Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

© 2014 Fujikawa et al. Background: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Results: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. Conclusions: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome

ONO-1301, a Sustained-Release Prostacyclin Analog, Ameliorates the Renal Alterations in a Mouse Type 2 Diabetes Model Possibly Through Its Protective Effects on Mesangial Cells

Diabetic nephropathy is the most common pathological disorder predisposing patients to end-stage renal disease. Considering the increasing prevalence of type 2 diabetes mellitus worldwide, novel therapeutic approaches are urgently needed. ONO-1301 is a novel sustained-release prostacyclin analog that inhibits thromboxane A2 synthase. Here we examined the therapeutic effects of the intermittent administration of slow-release ONO-1301 (SR-ONO) on diabetic nephropathy in obese type 2 diabetes mice, as well as its direct effects on mesangial cells. The subcutaneous injection of SR-ONO (3mg/kg) every 3 wks did not affect the obesity or hyperglycemia in the db/db obese mice used as a model of type 2 diabetes, but it significantly ameliorated their albuminuria, glomerular hypertrophy, glomerular accumulation of type IV collagen, and monocyte/macrophage infiltration, and also the increase of TGF-β1, α-smooth muscle actin (α-SMA) and MCP-1 compared to vehicle treatment. In cultured mouse mesangial cells, ONO-1301 concentration-dependently suppressed the increases in TGF-β, type IV collagen, α-SMA, MCP-1 and fibronectin induced by high ambient glucose, at least partly through prostacyclin (PGI2) receptor-mediated signaling. Taken together, these results suggest the potential therapeutic efficacy of the intermittent administration of SR-ONO against type 2 diabetic nephropathy, possibly through protective effects on mesangial cells

Citrus sudachi Peel Extract Suppresses Cell Proliferation and Promotes the Differentiation of Keratinocytes through Inhibition of the EGFR–ERK Signaling Pathway

Citrus sudachi is a well-known fruit in Tokushima Prefecture, Japan, and its peels are rich in phytochemicals, including phenolic compounds. Although it is expected that the extract of the C. sudachi peel elicits various beneficial physiological activities, the effect on the skin has not been investigated. In this study, we report that the aqueous extract from the peel of C. sudachi suppresses cell proliferation of the immortalized human keratinocyte cell line, HaCaT, and primary normal human epidermal keratinocytes. The extract of C. sudachi peel suppressed epidermal growth factor (EGF)-induced EGF receptor activation and tumor necrosis factor (TNF)-α-induced extracellular regulated kinase (ERK) 1/2 activation, which suggests that the extract exerts its inhibitory effect through inhibition of both the EGF receptor (EGFR) and its downstream molecules. Additionally, the extract of C. sudachi peel potentiated calcium-induced keratinocyte differentiation. These results suggest that the extract of C. sudachi peel may have beneficial effects against skin diseases that are characterized by hyperproliferation of epidermal keratinocytes, such as those seen in psoriasis and in cutaneous squamous cell carcinoma

Renal Distribution of Vasohibin-1 in Patients with Chronic Kidney Disease

Experimental studies have demonstrated the involvement of angiogenesis-related factors in the progression of chronic kidney disease (CKD). There have so far been no reports investigating the distribution and clinical roles of Vasohibin-1 (VASH-1), a negative feedback regulator of angiogenesis, in CKD. We recruited 54 Japanese CKD patients and 6 patients who had normal renal tissues excised due to localized renal cell carcinoma. We evaluated the correlations between the renal expression level of VASH-1 and the clinical/histological parameters. VASH-1 was observed in renal endothelial/mesangial cells, crescentic lesions and interstitial inflammatory cells. Significant positive correlations were observed between 1) crescent formation and the number of VASH-1+ cells in the glomerulus (r＝0.48, p＝0.001) or cortex (r＝0.64, p＜0.0001), 2) interstitial cell infiltration and the number of VASH-1+ cells in the cortex (r＝0.34, p＝0.02), 3) the glomerular VEGFR-2+ area and the number of VASH-1+ cells in the glomerulus (r＝0.44, p＝0.01) or medulla (r＝0.63, p＝0.01). These results suggest that the renal levels of VASH-1 may be affected by local inflammation, crescentic lesions and VEGFR-2

慢性関節リウマチをもつ者の在宅におけるセルフケアの実態

在宅で生活する慢性関節リウマチ患者のセルフケア行動を知るために質問紙調査を行った。また、病気の受容がセルフケアに与える影響を知るためにクロス集計を行い比較分析した。その結果多くのリウマチ患者はライフスタイルを守り生活する行動、病気治療への対処行動、自己概念の修正行動に積極的であった一方、セルフケア資源の活用範囲は狭いことが明らかになった。また病気の受容は上記のいずれの行動にも影響していると考えられた