Sex-inducing effects toward planarians widely present among parasitic flatworms

Summary Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.journal articl

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

<div><p>Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm²), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca²⁺-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors.</p></div

Involvement of Akt activation in UVA-induced IL-6 production.

<p>(A) Cells were pre-incubated with wortmannin (500 nM) or LY294002 (5 μM) for 30 min, and irradiated with 2.5 J/cm². After incubation for 24 h, the culture supernatants were harvested and the concentration of IL-6 was measured. Each value represents the mean ± SD (n = 4). (B, C) Cells were irradiated with 2.5 J/cm² and incubated for the indicated times (B). Cells were pre-incubated with MRS2211 (100 μM) for 30 min, and irradiated with 2.5 J/cm², and then proteins were extracted after incubation for 10 min (C). In each experiment, phosphorylation of Akt was detected by immunoblotting as described in Materials and Methods. Equal protein loading was confirmed using anti-Akt antibody. The results are typical of those obtained in three independent experiments. (D) Cells were pre-incubated with suramin (100 μM), MRS2211 (100 μM) or LY294002 (5 μM) for 30 min, and stimulated with ADP (100 μM). After incubation for 24 h, the culture supernatants were harvested and the concentration of IL-6 was measured. Each value represents the means ± SD (n = 4). Significant differences between the irradiated or ADP-treated control groups and the indicated groups are indicated by *** (P<0.001).</p

Involvement of P2 receptors in UVA-induced IL-6 production.

<p>Effects of P2 receptor antagonists on IL-6 production. (A) Cells were pre-incubated with PPADS (100 μM), 5-BDBD (2 μM), oxATP (100 μM), suramin (100 μM), MRS2179 (100 μM), MRS2578 (100 μM), NF157 (50 μM), clopidogrel (30 μM) or MRS 2211 (100 μM) for 30 min, and irradiated with 2.5 J/cm². (B) Cells were pre-incubated with NF157 (50 μM) and/or MRS 2211 (100 μM) for 30 min, and irradiated with 2.5 J/cm². In each experiment, the culture supernatants were harvested after incubation for 24 h and the concentration of IL-6 was measured. Each value represents the mean ± SD (n = 8–16). Significant differences between the irradiated control groups and the indicated groups are indicated by *** (P<0.001). (C) Cells were irradiated with 2.5 J/cm² and incubated for 24 h. Protein expression of P2Y11 and P2Y13 receptors in non-irradiated cells and irradiated cells was compared by means of immunoblotting.</p

Involvement of elevation of intracellular Ca²⁺ in UVA-induced IL-6 production.

<p>Cells were pre-incubated with (A) U73122 (10 μM) or BAPTA-AM (50 μM) or (B) SQ22536 (10 μM), and irradiated with 2.5 J/cm². After incubation for 24 h, the culture supernatants were harvested and the concentration of IL-6 was measured. Each value represents the mean ± SD (n = 4). (C) Cells loaded with Fluo-4 were irradiated with 2.5 J/cm² in the absence or presence of NF 157 (50 μM). The fluorescence was analyzed with a fluorometer at the indicated times. Each value represents the mean ± SD (n = 6). (D) Cells were pre-incubated with suramin (100 μM), NF 157 (50 μM), U73122 (10 μM) or BAPTA-AM (50 μM), and stimulated with ATP (100 μM). After incubation for 24 h, the culture supernatants were harvested and the concentration of IL-6 was measured. Each value represents the mean ± SD (n = 4). Significant differences between the irradiated or ATP-treated control groups and the indicated groups are indicated by ** (P<0.01) and *** (P<0.001).</p

Effect of UVA irradiation on cell viability in HaCaT cells.

<p>Cells were irradiated with 2.5–10 J/cm² UVA and incubated for 24 h. At the end of incubation, cell viability was determined by MTS assay as described in Materials and Methods. Each value represents the mean ± SD (n = 6). Significant differences between the irradiated groups and the non-irradiated groups are indicated by *** (P<0.001).</p

ATP release from UVA-irradiated HaCaT cells.

<p>(A) Cells were irradiated with 2.5 J/cm² UVA and then incubated for the indicated times. (B) Cells were irradiated with 2.5 J/cm² UVA after pretreatment with GdCl₃ (50 μM), glibenclamide (100 μM), bafilomycin A1 (50 nM), CBX (20 μM), or AZ11645373 (10 μM) for 30 min, and culture supernatants were collected at 1 min after UVA irradiation. (C) Cells were irradiated with 2.5 J/cm² UVA after pretreatment with FFA (50 μM), LaCl₃ (200 μM), probenecid (500 μM), or trovafloxacin (30 μM) for 30 min, and culture supernatants were collected at 1 min after UVA irradiation. In each experiments, the concentration of ATP was measured as described in Materials and Methods; each value represents the mean ± SD (n = 8–12). Significant differences between the time point 0 or irradiated control groups and the indicated groups are indicated by ** (P<0.01) and *** (P<0.001). (D) Intracellular localization of Cx43 and PANX1 were detected by immunostaining (green, Cx43 or PANX1; blue, nuclei). (E) Cells were irradiated with 2.5 J/cm² and incubated for 24 h. Protein expression of P2X7 receptor, Cx43 and PANX1 in non-irradiated and irradiated cells was compared by means of immunoblotting.</p

Involvement of released ATP in UVA-induced IL-6 production.

<p>(A) Cells were pre-incubated with apyrase (10 U/mL), CBX (20 μM), FFA (50 μM), probenecid (500 μM), LaCl₃ (200 μM), trovafloxacin (30 μM) or AZ11645373 (10 μM) for 30 min, and irradiated with 2.5 J/cm². (B) Cells were treated with 1–100 μM ATP at 1 min after UVA irradiation (2.5 J/cm²). After incubation for 24 h, the culture supernatants were harvested and the concentration of IL-6 was measured. Each value represents the mean ± SD (n = 4). Significant differences between the irradiated control groups and the indicated groups are indicated by *** (P<0.001). Significant differences between the control groups and the ATP-treated groups are indicated by # (P<0.001).</p

One-Pot Synthesis of Imines from Nitroaromatics and Alcohols by Tandem Photocatalytic and Catalytic Reactions on Degussa (Evonik) P25 Titanium Dioxide

Photoirradiation (λ > 300 nm) of Degussa (Evonik) P25 TiO₂, a mixture of anatase and rutile particles, in alcohols containing nitroaromatics at room temperature produces the corresponding imines with very high yields (80–96%). Other commercially available anatase or rutile TiO₂ particles, however, exhibit very low yields (<30%). The imine formation involves two step reactions on the TiO₂ surface: (i) photocatalytic oxidation of alcohols (aldehyde formation) and reduction of nitrobenzene (aniline formation) and (ii) condensation of the formed aldehyde and aniline on the Lewis acid sites (imine formation). The respective anatase and rutile particles were isolated from P25 TiO₂ by the H₂O₂/NH₃ and HF treatments to clarify the activity of these two step reactions. Photocatalysis experiments revealed that the active sites for photocatalytic reactions on P25 TiO₂ are the rutile particles, promoting efficient reduction of nitrobenzene on the surface defects. In contrast, catalysis experiments showed that the anatase particles isolated from P25 TiO₂ exhibit very high activity for condensation of aldehyde and aniline, because the number of Lewis acid sites on the particles (73 μmol g^–1) is much higher than that of other commercially available anatase or rutile particles (<15 μmol g^–1). The P25 TiO₂ particles therefore successfully promote tandem photocatalytic and catalytic reactions on the respective rutile and anatase particles, thus producing imines with very high yields