are not required for the formation of centrioles or cilia. Diatoms, for example, make both of these organelles Given our phylogenetic results, ciliary and centrosomal Nek functions are likely to be ancestral and thus may be retained in phylogenetically distant organisms. The wide distribution of the Neks from both unikonts and bikonts suggests strongly that the Neks had already expanded in the eukaryotic LCA. We suggest deviations of the topologies of the Nek subfamilies from our analysis from the species tree may represent different evolutionary constraints in the various lineages.y correlated with the presence of ciliated cells which divide. The cell biology of the eukaryotic LCA can be partially inferred by applying the principal of parsimony to diverse extant eukaryotes: it is thus likely that the LCA was ciliated with a 9+2 axoneme and centrioles with triplet microtubules, and that the LCA could undergo both mitosis and meiosis Our conclusion that the LCA, which must have been a non-terminally differentiated ciliated cell, had at least five Neks, should be interpreted in two ways: first, we are discerning the nature of the eukaryotic LCA, which is distinct from the first eukaryote; second, the presence of several Neks in the LCA conforms well with our idea that this kinase family is ancestrallAlthough the relationship between cilia and the cell cycle is a long-standing problem in cell biology Table S1(0.03 MB DOC)Click here for additional data file.Figure S1Phylogram of the largest and most diverse dataset containing Leishmania and Trypanosoma sequences that was able to converge. This tree is rooted on HsCdk2. Please note that the majority of Leishmania and Trypanosoma Neks are members of a well-supported clade that includes HsNek8 (posterior probability = 0.90).(1.05 MB TIF)Click here for additional data file.Figure S2Phylogram of Nek kinase domains from species listed in (1.16 MB TIF)Click here for additional data file.


Deep Sclerectomy is a non penetrating surgical procedure for the treatment of open angle glaucoma. In this article we will describe the surgical technique, the indications for surgery and will review the scientific literature on surgical outcome following this procedure. We will also discuss the important role played by antimetabolites, implants and the use of gonipuncture to achieve the desired IOP reduction. Over the past decades, non penetrating surgical procedures have been developed to improve the safety of glaucoma surgery. Deep sclerectomy (DS) has emerged as one of the more established non penetrating procedures and a growing body of evidence on its safety and efficacy has become available. Following DS, the aqueous outflow is enhanced by removing the inner wall of Schlemm's canal and juxta-canalicular trabecular meshwork, the structures responsible for most of the outflow resistance in open angle glaucoma. In this procedure a trabeculo-Descemet's membrane (TDM) is left intact to control aqueous outflow through the filtration site. This controlled pressure reduction is responsible for a better safety profile of DS with lower rate of complications related to over drainage and hypotony. The superior safety profile has been confirmed by several clinical studies and is now wildly accepted.The earliest reports on non penetrating filtering procedures were published in 1964 by KrasnovNon penetrating procedures were further refined in 1980s and 1990s. ZimmermannTo improve long term efficacy, DS has been further developed with the use of anti metabolites to reduce postoperative scarring and use of space-maintainer implants to keep the scleral lake open.Traditional glaucoma teaching places surgery as a procedure of last resort after medical and laser therapies. This is largely due to the relatively high complication rates and unpredictability of trabeculectomy.DS targets the site of highest outflow resistance in open angle glaucoma, namely the inner wall of Schlemm's canal and juxtacanalicular trabecular meshwork.12Neovascular glaucoma is an absolute contraindication to DS, as a fibro vascular membrane is formed over the irido-corneal angle making filtration through a TDM impossible. Cases of ICE syndrome are contraindicated for the same reason. A narrow angle is a relative contraindication to DS as proximity of the iris can lead to anterior synechia formation or iris incarceration following surgery. DS is also relatively contraindicated in eyes with damaged trabeculum  as surgical success relies on intact angle structures.1μm thick scleral bed. This flap should be 4 × 4 mm leaving a step for closure of the superficial flap. Defining the correct tissue plane is critical, as dissecting in this plane Schlemm's canal gets deroofed as the dissection is advanced anteriorly [The aim of DS is to create a TDM which functions as site of controlled aqueous outflow as well as to form an intrascleral space to act as a reservoir.teriorly . The TDMteriorly . The supteriorly  and 4.DS appears to be a safe surgical procedure to reduce IOP. The complication rate appears to be lower; flat anterior chamber, choroidal detachment, hypotonic maculopathy, and postoperative infections are rare.et al.DS achieves good IOP control in the early postoperative period. However, without the use of adjunctive therapies an increasing proportion fails over the long term. Khairy et al.et al. report 10-year postoperative outcomes after DCSI in 105 eyes and find 47.7% complete success (IOP less than 21 mmHg without glaucoma medications) and 88.9% qualified success (IOP less than 21 mmHg with or without glaucoma medications) with laser gonio puncture in 59.8% of patients. Five-fluotouracil (5-FU) injections were required in 24.5% of cases to treat bleb fibrosis or encapsulation.The use of implants to maintain the intrascleral space improved the long-term results.et al. study compares DS and DS augmented by MMC (0.2 mg/ml/2.5 min) with 36 months follow-up. The use of MMC has been associated with a larger IOP reduction and improved success rate, with 72.5% of DS achieving qualified success versus 95% of DS-MMC. Anandet al. study DS with low dose MMC (0.25 mg/ml/2 min) in a Nigerian population, and fail to show a significant benefit of using an antimetabolite, and had low success rate in both DS and DS MMC groups in this west African population.Several studies show that use of antimetabolites improves success rates. They act by reducing scaring at the filtration site. Kozoboliset al.21et al.et al. find no significant difference in outcomes between DS and trabeculectomy.Several randomized prospective studies compare DS with trabeculectomy, and most find only small differences in their efficacy to control IOP. El Sayyad DS has evolved over the past decades, and now long term data on its safety and efficacy is becoming available. There is consensus that complications related to over-filtration and infections are significantly lower than in trabeculectomy. The studies summarized above showed that the use of implants, antimetabolites and gonio puncture could ensure good long term IOP control.The results of El Sayyad from Saudi Arabia are encouraging for the population of that region, however Anand's results from Nigeria were disappointing for a predominantly Afro-Caribbean population due to aggressive scaring. These results are worth bearing in mind when selecting patients for the procedure from different racial backgrounds.As for any technically challenging procedure the learning curve for DS is long, can take up to 20 cases. However the most common intra-operative complication is TDM perforation, which essentially converts DS to a trabeculectomy, a procedure most surgeons would be familiar with. However we recommend DS for surgeons with a sufficiently high glaucoma surgical caseload that allows for developing and maintaining their technical skills.The role of DS in the management of open angle glaucoma is still being defined. As it appears to have a superior safety profile than penetrating procedures it could be considered at an earlier stage of the disease, especially in case of medical/laser therapy intolerance or unavailability.


Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of dioleoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two extrinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Förster resonance energy transfer (FRET) was observed between the protein and pyrene-PC and between pyrene-PC and NBD-cholesterol, leading to overlapping emission bands. Partial least squares analysis was applied to fluorescence spectra of pyrene-PC in liposomes with different DOPC/DOPA ratios, generating a model that was tested by an internal validation  and was further used to predict the apparent lipid molar ratio in AChR-containing samples. The values predicted for DOPA, the lipid with the highest Tm, indicate that the protein exerts a rigidifying effect on its lipid microenvironment. A similar conclusion was reached from excimer formation of pyrene-PC, a collisional-dependent phenomenon. The excimer/monomer ratio (E/M) at different DOPC/DOPA molar ratios revealed the restricted diffusion of the probe in AChR-containing samples in comparison to pure lipid samples devoid of protein. FRET from the AChR (donor) to pyrene-PC (acceptor) as a function of temperature was found to increase with increasing temperature, suggesting a shorter distance between AChR and pyrene PC. Taken together, the results obtained by MA on complex spectra indicate that the AChR rigidifies its surrounding lipid and prefers DOPA rather than DOPC in its immediate microenvironment. Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at different wavelengths. We applied MA to spectral data of purified PACS Codes: 32.50.+d, 33.50.Dq Torpedo postsynaptic membrane. The lipid bilayer beyond the lipid annulus has also been reported to be affected, albeit to a lesser extent, by the presence of the AChR protein . The id state , similarid state . Incorpoid state , suggestFluorimetric methods are extensively used to study the lipid organization in bilayers in combination with appropriate (intrinsic or extrinsic) fluorescent probes. Even after careful design and correct experimental performance, however, with suitable fluorescence spectra being recorded, the experimentalist is often frustrated by the paucity of information that can be extracted by direct examination of spectral data. Analysis of fluorescent spectra from complex biological systems containing various components and fluorescent probes with overlapping emission bands and/or interfering signals is therefore not an easy task . ValuablIn the present work we make use of Partial Least Squares (PLS-1)  as a mulTorpedo californica were obtained from Aquatic Research Consultants  and stored at -70°C until further use. DOPA, DOPC and Chol were obtained from Avanti Polar Lipids, Inc . NBD-Chol and cholic acid were purchased from Sigma Chemical . PyPC was obtained from Molecular Probes Inc. . Mid-range protein molecular weight markers were purchased from Promega . All other reagents were analytical quality. Lipid stock solutions were prepared in chloroform/methanol (2:1 v/v) at a concentration of 0.5 mg/ml and 0.05 mg/ml for PyPC. All solutions were stored at -20°C before use.Frozen electric organs from Torpedo californica were prepared as described previously  values in the control samples as temperature increases. These findings validate the procedure for calculating FRET and confirm that energy transfer increases with increasing temperature.In order to check that the increase in FRET = 1- enabled us to extract otherwise masked spectral fluorescence data on protein-lipid interactions under conditions known to affect the functionality of the former [reviewed in ].m (DOPA).The PLS-1 model predicted higher DOPA molar fractions (here termed "apparent") than their actual values in lipid bilayers containing the AChR. These predictions emerge from the MA applied over a selected portion of the analyzed spectra corresponding principally to the PyPC excimer band, which reports on membrane fluidity. The obtained information proved extremely difficult to extract from the raw overlapping spectra Figs.  by utilim of each lipid; -8° for DOPA) it is also unlikely that lateral phase segregation occurred at any of the assayed lipid molar ratios. Conversely, the drop in the E/M upon incorporation of the AChR indicates that the protein decreases the diffusion rate/fluidity of its surrounding lipid matrix, in agreement with the results of the apparent DOPA molar fraction experiments. E/M was also found to decrease in studies of AChR reconstituted in a single-phase DPPC bilayer and in coexisting liquid-solid bilayers composed of POPA/POPC[The series of experiments using E/M in bilayers lacking the AChR revealed no appreciable changes in the diffusion rate/fluidity with changing DOPA/DOPC molar ratios Fig. . This isPOPA/POPC.m's above and below the working temperature, this enhanced affinity could lead to segregation of PA-enriched domains from the bulk lipid. In a liquid system such as the one used in this work, where no liquid-solid phase segregation is likely to occur, the minor increase in FRET with increasing temperature can most economically be interpreted in terms of a migration and re-distribution of the probe. The mixing of lipids upon heating, and the resulting diffusion of DOPC to regions near the AChR were mirrored by the diffusion of PyPC, which partitions favorably in DOPC-enriched domains. Thus, increasing temperature causes a dispersion of DOPA and a concurrent enrichment of DOPC and PyPC molecules in the immediate vicinity of the AChR. AChR incorporation into PA-containing membranes leads to an increase in both the lateral packing densities and the Tm's of POPC/DOPA or POPC/POPA mixtures, an effect which is absent in pure POPC bilayers [m's of the lipid system. PA is required for proper receptor function [m is higher than that of non-functional bilayers composed of PC only [AChR-PyPC FRET experiments in DOPA/DOPC bilayers revealed the preference of the AChR protein for DOPA over DOPC, in agreement with previous results showing favored interactions between PA and the AChR relative to PC, PG and PS ,7,30,29.bilayers . In the bilayers . The profunction ,33, and The ability of the AChR to selectively modulate the physical properties of the bilayer could stem essentially from a hydrophobic mismatch between the AChR and the fatty acyl chains and/or polar head groups. Such mismatch could also play an important role in the modulation of AChR function. The present results, obtained by application of MA to complex spectra obtained under various fluorescence spectroscopy modalities  and exploiting the intrinsic fluorescence of the AChR protein or extrinsic fluorescent derivatives of phosphatidylcholine or cholesterol, support the more general notion that the AChR organizes its immediate environment with increased lateral packing density and rigidity. The functional counterpart of this finding is that ordered lipid membranes stabilize the receptor in a resting, activatable state, whereas fluid or disordered membranes favor a desensitized conformation of the receptor protein.m: transition temperature.MA: multivariate analysis; AChR: acetylcholine receptor; Chol: cholesterol; DOPA: dioleoylphosphatidic acid; DOPC: dioleoylphosphatidylcholine; DPPC: dipalmitoylphosphatidylcholine; DTNB: 5,5'-dithiobis (2-nitrobenzoic acid); FRET: Förster resonance energy transfer; NBD-Chol: amino)-23,24-bisnor-5-cholen-3-ol; PA: phosphatidic acid; POPC: 1-palmitoyl-2-oleoylphosphatidylcholine; PC: phosphatidylcholine; POPA: 1-palmitoyl-2-oleoylphosphatidic acid; PyPC: 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine; RMSEP: root mean square error of prediction; TReference spectra of AChR, PyPC and NBD-Chol. Fig. Click here for fileREMSEP and explained y-variance. Fig. Click here for fileFRET controls. Fluorescence emission of AChR-only and PyPC-only samples as a function of temperature. Fig. Click here for file


