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The aim of the study is to clarify the participation of extracellular vesicles (EV) secreted by murine primary retinal pigment epithelial (mpRPE) cells in the cell to cell communication with macrophages (Mps), firstly described by the authors in 2016. In ocular inflammation, Mps act as sources of tumor necrosis factor-α (TNF-α), an activator of RPE cells. TNF-α stimulates the production of monocyte chemotactic protein (MCP-1) by RPE cells, thereby causing greater recruitment of Mps to the sub-RPE space. Murine RAW 264.7 Mps cells were co-cultured with C57BL/6 mouse mpRPE cells, either together or separated in transwells, vertically or horizontally connectable, with 0.40 or 0.03 μm membrane filters. The association of EV with mpRPE or RAW 264.7 was quantified by fluorescence cell sorting (FACS) using Qdot655 streptavidin-conjugated biotinylated EV. Increased levels of CD63

The aim of the study is to clarify the participation of extracellular vesicles (EV) secreted by murine primary retinal pigment epithelial (mpRPE) cells in the cell to cell communication with macrophages (Mps), firstly described by the authors in 2016. In ocular inflammation, Mps act as sources of tumor necrosis factor-α (TNF-α), an activator of RPE cells. TNF-α stimulates the production of monocyte chemotactic protein (MCP-1) by RPE cells, thereby causing greater recruitment of Mps to the sub-RPE space. Murine RAW 264.7 Mps cells were co-cultured with C57BL/6 mouse mpRPE cells, either together or separated in transwells, vertically or horizontally connectable, with 0.40 or 0.03 μm membrane filters. The association of EV with mpRPE or RAW 264.7 was quantified by fluorescence cell sorting (FACS) using Qdot655 streptavidin-conjugated biotinylated EV. Increased levels of CD63+ EV were detected in co-cultures by western blotting or FACS analysis, in accordance with the increased production of nanoparticles (50–150 nm) detected by Nanosight tracking analysis. The gene expressions of cytokines, MCP-1, IL-6, IL-8, and VEGF in mpRPE cells and the corresponding proteins were increased in co-cultures even in transwells, vertically connected with 0.40 μm membrane filters, while the repressed TNF-α protein production was not affected. Most of the CD63+ EVs produced by mpRPE cells in co-cultures were associated with Raw264.7, but not with mpRPE cells. Semi-purified CD63+ EV secreted from mpRPE cells, increased the secretion of MCP-1, IL-6, and VEGF in co-cultures with RAW 264.7. Culture chamber separation horizontally connected with 0.03 μm membrane filters reduced this increased secretion. Collectively, mpRPE derived CD63+ EV partly participate in the sub-retinal innate inflammation. To evaluate the functional damage of RPE cells upon chronic exposure to here defined EVs will be the critical issue to uncover their roles in chronic retinal degenerative diseases.

Purpose The purpose of this study was to clarify the interactions between macrophages (MPs) and RPE cells in coculture systems to investigate the functional plasticity of RPE cells. Methods Adherent peritoneal cells or murine MP cell line Raw 264.7 was cocultured with primary RPE cells taken from C57BL/6 mice, with or without lipopolysaccharide (LPS) or TNF-α stimulation. The cytokine levels of the culture supernatants (CSs) were then analyzed with the Bio-Plex murine 23-Plex Panel Assay Kit (Bio-Rad Laboratories). Monocyte chemoattractant protein-1 (MCP-1), IL-6, VEGF, and TNF-α in CS were further quantified by ELISA. The expression profiles, in cocultures, of complement-associated genes, TNF-α, and angiogenesis-associated genes were analyzed by quantitative real-time PCR. Results The production of MCP-1, IL-6, and VEGF was synergistically elevated when primary MPs or RAW264.7 cells and RPE cells were cocultured compared with those derived from sole cultures of MPs and RPE cells. The synergistic effect was confirmed without direct cell contact and was more prominent in the presence of LPS or TNF-α. TNF-α production by MPs was suppressed by RPE cells. Coculture of RPE cells with RAW264.7 cells increased the gene expression of C3, CFB, and VEGF genes, whereas it reduced those of complement regulatory factors CFH, CD59, clusterin, and TNF-α and antiangiogenic pigment epithelium-derived factor (PEDF). Conclusions Our findings indicate the presence of ingenious interactions between MPs and RPE cells that forces the inflammation and complement activation in the vicinity of RPE cells under pathologic conditions.

