Brassinolide induces stem growth of water spinach (Ipomoea aquatica Forsk.)

This report is a preliminary study to clarify the effects of exogenous brassinolide (BL) on the stem tissue growth of water spinach. Local varieties in Taiwan were used for the indoor experiments. The clones were propagated and grown under fluorescent lamps for plant growth, and the clones with 5 leaves were cut off and grown in a water culture for 7 days. Distilled water was used for diluting the concentration of BL, and the BL concentrations used were 0, 1, 2.5, 5, and 10 ppb (w/v). Each clone was cultured in 80 ml of the water solution. The plant height and stem diameter at the centre of the first internode above the point of cutting were measured after 5 days of treatment. The stem diameter and pith thickness increased significantly as the BL concentration in the water culture increased, whereas the cortex thickness was not affected by the BL concentration. The cavity diameters in the 5 and 10 ppb plots were significantly larger than those in the other plots. There was no significant difference in plant height among the treatments, although the plant height tended to be higher with increasing BL concentration.Article信州大学農学部AFC報告 11（1-2）: 1-4(2013)departmental bulletin pape

Effects of fruit skin and water temperature during soaking before germination on the emergence rates of common buckwheat

The studies focused effects of the presence of fruit skin (hull or pericarp) and water temperature during water soaking before germination on the emergence rates of common buckwheat (Fagophyrum esculentum Moench) after seeding. We aimed to understand the mechanisms underlying the poor emergence rates that have been observed after water flooding before germination. Shinano No.1 was exposed to water soaking treatments at temperatures of 10,15,20, or 25℃ for 3 days. After soaking, the seeds were grown at 20℃, and the percentages of emerged seedlings were investigated 7 days after seeding. Dehulled seeds and normal seeds were soaked for 4 days at 25℃, and the contents of dissolved oxygen were measured in the water. The emergence rates of normal intact seeds (fruits)decreased significantly (p＜0.01)with an increase in soaking water temperature before germination. The emergence rates of dehulled seeds improved after removing the fruit skin, and it reached 70%,even at 25℃.Soaking treatment for 96 h at 25℃ decreased the emergence rates of normal seeds and dehulled seeds. The emergence rate of normal seeds was significantly lower than that of dehulled seeds (p＜0.001). Moreover, there was no difference in the emergence rates of dehulled seeds between plots with or without daily water replacement. The contents of dissolved oxygen in the water at 25℃ did not differ between normal seeds and dehulled seeds after 50h. These results suggested that high temperatures affected embryo viability and that the relative mechanical resistance of the fruit skin on the embryo directly affected the seed emergence rate.Article信州大学農学部AFC報告 13: 113-118 (2015)departmental bulletin pape

Influence of organic fertilizer on rhizome yield and α-tocopherol content of Codonopsis lanceolata

Codonopsis lanceolata (Sieb. et Zucc.) Trautv. is a valuable wild vegetable in East Asian countries, especially Korea. We investigated effects of organic fertilizer application on rhizome yield and quality under Andosol soil conditions in the AFC field of Shinshu University. Commercial barnyard manure, which was fermented with crushed bark and beef cattle dung, and Japanese oak leaf mold were applied at the rates of 0, 5, 10, 15, and 20 g m-2. Barnyard manure increased the fresh weight yield of rhizomes but decreased the DL-α-tocopherol (vitamin E) content. The rhizome yield was drastically influenced by the C/N ratio of the soil from the surface to 30 cm below ground level that was controlled by the application ratio of barnyard manure to leaf mold. Total N, soluble P, exchangeable K, Ca, and Mg did not significantly affect the rhizome yield. There was a significant negative correlation between the rhizome fresh weight and vitamin E content at harvest time. The highest vitamin E content of 9-10 mg 100 g-1 dry matter was obtained at a leaf mold application rate of 10 g m-1. Nitrogen supply from the soil to plants primarily influenced the yield and quality of rhizomes as vegetables or drugs. The vitamin E content under a higher soil C/N ratio was the highest among commercial vegetables available in common Japanese food markets.Article信州大学農学部AFC報告 10: 1-7(2012)departmental bulletin pape

Dual Programmed Cell Death Pathways Induced by p53 Transactivation Overcome Resistance to Oncolytic Adenovirus in Human Osteosarcoma Cells