Trypanosoma brucei (T.b.).There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug.Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT. Trypanosoma brucei. We exploited the inability of trypanosomes to engage in de novo purine synthesis as a therapeutic target. Cordycepin was selected from a trypanocidal screen of a 2200-compound library. When administered together with the adenosine deaminase inhibitor deoxycoformycin, cordycepin cured mice inoculated with the human pathogenic subspecies T. brucei rhodesiense or T. brucei gambiense even after parasites had penetrated into the brain. Successful treatment was achieved by intraperitoneal, oral or subcutaneous administration of the compounds. Treatment with the doublet also diminished infection-induced cerebral inflammation. Cordycepin induced programmed cell death of the parasites. Although parasites grown in vitro with low doses of cordycepin gradually developed resistance, the resistant parasites lost virulence and showed no cross-resistance to trypanocidal drugs in clinical use. Our data strongly support testing cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.There is an urgent need to substitute the highly toxic arsenic compounds still in use for treatment of the encephalitic stage of African trypanosomiasis, a disease caused by infection with  Following a hemo-lymphatic stage with waves of parasitemia, the nervous system is involved manifested as a leukencephalitis, invariably lethal if left untreated. Drugs, which are effective at an early stage of the disease, but poorly penetrate the blood-brain barrier, are ineffective for the second stage. For treatment of the second encephalitic stage of HAT, arsenic compounds such as melarsoprol, which are associated with severe and even lethal side-effects, are still widely used. Moreover, there has been an alarming increase in melarsoprol-refractory sleeping sickness cases T.b. gambiense. However, this drug is given by intravenous injections, is expensive, and is not effective against T.b. rhodesiense that causes the East African form of HAT. Since the development of new drugs for HAT is not likely to occur in the immediate future, the strategy of testing drugs approved or in clinical use for other diseases should be pursued in order to identify less toxic or alternative drugs to cure second stage HAT Human African trypanosomiasis (HAT), also known as sleeping sickness, is a neglected tropical infectious disease that has re-emerged in sub-Saharan Africa in the late 1900de novo. Instead they depend on the salvage pathway of nucleosides from the body fluids of the host de novo purine synthesis has been exploited as a therapeutic target. The trypanocidal potential of cordycepin (3′-deoxyadenosine), a metabolite from the fungi Cordyceps spp., was noted in experiments performed in the 1970's in vivoIn contrast to most mammalian cells, trypanosomes cannot synthesize purines T. brucei where its intracellular cleavage to adenine is followed by phosphoribosylation to AMP, or by a high affinity adenosine kinase Trypanosoma sp.Cordycepin is probably taken up by trypanosomes through transporters. Nucleoside transport systems, P1 and P2, are able to concentrate cordycepin inside the cells and has turned out to play an important role in the uptake of trypanocides T.b. brucei infections in mice T.b. brucei. Cordycepin and deoxycoformycin treatment did also cure second stage infection with human pathogenic T.b. gambiense and T.b. rhodesiense, and oral administration of the duplet when combined with a proton pump inhibitor was effective. Cultivation of T.b. brucei with low doses of cordycepin allowed resistance to cordycepin to develop, but resistant parasites lost virulence, and no cross-resistance between cordycepin and melasoprol. suramin and pentamidine was noted. Importantly treated mice showed diminished levels of pro-inflammatory cytokines in the brain.We have previously shown that intraperitoneal (i.p.) injection of cordycepin, together with coformycin or deoxycoformycin, can cure Altogether, our findings encourage the possible use of cordycepin and deoxycoformycin as an alternative for treatment of second stage HAT or melasoprol-resistant HAT.Cordycepin, suramin, pentamidine, omeprazole and EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine) were all purchased from Sigma . Deoxycoformycin (pentostatin) was a kind gift of R. McCaffrey . Melarsoprol  was a gift from P. Simarro . Unless otherwise indicated, all reagents were diluted in PBS, fractioned and stored at −20°C until further use.The library of nucleoside analogs used consisted of compounds design to be inhibitors of various viral and microbial enzymes and is property of Medivir AB.−/−ad libitum under specific pathogen-free conditions. All experiments were conducted following protocols that received institutional approval and authorization by the Stockholm Region's animal protection committee.BALB/c, C57Bl/6 and RAG1T.b. brucei (AnTat1.1E), T.b. rhodesiense (STIB 851) and T.b. gambiense (MBA) were kindly provided by P. Büscher . Tbat1 null parasites constructed by sequential homologous recombination of T.b. brucei Lister 427 T.b. brucei, T.b. gambiense and T.b. rhodesiense freshly isolated from infected C57BL/6 mice (3 days post infection with 1×107 parasites/mouse) were separated by DEAE-cellulose chromatography under sterile conditions T. brucei parasites were incubated in D-MEM containing 10% heat-inactivated calf serum, 28 mM HEPES, 0.14% glucose, 1.5% NaHCO3, 2 mM L-glutamate, 0.14 mg/ml gentamycin, 0.3 mM dithiothreitol, 1.4 mM sodium pyruvate, 0.7 mM L-cysteine, 28 µM adenosine, 14 µM guanosine at 37°C.Bloodstream forms of 4 parasites were cultured in 96 flat-bottomed well culture plates with serial drug dilutions for 72 h at 37°C. Cultures (100 µl) were incubated for 2 h with 10 µl of WST-1 reagent . Viability was measured by the conversion of WST-1 reagent to formazan, recorded by multiwell scanning spectrophotometer at an excitation wavelength of 450 nm. A direct correlation between WST-1 reagent oxidation and parasite numbers was confirmed (data not shown).To measure drug sensitivity, 2.5×10Mice were deeply anesthetized with isoflurane, sacrificed and brains were dissected and snap frozen. To examine presence of trypanosomes within and outside the blood vessels in the brain, cryostat sections were cut and immunolabelled with anti-AnTat1.1 VSG  and goat polyclonal anti-glucose