Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena

Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2-50 μmol m-2 s-1 (total fluence of 1200 μmol m-2). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response

Origins of a cyanobacterial 6-phosphogluconate dehydrogenase in plastid-lacking eukaryotes

BackgroundPlastids have inherited their own genomes from a single cyanobacterial ancestor, but the majority of cyanobacterial genes, once retained in the ancestral plastid genome, have been lost or transferred into the eukaryotic host nuclear genome via endosymbiotic gene transfer. Although previous studies showed that cyanobacterial gnd genes, which encode 6-phosphogluconate dehydrogenase, are present in several plastid-lacking protists as well as primary and secondary plastid-containing phototrophic eukaryotes, the evolutionary paths of these genes remain elusive. ResultsHere we show an extended phylogenetic analysis including novel gnd gene sequences from Excavata and Glaucophyta. Our analysis demonstrated the patchy distribution of the excavate genes in the gnd gene phylogeny. The Diplonema gene was related to cytosol-type genes in red algae and Opisthokonta, while heterolobosean genes occupied basal phylogenetic positions with plastid-type red algal genes within the monophyletic eukaryotic group that is sister to cyanobacterial genes. Statistical tests based on exhaustive maximum likelihood analyses strongly rejected that heterolobosean gnd genes were derived from a secondary plastid of green lineage. In addition, the cyanobacterial gnd genes from phototrophic and phagotrophic species in Euglenida were robustly monophyletic with Stramenopiles, and this monophyletic clade was moderately separated from those of red algae. These data suggest that these secondary phototrophic groups might have acquired the cyanobacterial genes independently of secondary endosymbioses. ConclusionWe propose an evolutionary scenario in which plastid-lacking Excavata acquired cyanobacterial gnd genes via eukaryote-to-eukaryote lateral gene transfer or primary endosymbiotic gene transfer early in eukaryotic evolution, and then lost either their pre-existing or cyanobacterial gene

Photoactivated Adenylyl Cyclase Controls Phototaxis in the Flagellate Euglena gracilis

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNA) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S, Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no photo taxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB

Paeoniflorin ameliorates acquisition impairment of a simple operant discrimination performance caused by unilateral nucleus basalis magnocellularis lesion in rats

The effect of paeoniflorin on learning impairment of operant brightness discrimination performance　was investigated in rats with unilateral nucleus basalis magnocellularis（NBM）lesion. The animals　with unilateral NBM lesion exhibited a significant acquisition impairment of brightness discrimination task during the early phase of a training period（1-5days after starting the training session). When　administered daily during a training period，paeoniflorin significantly improved the learning impairment　of unilateral NBM-lesioned rats at 0.01 but not 0.1mg／kg／day（p.o.）. Tacrine （0.3mg／kg／day，p.o.）， a　cholinesterase inhibitor，also significantly ameliorated the learning deficit. These results suggest that　paeoniflorin improves the impairment of non-spatial learning performance caused by cholinergic　dysfunction in rats and that it may have a beneficial effect on senile dementia

Identification and characterization of a fluorescent flagellar protein from the brown alga Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae): A flavoprotein homologous to Old Yellow Enzyme

The posterior flagellum of the zoospore of the brown alga Scytosiphon lomentaria exhibits bright green autofluorescence. To identify the fluorescent flagellar substance(s), we isolated flagella from zoospores and partially purified a flavoprotein by anion-exchange and gel-filtration chromatography. Spectrofluorometric and chromatographic analyses showed that the flavoprotein had an apparent molecular mass of 41 kDa and a non-covalently bound flavin mononucleotide as a chromophore. Based on partial amino acid sequences of the protein, a cDNA of the 41-kDa flavoprotein was cloned and sequenced. The deduced amino acid sequence of the cDNA was homologous to that of the Old Yellow Enzyme family distributed in proteobacteria, yeasts and vascular plants

The protective effect of CD40 ligand–CD40 signalling is limited during the early phase of Plasmodium infection

Abstractγδ T cells are essential for eliminating Plasmodium berghei XAT. Because administration of the agonistic anti-CD40 antibody can induce elimination of P. berghei XAT parasites in γδ T cell-deficient mice, we considered that γδ T cells might activate dendritic cells via CD40 signalling during infection. Here we report that administration of the anti-CD40 antibody to γδ T cell-deficient mice 3–10days post-P. berghei XAT infection could eliminate the parasites. Our data suggest that dendritic cell activation via γδ T cells expressing CD40 ligand is critical during the early phase of infection

Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis

Photoactivated adenylyl cyclase (PAC) is a recently discovered blue-light photoreceptor that mediates photomovement in Euglena gracilis (Iseki et al., Nature, 2002, 415, 1047-1051). PAC appears to be a heterotetramer composed of two FAD-binding subunits (PACα and PACβ). Both subunits have a pair of homologous regions (F1 and F2) which show homology with prokaryotic "sensors of blue-light using FAD" (BLUF) domains. The F1 and F2 domains of PAC are the only eukaryotic BLUF domains found thus far. We obtained soluble recombinant F1 and F2 proteins in PACα by heterologous expression with fused glutathione-S-transferase (GST) in E. coli. The expressed F1 samples did not bind flavins, but the F2 samples contained both FAD and FMN with trace amounts of riboflavin. We also assembled the histidine-tagged recombinant F2 (6His-F2) from inclusion bodies in E. coli with exogenous FAD or FMN. Blue-light-induced changes in absorption spectra of these assembled samples were highly similar to those reported for prokaryotic BLUF domains. The FAD- or FMN-assembled 6His-F2 photocycled with nearly the same rate constants of light-reaction and dark-relaxation, which were slightly lower than those of GST-cleaved F2. The estimated quantum efficiency for the phototransformation was 0.28-0.32, and the half-life was 34-44 s at 25 °C for the recombinant PACα F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tl10078) to ca. 900 s (AppA). The mutated recombinant Y472F and Q514G of PACα F2 and the F2 domain of the PACα homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation

Investigating the complex X-ray spectrum of a broad-line 2MASS red quasar: XMM-Newton observation of FTM 0830+3759

We report results from a 50 ks XMM-Newton observation of the dust-reddened broad-line quasar FTM 0830+3759 (z=0.413) selected from the FIRST/2MASS Red Quasar survey. For this AGN, a very short 9 ks Chandra exposure had suggested a feature-rich X-ray spectrum and HST images revealed a very disturbed host galaxy morphology. Contrary to classical, optically-selected quasars, the X-ray properties of red (i.e. with J-Ks> 1.7 and R-Ks> 4) broad line quasars are still quite unexplored, although there is a growing consensus that, due to moderate obscuration, these objects can offer a unique view of spectral components typically swamped by the AGN light in normal, blue quasars. The XMM-Newton observation discussed here has definitely confirmed the complexity of the X-ray spectrum revealing the presence of a cold (or mildly-ionized) absorber with Nh ~10^{22} cm^-2 along the line of sight to the nucleus and a Compton reflection component accompanied by an intense Fe K emission line in this quasar with a Lum(2-10) ~5 x 10^{44} erg/s. A soft-excess component is also required by the data. The match between the column density derived by our spectral analysis and that expected on the basis of reddening due to the dust suggests the possibility that both absorptions occur in the same medium. FTM 0830+3759 is characterized by an extinction/absorption-corrected X-ray-to-optical flux ratio alphaox = -2.3, that is steeper than expected on the basis of its UV luminosity. These findings indicate that the X-ray properties of FTM 0830+3759 differs from those typically observed for optically-selected broad line quasars with comparable hard X-ray luminosity.Comment: Accepted for publication in Ap