Tumor suppressor p53 is a multifunctional transcription factor that regulates diverse cell fates, including apoptosis and autophagy in tumor biology. p53 overexpression enhances the antitumor activity of oncolytic adenoviruses; however, the molecular mechanism of this occurrence remains unclear. We previously developed a tumor-specific replication-competent oncolytic adenovirus, OBP-301, that kills human osteosarcoma cells, but some human osteosarcoma cells were OBP-301-resistant. In this study, we investigated the antitumor activity of a p53-expressing oncolytic adenovirus, OBP-702, and the molecular mechanism of the p53-mediated cell death pathway in OBP-301-resistant human osteosarcoma cells. The cytopathic activity of OBP-702 was examined in OBP-301-sensitive (U2OS and HOS) and OBP-301-resistant (SaOS-2 and MNNG/HOS) human osteosarcoma cells. The molecular mechanism in the OBP-702-mediated induction of two cell death pathways, apoptosis and autophagy, was investigated in OBP-301-resistant osteosarcoma cells. The antitumor effect of OBP-702 was further assessed using an orthotopic OBP-301-resistant MNNG/HOS osteosarcoma xenograft tumor model. OBP-702 suppressed the viability of OBP-301-sensitive and -resistant osteosarcoma cells more efficiently than OBP-301 or a replication-deficient p53-expressing adenovirus (Ad-p53). OBP-702 induced more profound apoptosis and autophagy when compared with OBP-301 or Ad-p53. E1A-mediated miR-93/106b upregulation induced p21 suppression, leading to p53-mediated apoptosis and autophagy in OBP-702-infected cells. p53 overexpression enhanced adenovirus-mediated autophagy through activation of damage-regulated autophagy modulator (DRAM). Moreover, OBP-702 suppressed tumor growth in an orthotopic OBP-301-resistant MNNG/HOS xenograft tumor model. These results suggest that OBP-702-mediated p53 transactivation is a promising antitumor strategy to induce dual apoptotic and autophagic cell death pathways via regulation of miRNA and DRAM in human osteosarcoma cells. Mol Cancer Ther; 12(3); 314-25

Preclinical evaluation of telomerase-specific oncolytic virotherapy for human bone and soft tissue sarcomas

骨・軟部肉腫は, 一部に治療抵抗性で予後の悪い症例が存在するため, 新たな治療法の確立が重要な課題である.　我々は, 5型アデノウイルスを基本骨格として, テロメラーゼ活性に依存して増殖する腫瘍融解ウイルス（OBP-301）や, coxsackie and adenovirus receptor（CAR）陰性の腫瘍細胞に感染するファイバー改変型ウイルス（OBP-405）を用い, 骨・軟部肉腫細胞に対する抗腫瘍効果を検討した.　 　14種類の骨・軟部肉腫細胞株に対してOBP-301の細胞障害活性を検討し, 12種類の細胞株でOBP-301に感受性を認めた.　また, OBP-301の細胞障害活性はCARの発現と相関していた.　さらに, テロメラーゼ活性の低い細胞に対しても, 5型アデノウイルスの複製に必須のE1Aによりテロメラーゼ活性の増強効果がおこり, 強い抗腫瘍活性を示すことを明らかにした.　次に, 骨肉腫脛骨同所性移植動物モデルを作成しOBP-301を投与したところ, OBP-301投与群では対象群と比べて有意に腫瘍増殖を抑制した.　最後に, OBP-301に感受性を認めなかったCAR陰性細胞株に対してOBP-405を用いて検討し, OBP-405が有効に作用することを確認した.　 　OBP-301やOBP-405を用いたウイルス療法は, 骨・軟部肉腫に対する新たな治療法となる可能性がある.

Mechanism of resistance to trastuzumab and molecular sensitization via ADCC activation by exogenous expression of HER2-extracellular domain in human cancer cells

Trastuzumab, a humanized antibody targeting HER2, exhibits remarkable therapeutic efficacy against HER2-positive breast and gastric cancers; however, acquired resistance presents a formidable obstacle to long-term tumor responses in the majority of patients. Here, we show the mechanism of resistance to trastuzumab in HER2-positive human cancer cells and explore the molecular sensitization by exogenous expression of HER2-extracellular domain (ECD) in HER2-negative or trastuzumab-resistant human cancer cells. We found that long-term exposure to trastuzumab induced resistance in HER2-positive cancer cells; HER2 expression was downregulated, and antibody-dependent cellular cytotoxicity (ADCC) activity was impaired. We next examined the hypothesis that trastuzumab-resistant cells could be re-sensitized by the transfer of non-functional HER2-ECD. Exogenous HER2-ECD expression induced by the stable transfection of a plasmid vector or infection with a replication-deficient adenovirus vector had no apparent effect on the signaling pathway, but strongly enhanced ADCC activity in low HER2-expressing or trastuzumab-resistant human cancer cells. Our data indicate that restoration of HER2-ECD expression sensitizes HER2-negative or HER2-downregulated human cancer cells to trastuzumab-mediated ADCC, an outcome that has important implications for the treatment of human cancers