transporter 1  as described previously Renilla luciferase expressing parasites were generated as recently described . The use of such luciferase tagged T. brucei for real time studies of parasite dynamics was validated in vitro and ex vivo . BABLB/c mice were infected i.p. with 2×103 luciferase tagged T.b. brucei AnTat1.1. At different days after infection, mice were anesthetized with 2.3% isoflurane, injected intraperitoneally with 100 µL of coelenterazine (2 µg/µl dissolved in methanol) (Synchem) diluted with 90 µL PBS pH 7, and light emission in photons/second/cm2/steradian (p/sec/cm2/sr) was recorded in an IVIS Imaging System 100 (Xenogen LifeSciences) and Living Image 2.20.1 software (Xenogen) for 180 seconds. Measurements started 3–5 minutes after substrate injection to allow the spread of the coelenterazine.Stable recombinant Briefly, following a 30 min permeabilization of trypanosomes in 10 mM phosphate buffer containing 6 µM digitonin, nuclei were stained with a 10 µg/ml propidium iodide solution in 10 mM phosphate buffer and kept on ice until measurement by flow cytometry.DNA fragmentation was assessed by using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling method (Roche) as described Exposed phosphatidylserine was detected on the outer membrane of cells using Annexin-V-Fluor (Roche) following instructions by the manufacturer. Fluorescence was measured using FACS analysis as described before In order to analyze disruption of the plasma membrane after cordycepin treatment, nuclei were stained with propidium iodide (5 µg/ml) in the absence of a cell permeant and analyzed by flow cytometry.t). The relative amount of target gene transcripts was calculated using the 2−ΔΔCt method as described Gene transcripts of several pro-inflammatory cytokines were quantified in brains from cordycepin-treated and PBS-treated, uninfected and infected mice by real time PCR. Total RNA was extracted from half of the fresh frozen brains, reverse-transcribed, and the transcripts levels quantified on an ABI Prism 7000 sequence detection system (Applied Biosystems) as described previously senso latu T. brucei primers Total DNA was extracted from brains from treated and untreated mice using a Dneasy tissue kit . The amount of parasite DNA was quantified by real time PCR as described above using T. brucei genome project (www.ebi.ac.uk) and Blast2seq (www.ncbi.nlm.nih.gov). Single nucleotide polymorphisms between clones were taken into account.Total DNA from parental and resistant strains was extracted using the DNeasy tissue kit (Qiagen). Candidate genes suspected to be involved in the resistance to cordycepin were PCR amplified from total DNA using forward and reverse specific primers designed over the following annotated genes by the  project : AdenosiT.b. brucei was first studied. For this purpose we screened a library of 2200 drug-like nucleosides compounds for toxicity against T.b. brucei AnTat1.1. The screening revealed 14 hits showing toxicity for T.b. brucei at less than 1 µM concentration. Ten of these compounds were found to also be toxic for mammalian cells at these concentrations. One of the remaining compounds was identified as cordycepin. A second one was tubercidin, a nucleoside previously shown to inhibit the respiratory chain in T.b. brucei, but that induced adaptation of parasites to a glucose-independent metabolism We have previously suggested that cordycepin could be used for treatment of second stage African trypanosomiasis. Whether other nucleoside analogues could have a more selective toxic effect on T.b. brucei infections. Three out of 22 tested compounds showed at least 3 log higher toxicity for T.b. brucei as compared to mammalian HL-cells (T.b. brucei (data not shown).Subsequently, the trypanocidal activity of cordycepin was compared with that of adenosine deaminase resistant nucleoside analogues. Such nucleosides could potentially act as stand-alone drugs in treatment of in vivo. Cordycepin 116, but not cordycepin 110, showed anti-trypanosomal effect during the early stage of infection  were analyzed. Parasite DNA in whole blood or brain tissues was measured by real time PCR.Experiments were then performed to confirm, using more extended protocols, the curative effect of cordycepin and deoxycoformycin on the second stage murine infection with 3T.b. brucei. displayed parasites at 85 days of infection (65 days after treatment). No parasites were detected in the blood of cordycepin- and deoxycoformycin-treated mice. On the contrary, although no parasites were detected directly after treatment with 20 mg/kg suramin, the presence of parasites was recorded at least once in the blood of all suramin-treated mice before sacrifice (65 days after treatment), although only in a fraction of mice at a given time.In a first experiment we showed that none of the C57Bl/6 mice (n = 9) treated i.p. with 2 mg/kg body weigth (kg)/day (d) cordycepin and 0.2 mg/kg/d deoxycoformycin for 7 consecutive days, starting at day 20 after infection with 2×10−/− mice inoculated with blood or brain homogenates from suramin-treated mice showed parasites early after infection, no parasites were detected in mice inoculated with either blood or brain homogenates from cordycepin- and deoxycoformycin-treated mice. Parasite DNA could not be amplified and parasite antigens were not detected in the brain tissues of cordycepin- and deoxycoformycin-treated mice. Infected and non-treated mice showed signs of morbidity before day 32 after infection. No mortality was recorded in the cordycepin-treated group and 1 out of 8 suramin-treated mice died 79 days after infection.Whereas all RAG1In a second experiment the minimal effective concentration of cordycepin was determined. For this purpose, we compared the treatment of mice given different doses of cordycepin and a constant dose of deoxycoformycin. Mice were inoculated with 2, 1.2 and 0.6 mg/kg/d cordycepin plus 0.2 mg/kg/d deoxycoformycin for 7 days starting at day 20 after infection. Whereas the trypanocidal effect was evident in all groups of mice (n = 9), a dose-dependent effect was observed, with doses lower than 2 mg/kg/d not sufficient to completely clear parasitemia . One outNext, groups of mice (n = 7) were dosed with 2 mg/kg/d cordycepin and 0.2 mg/kg/d deoxycoformycin during 3, 5 or 7 days starting 20 days after infection. Again, a dose-dependent trypanocidal effect was observed also in this experiment. Five or three doses were not enough to completely clear infection, whereas in the group treated with 7 doses only one animal remained infected . Since, 3T.b. brucei. Treatment with either 0.1 or 0.2 mg/kg/d deoxycoformycin resulted in complete cure of second stage infection, while no curative effect was observed when 0.02 mg/kg/d deoxycoformycin was used for treatment  were treated for 10 days with 2 mg/kg/d cordycepin and different concentrations of deoxycofomycin starting 20 days after infection with 2×10reatment . We alsoStarting treatment with cordycepin and deoxycoformycin at 20 days after infection resulted in loss of weight of infected mice. Surprisingly, toxicity was largely independent of the dose of cordycepin and deoxycoformycin