The factors affecting the phytotoxicity of imazosulfuron on rice plants such as an application time, flooding water depth, temperature after application, water leaching and transplanting depth were studied under greenhouse conditions. Applications of imazosulfuron ranging from 75g a.i./ha to 150g a.i./ha caused no significant difference in dry weight of rice plants irrespective of variation in application time between 1 day and 13 days after transplanting or in flooding water depth between 1cm and 5cm. No significant reduction in dry weight of rice plants transplanted at a depth of -0.5cm or 2cm was recognized irrespective of temperature (20°C/13°C, 26°C/19°C, 32°C/25°C; day/night) after treatment application of 100g a. i./ha. There was no significant difference in dry weight between the -0.5cm and the 2cm transplanting depths under any temperature condition, although the dry weight of rice plants transplanted at the -0.5cm depth under the highest temperature was significantly different from the untreated control. When either basal leaf sheaths or root tips of the rice plants were exposed to 90g a.i./ha of imazosulfuron, the dry weights of the shoots were significantly different from untreated control. With water leaching at the rate of 2cm/day for 3 days after applications, the dry weight of rice plants transplanted at the 1cm depth, which was significantly different from the untreated control, was reduced more than those transplanted at the 3cm depth by application of 150g a.i./ha.

Factors affecting the herbicidal activity of imazosulfuron against weeds in paddy fields and movement of this herbicide in paddy soil were studied by bioassay method under greenhouse conditions. Reduction of more than 90% in dry weight was observed in Cyperus serotinus, Scirpus juncoides var. ohwianus and Echinochloa oryzicola treated with 90g a.i./ha of imazosulfuron for 1 day or more, 7 days or more and 14 days or more, respectively. Application of 90g a.i./ha at the sprouting stage of Sagittaria pygmaea suppressed the growth in dry weight irrespective of tuber size. However, at 3g a.i./ha growth of this plant sustained less reduction with increasing tuber size. Differences in flooding water depth and temperature had no appreciable influence on the herbicidal activity. Growth inhibition of S. juncoides var. ohwianus by pre-emergence or 1.5-leaf stage applications under nonleaching conditions was significantly higher than that under water leaching conditions. Under non-leaching condition, there was no remarkable difference in this inhibition between pre-emergence and 1.5-leaf stage applications, while under water leaching conditions the growth reduction with the latter application was significantly larger. Growth inhibition of S. juncoides var. ohwianus and S. pygmaea planted at a depth of 0.5cm and 2cm, respectively, was significantly higher than that planted deeper with the preemergence applications. With application at the 2-leaf stage of S. juncoides var. ohwianus, however, the difference in planting depth did not significantly affect the herbicidal activity. Bioassay method showed that movement in paddy soil, where the water leaching was conducted at the rate of 1.5cm/day from the 4cm flooding water depth following application of 150g a.i./ha, was limited to within 2cm of the soil layer under the soil surface.

イミダゾ [1,2-a] ピリジン環を有するスルホニル尿素系化合物の畑地雑草に対する除草活性を調べたところ, 縮合環上2位にメチルスルホニル基を有する化合物が興味ある活性を示した. そこでその類縁化合物を合成し, それらの除草活性を調べた. また, 類縁体合成の過程で鍵中間体の簡便な合成法を見出した. 合成したスルホニル尿素系化合物の中で1-(2-エチルスルポニルイミダゾ [1,2-a] ピリジン-3-イルスルポニル)-3-(4,6-ジメトキシピリミジン-2-イル) ウレア (TKM-19) は出芽前, 出芽後処理のどちらでも高い除草活性を示し, 小麦に対して選択性を有していた. また, このものは既存の除草剤では防除が困難なウマノチャヒキに対しても卓効を示した.