used, since mice treated either for 15, 10, 7, 5 or 3 days with 2 mg/kg/d cordycepin and 0.2 mg/kg/d deoxycoformycin or treated with 7 doses of 2, 1.2 or 0.6 mg/kg/d cordycepin and 0.2 mg/kg/d deoxycoformycin all showed similar weight loss . Whereas mice inoculated i.p. with 2×105T.b. gambiense (MBA stabilates) showed parasitemia up to at least 70 days after infection, mice inoculated 2×103 or 2×104T.b. gambiense showed no parasites in the blood. In contrast to T.b. rhodesiense, morbidity was only observed in T.b. gambiense-infected mice at the time of sacrifice (80 days after infection) with small numbers of parasites observed in brain parenchyma, with elevated parasite densities observed only in the septum (data not shown). Treatment of T.b. gambiense infected mice with 2 mg/kg/d cordycepin and 0.2 mg/kg/d deoxycoformycin for 10 days, starting at 30 days after infection, when parasites where first detected in the brain parenchyma, completely eliminated parasitemia for at least 50 days after treatment  of cordycepin for 72 h at 37°C. Parasites surviving the highest concentrations of cordycepin, were then further cultured for 3 days with similar or higher concentrations of cordycepin. This procedure was repeated for four months, after which a 40–60 fold increased resistance to cordycepin, compared to the parental strain was achieved  and pleomorphic (AnTat1.1) parasites displayed mutations in the TbAT1 gene, we tested whether this transporter affected the susceptibility of parasites to cordycepin by using TbAT1 knockout parasites. We found that resistance of cordycepin by knockout parasites was only slightly affected by the mutation, suggesting that the absence of the P2 transporter is not sufficient to explain the observed increased resistance of parasites to cordycepin .T. brucei. In brief, cordycepin was selected from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal effective concentrations of cordycepin and deoxycoformycin for late stage treatment in vivo were determined. Of importance, the dose of deoxycoformycin used is similar or lower to that employed in leukemia patients, and cordycepin and deoxyformycin have been tested in clinical trials in humans  and in preclinical studies in dogs at higher concentrations to the ones here presented in vivo will improve efficacy of treatment by reducing the doses, and that enteric coating might reduce the problem of acid lability of deoxycoformycin. The nucleoside analogue treatment was also effective for treatment of late stage infections with both T.b. rhodesiense and T.b. gambiense, different to some drugs in clinical use such as DFMO (eflornithine), which is only effective against T.b. gambiense. Cordycepin and deoxycoformycin treatment diminished the inflammatory cytokine accumulation in the brains of infected mice. In agreement, the number of CD45+ inflammatory cells in the brain parenchyma of T. brucei-infected mice was also reduced after treatment cordycepin and deoxycoformycin T. brucei to be similar to those described in pluricellular organisms. Programmed cell death was followed by secondary necrosis. Interestingly, programmed cell death processes have previously been shown to be activated during the life cycle of trypanosomatids, regulating parasite number in certain host tissues, in the maintenance of clonality, as a mechanism of immunomodulation and of parasite differentiation We here report a preclinical evaluation of the chemotherapeutic efficiency of cordycepin and deoxycoformycin for late stage infection with T.b. brucei developed resistance to cordycepin upon prolonged culture with low doses of the compound, such resistant parasites showed diminished virulence and reduced growth in vivo. Moreover, resistant parasites showed no cross resistance to melarsoprol, suramin and pentamidine in clinical use for early or late stage treatment of HAT, suggesting that cordycepin targets are different to those of the other drugs. This is of importance since a strategy to delay or prevent the development of resistance is to combine drugs that act on different targets and therefore are unlikely to show cross-resistance. Additionally, synergistic effects may arise from a combination of two drugs that act differently. In such a situation, the concentrations of the combination partners can be reduced compared with those used in a mono-therapy, which leads to better safety.The development of this new drug combination with reduced toxicity, high activity and oral availability gives rise to renewed hope of new and safer treatments of HAT. However, the utility and longevity of these new treatments will, to a large degree, be determined by the development of parasite resistance to the drug. We found that although Since resistance to cordycepin was slowly increased during the four months-long culture of the parasites with the drug (data not shown), it is likely that a number of mutations had accumulated during the process. After cloning and sequencing 11 candidate genes from cordycepin-induced resistant and parental strains, we found that parasites showed altered functional mutations only in the P2 transporter encoded by the TbAT1 gene. Although mutations found in the other examined genes were present, they did not show any predicted deleterious or altered effect for the respective protein function. Cordycepin uptake has been reported to be mediated by both the P1 and P2 transporters T. brucei showed two- and three-fold increases resistance to melarsoprol and melarsen Since cordycepin-resistant parasites show a genetic defect in TbAT1, it was surprising that no cross resistance to pentamidine, suramin and melarsoprol was not observed: the P2 transporter has been shown to be responsible for uptake of different trypanocides such as melaminyl arsenicals, pentamidine, diminazenes, cordycepin and tubercidin Most drugs for treatment of HAT were developed in the first half of the twentieth century, and some of them would not pass current high safety standards. A safe drug that is effective in the treatment of the encephalitic stage of HAT would significantly change the control and management of sleeping sickness. The data here presented strongly suggest that the combination of cordycepin and deoxycoformycin can be used in clinical tests for treatment of African trypanosomiasis. Besides the results from the preclinical experiments described in our manuscript, cordycepin fulfills a number of requirements for to be considered a candidate drug: It is available for other purposes and cheap, it can be given orally, clinical trials of the doublet have been performed and toxicity in man at the same concentrations used in animals seems not to pose a problem.Alternative Language Abstract S1Spanish translation of the abstract by MER.(0.04 MB DOC)Click here for additional data file.Figure S1T.b. brucei incubated with 1 µM cordycepin was measured by incubating T. brucei with TMRE.The depolarization of mitochondrial membrane in (0.59 MB PPT)Click here for additional data file.Table S1Sequencing primers list(0.06 MB DOC)Click here for additional data file.Text S1Supplementary methods.(0.03 MB DOC)Click here for additional data file.


The phenotype of an organism is an outcome of both its genotype, encoding the primary sequence of proteins, and the developmental orchestration of gene expression. The substrate of gene expression in eukaryotes is the chromatin, whose fundamental units are nucleosomes composed of DNA wrapped around each two of the core histone types H2A, H2B, H3 and H4. Key regulatory steps involved in the determination of chromatin conformations are posttranslational modifications (PTM) at histone tails as well as the assembly of histone variants into nucleosomal arrays. Although the mechanistic background is fragmentary understood, it appears that the chromatin signature of metazoan cell types is inheritable over generations. Even less understood is the conservation of epigenetic mechanisms among eukaryotes and their origins.In the light of recent progress in understanding the tree of eukaryotic life we discovered the origin of histone H3 by phylogenetic analyses of variants from all supergroups, which allowed the reconstruction of ancestral states. We found that H3 variants evolved frequently but independently within related species of almost all eukaryotic supergroups. Interestingly, we found all core histone types encoded in the genome of a basal dinoflagellate and H3 variants in two other species, although is was reported that dinoflagellate chromatin is not organized into nucleosomes.Trichoplax. H3.1 and H3.1t are probably restricted to mammals.Most probably one or more animal/nuclearid H3.3-like variants gave rise to H3 variants of all opisthokonts . H3.2 and H3.1 as well as H3.1t are derivatives of H3.3, whereas H3.2 evolved already in early branching animals, such as We deduced a model for protoH3 of the last eukaryotic common ancestor (LECA) confirming a remarkable degree of sequence conservation in comparison to canonical human H3.1. We found evidence that multiple PTMs are conserved even in putatively early branching eukaryotic taxa (Euglenozoa/Excavata).At least a basal repertoire of chromatin modifying mechanisms appears to share old common ancestry and may thus be inherent to all eukaryotes. We speculate that epigenetic principles responsive to environmental triggers may have had influenced phenotypic variation and concomitantly may potentially have had impact on eukaryotic diversification. The regulation of eukaryotic gene expression occurs in the context of DNA fibres compacted by interactions with proteins - the chromatin, where on the first level of compaction the DNA is wrapped around a protein octamer composed of the four core histone proteins types H2A, H2B, H3 and H4 forming the nucleosome. This fibre becomes further compacted by the interaction with linker histone H1 and other proteins, forming a 30 nm fibre . In eukaChromatin modifying mechanisms play a superordinate role in the orchestration of developmental processes, thus influencing phenotypic differentiation. Recent studies have also shown that some chromatin modifying mechanisms are responsive to environmental agents -14, whicHowever, the diversity of epigenetic mechanisms, their common themes as well as differences, and finally their potential impact to the evolution of eukaryotes remains largely unexplored. From all core histone types, variants of histone H3 with their dynamic posttranslational modifications have been most extensively studied in selected model organisms to date. In order to discover the evolutionary history of basal chromatin modifying mechanisms, we decided to undertake combined phylogenetic and molecular biological analyses focusing on variants of the core histone H3 and its PTM signature.Entamoeba, were usually used to represent putatively early branching eukaryotic supergroups [To date phylogenetic analyses of histone H3 variants were limited due to poor data availability of sequences from species representing putatively early branching eukaryotes. If not neglected totally parasitic organisms, such as ergroups -19. On tMonosiga brevicollis  and Arabidopsis thaliana  vary in only 2 out of 135 residues . Similarly, histone H3 from Nuclearia simplex  deviates in only 4 out of 135 residues from histone H3 in the green alga Ostreococcus lucimarinus  (~97% identity) . We focused on the identification of H3 sequences from non-parasitic organisms representing putatively early branching Euglenozoa and Excavata. Importantly, we were able to assemble multiple new histone H3 sequences from various species of putatively early branching eukaryotes . We also obtained sequences of histone H3 variants from a ciliated protist, Stylonychia lemnae   using vaInterestingly, we found extremely conserved histone H3 sequences and remarkable examples of divergent H3 variants in all eukaryotic supergroups  as well as CenH3 variants , which showed tendency to be positioned at the bottom of trees, including all characterized and putative CenH3 derivatives. Although the bootstrap support for such unrooted trees was weak for numerous clades, the monophyly of many groups was well represented , whereas the monophyly of Amoebozoa, Chromista  and Archaeplastidae could only weakly or not be resolved, probably due to the generally very high degree of H3 sequence similarity in those groups , suggesting a cell cycle dependent regulation by phosphorylation of specific H3 variants. Since phosphorylation of serine, or presumably also threonine, can prevent or even disrupt binding of effector proteins  at adjacent methylated lysine residues, it can be assumed that such sites could also function as switches regulating the chromatin signature ,27. The Xenopus, Branchiostoma, Drosophila) or slightly different protein sequence . Notably, in our phylogenetic analyses the only H3 from Nuclearia simpex we could identify occurs near the root of the animal H3.3 clade. This H3.3-like variant deviates from human H3.3 only in one substitution . In Opisthokonta S87 occurs predominantly in fungal H3 variants , whereas A87 is found in animals, choanoflagellates and most sequences of Amoebozoa. For example in two putative stem Amoebozoa species A87 is found in a H3 variant of Mastigamoeba balamuthi , whereas S87 is found in a 118 aa H3 sequence fragment of Hyperamoeba dachnya . Remarkably, S87 dominates in H3 variants of Bikonta. We conclude that one or more H3 variants very similar to animal/nuclearid H3.3 gave rise to all H3 variants found in extant opisthokonts.Our data suggest, that a variant similar to the replication-independent mammalian histone H3.3 - but not the  H3.1 - was likely ancestral to H3 variants of fungi and their sister group nuclearids as well as choanozoa and animals. In many metazoan species H3.3 occurs with identical * . However, the occurrence of numerous H3 variants is not restricted to animals. Our analyses strongly suggest that they have evolved frequently and independently in many eukaryotic taxa. For example, we characterized the macronuclear genomic sequences of at least 7 core histone H3 variants and one putative CenH3 variant (mdp64) in the spirotrichous ciliate species Stylonychia lemnae, which have been partially identified before by Bernhard [Stylonychia H3 variants are within sequence motifs known to be involved in 'writing' or 'reading' the histone PTM signature, thus determining chromatin higher order structure  and the somatic (macronuclear) genomes resulting in a selection of macronucleus-destined sequences . In spiiewed in , which ciewed in . We assuCandida albicans), Amoebozoa (3 in Entamoeba sp. and Dictyostelium discoideum), Plants , Apicomplexa/Chromista , Heterokonta/Chromista , also in excavates  and more divergent H3 variants in euglenozoa (2 in Trypanosoma sp.). Since we could identify only one H3 sequence from a Rhizaria species, we can make no statement for this group, whether H3 variants exist or not.Examples of increased H3 variant numbers within related species could be found in almost all eukaryotic supergroups by our analyses  as well as in the dinoflagellates Pyrocystis lunula and Karlodinium micrum , whereas K9/K27 motifs are absent in Karlodinium micrum and Pyrocystis lunula. Furthermore the K36 motif lacks in Pyrocystis lunula. Since the Pyrocystis H3 variant seems to be among numerous genes whose expression profiles are affected by oxidative stress [Using nuclease digestion experiments and electron microscopic approaches it has been observed, that the most portion of chromatin in dinoflagellates is not organized into nucleosomal repeats -36. In ae stress , evidencPerkinsus marinus we found also sequences encoding core histones H4 (GeneBank XM_002777579), H2A (GeneBank XM_002772145) and H2B (GeneBank XM_002787339) but no sequences homologous to other dinoflagellate histone-like proteins [Remarkably, in proteins , suggestKarlodinium micrum or Pyrocystis lunula, whereas we found one H2A-family sequence fragment in another dinoflagellate, Alexandrium tamarense (GeneBank AY849372). Our observation suggests that these histone variants are the only core histone types involved in chromatin organization in these species, raising the question what could be the consequences on chromatin structure. Without experimental evidence we can only speculate that in dinoflagellates like Karlodinium micrum or Pyrocystis lunula, which apparently do not possess a complete set of core histone types, H3 variants could be involved into chromatin organization of a fraction of the genome, similar to spermatozoa in many species, which replace most histones by protamines but retain nucleosomal chromatin organization at some genomic loci (reviewed in [On closer look we could not identify further core histone types in the genomes of iewed in ). It canPerkinsus marinus and also the evidence for single core histone types in other species strongly support the view that the alternative chromatin organization in most representatives of that phylum is a derived, not an ancestral feature in dinoflagellate chromatin evolution.However, the presence of all four core histone types in a basal dinoflagellate like Due to the presence of conserved H3 variants with high similarity to animal/nuclearid H3.3-like variants in selected species of all eukaryotic supergroups, it may be concluded that ancestral states are unlikely to be represented by the more divergent H3 variants in various supergroups. Following these assumptions we deduced ancestral states for selected clades well supported in our phylogenetic analyses and subsequently a putative protoH3 sequence, which exhibits 87% sequence identity compared with human  H3.1 Figure , confirmTrypanosoma and Leishmania is almost identical to canonical H3  alter between the aromatic amino acids tyrosine (Y) or phenylalanine (F). The observed presence or absence of putative phosphorylation sites at various positions as a character state of many histone H3 variants is nicely confirmed by the reconstruction of ancestral H3 s of all supergroups as well as LECA's protoH3 Figure . It seemEuglena gracilis (Euglenozoa/non-parasitic) and Trichomonas vaginalis (Excavata/parasitic) as representative species, since Euglena histone H3 differs in only one amino acid (A28) from human H3.1 (S28) in the N-terminal 40 residues (~98% identity). Although the N-terminal sequence identity in comparison with human H3.1 does not exceed ~78% in two H3 variants of Trichomonas vaginalis the overall similarity of these variants is higher than in other parasitic Excavata/Euglenozoa model systems, such as Giardia, Leishmania or Trypanosoma. Moreover motifs adjacent to K4, K9, K14, K27 and K36 exhibit a high degree of conservation. Importantly, we performed competition assays as controls as described in [Stylonychia lemnae, since antibodies used have been extensively tested in this single cell organism before [Euglena gracilis as well as Trichomonas vaginalis. Histone H3 acetylation at K9 or K14, which in Stylonychia occurs in the transcriptionally highly active macronuclei (M), was detected in nuclei (n) of Euglena and in most undistinguished stages observed in Trichomonas. Using antibodies targeted to H3K36ac, which in Stylonychia is restricted to developing macronuclear anlagen (a), we could not detect this PTM in Euglena, whereas signals were prominent in nuclei of most stages of Trichomonas. H3K4me3 is mostly associated with transcriptional activity as highlighted by strong macronuclear signals in Stylonychia macronuclei (M). This PTM was detected in nuclei (n) of Euglena and many undistinguished stages of Trichomonas. H3 methylated at K9 or K27 in the context of ARKS/T sequence motifs frequently propagates binding of heterochromatin binding protein 1 (HP1)-like chromobox proteins, often resulting in heterochromatin formation. In Stylonychia H3K27me3 occurs in the heterochromatic micronuclei (m), which are silent in gene expression. Using an antibody which cross-reacts with H3K9me3 and H3K27me3 we observed nuclear signals corresponding to ARKme3S/T in both Euglena and Trichomonas. The assumption that ARKme3S/T within H3 tails could be involved in heterochromatin formation even in early branching eukaryotes is strengthened by the presence of numerous HP1-like chromodomain proteins encoded in the genomes of several representative species, with at least 8 chromodomain proteins being encoded in the genome of Trichomonas vaginalis  were scanned for H3 sequences using Stylonychia lemnae macronuclear genome encoded H3 variants using degenerate oligonucleotides in combination with telomere suppression PCR (TSP), a technique to amplify the 5'- or 3'-ends of Stylonychia nanochromosomes including their telomeric sequences [Stylonychia was initiated by mixing equal numbers of cells from different mating types. Samples for cDNA synthesis were taken periodically at time points as indicated. Total RNA was isolated as described earlier [We fully characterized equences . Sexual  earlier . SubsequAligments were performed using ClustalW included in MEGA 4.1  and werePhylogenetic tree calculations were conducted using MEGA 4.1  softwareThe evolutionary history of 159 H3 and CenH3 variants Figure  was infeThe evolutionary relationship of 128 non-redundant histone H3 variants Figure  was infeAncestral states represented by selected internal nodes from clades well supported by NJ tree topology were reconstructed  or alternatively in methanol:acetic acid (3:1) (Euglena), washed twice with phosphate buffered saline (PBS), and immobilized onto poly-L-lysine coated coverslips. Subsequently immunostaining with PTM-specific antibodies and in some experiments peptide competition assays were performed as described earlier [http://rsb.info.nih.gov/ij/, 1997-2004.) and Adobe Photoshop CS3 software.Cells were fixed in 2% paraformaldehyde  and aligned protein sequences of 128 H3 variants. Amino acid positions refer to human H3.1. Identical sites are shaded in black, similar residues are shaded in light grey. The positions of four helix motifs within the histone fold domain and the putative chaperone recognition domain are marked at the top of the alignment.Click here for filePhylogenetic tree of eukaryotic life (simplified after [). The position of selected species is highlighted.ed after ). The poClick here for fileFASTA formatted histone H3 variant sequence alignment.Click here for fileOverview about the most frequent residue alterations in various ancestral state sequences of histone H3 . Variable sites are highlighted (*); symbols beneath list the most frequent amino acid variations. Outgroup taxons used for ancestral state reconstruction a displayed within brackets for each clade.Click here for fileThe alignment contains some exemplary N-terminal chromodomain sequences of putative Hp1-like proteins from putatively early branching eukaryotes, which possess a set of conserved residues (*) formally required for ARKme3S/T binding. Residues identical in 85% of all sequences are shaded black; residues similar in 85% of all sequences are shaded grey. Notably, in three Trichomonas vaginalis sequences a C-terminal chromoshadowdomain could be recognized (C+CS).Click here for file


However, treated spheroids showed an increased uptake of doxorubicin and, consequently, an increased toxicity following electrical field exposure. The electrical field raised intracellular reactive oxygen species (ROS) as revealed using 2′,7′-dichlorofluorescein diacetate (H2DCFDA) as an indicator. ROS induced membrane lipid peroxidation since the lipid peroxidation end products malondialdehyde (MDA) and 4-hydroxy-2-(E)-nonenal (4-HNE) were detected after electrical field treatment. Moreover, lipid peroxidation decreased the lipid diffusion coefficient D from 4.2 × 10−10 cm2 s−1 to 2.7 × 10−10 cm2 s−1 in the control and treated sample, respectively, as revealed by fluorescence recovery after photobleaching (FRAP) experiments. The field effects could be mimicked by incubating spheroids with 100 nM hydrogen peroxide and were inhibited by the radical scavengers dehydroascorbate (DHA) and α-tocopherol (vitamin E), indicating that the increased uptake of doxorubicin after electrical field treatment is owing to lipid peroxidation and decreased membrane lipid mobility. Treatment of tumours with low intensity electrical fields may be useful to improve the cytotoxic capacity of anthracyclines. © 1999 Cancer Research CampaignElectrochemotherapy (ECT) is a new approach to the treatment of tumours. In the present study, multicellular prostate tumour spheroids were treated with non-lethal direct current (DC) electrical fields, and uptake and toxicity of doxorubicin were investigated. An electrical field with a field strength of 500 Vm


Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal− fraction. Furthermore, the incubation of the D-gal− fraction with anti-TNF-α plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal+ activity. Overall, these results suggest that the D-gal− inhibitory effect is partially mediated by TNF-α and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal+ fraction.In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-α and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal


Owners would pay ≈400–700 CFA francs (US $0.78–$1.36)/animal. To vaccinate  Canine rabies globally causes an estimated 55,000 human deaths each year; 23,750 (≈43%) of which occur in Africa . We then estimated the maximum amount that could be charged to owners (cost recovery) and still achieve a minimum of 70% of dogs vaccinated.One way to fund dog rabies vaccination programs is to charge owners a fee for each dog vaccinated. However, the higher the fee, the lower the compliance is likely to be. To estimate the association between the amount charged to dog owners and the probability of vaccination , we collected data from 3 observational studies  and 1 survey of dog owners (2006) . Both campaigns followed similar protocols. Each campaign covered the same 3 city quarters, which had high-density dog populations (www.oanda.com/convert/classic) For each campaign, the percentage of dogs vaccinated was estimated by using a capture–recapture method . For the 2002 free-to-owners campaign, 71%–87%  of all dogs (owned and unowned) were vaccinated in 2 of the zones (1 zone per quarter) included in the campaign . For this study, we used the latter estimates because we were interested in measuring owner compliance to charges for dog vaccination.We obtained direct observations of the association between compliance  and amount charged to owners from 2 pilot dog vaccination campaigns held in N’Djaména in 2002 and 2006 . We did not perform additional statistical analyses.We graphed the 3 observational data points (assuming a straight-line interpolation between points) and the reverse cumulative probability of the owner-stated amounts that they would be willing to pay for their animal to be vaccinated against rabies . An initInterviewed households provided 356 questionnaires from which we estimated owner-stated willingness-to-pay for pet vaccination and calculated the resultant reverse cumulative probability of having their animal vaccinated. When asked how much they would be willing to pay, 5 (1%) owners stated that they were against vaccination. We interpreted that response to indicate that such owners would, essentially, have to be paid to have their animals vaccinated.<1,500 CFA francs/animal vaccinated, owners were more likely to state that they would pay to have their pet vaccinated than they were to actually do it. The stated values and observed values were closest at 2,000 CFA francs  (When the proposed cost of vaccination was cinated) . This fiFew studies have compared what members of the general public state they are willing to pay for a public health intervention with their actual observed behavior . Full-cost recovery concepts will not ensure that enough dogs are vaccinated in Chad  to interrupt rabies transmission in dogs in urban areas. Clearly, to have Owner Valuation of Rabies Vaccination ofDogs, Chad