10CM短柱快速分离3-MH的实验研究

本研究在改进后短程序基础上,对氨基酸分离柱进行了改进。改进后的分离柱长为10cm。比原来20cm长柱分离3—MH的时间缩短了近1/2。实验所得的(回收率为97.59%,分离度0.89±0.02。变异系数1.17)这些指标较国外用其它方法所得的结果有良好的相关性。多次测定结果说明长柱与短柱比较无明显差异。证明了短柱对3—MH含量无影响。这一改进所建立的方法大大地缩短了样品的分析时间,节约了大量进口试剂,开展这方面的工作将有利益提高严重烧伤、创伤后蛋白质代谢和营养学等方面的研究水平。     

绿色木霉组蛋白去乙酰化酶编码基因TvRpd3在木霉生物防治作用中的功能分析

【目的】本文借助基因编辑技术在具有生物防治潜力的绿色木霉(Trichoderma viride)中敲除组蛋白去乙酰化酶编码基因TvRpd3,来研究TvRpd3基因及其编码蛋白在提高木霉病原菌拮抗能力中的作用。【方法】利用融合PCR和同源重组策略构建了TvRpd3基因缺失的突变菌株,通过对峙培养、表型观察、免疫组化检测、代谢组学分析等系统比较TvRpd3基因敲除前后菌株的组蛋白乙酰化修饰水平、次级代谢产物合成、病原菌拮抗能力以及田间防治效果等。【结果】与野生型菌株相比,缺失TvRpd3基因的木霉工程菌(?TvRpd3)对多种病原菌表现出了更强的对峙抑制效果,其所产的发酵液对小麦白粉病、烟草黑胫病和番茄枯萎病的防治效果分别提高了62.27%、57.45%和70.71%。同时,敲除TvRpd3基因也显著改变了木霉工程菌所产次级代谢产物的种类和产量,抗生性物质的产量大幅提高。【结论】绿色木霉TvRpd3基因及其介导的组蛋白乙酰化修饰在提高绿色木霉生物防治中起着重要作用。     

Calcineurin B-like interacting protein kinase 31 confers resistance to sheath blight via modulation of ROS homeostasis in rice

Sheath blight (ShB) severely threatens rice cultivation and production; however, the molecular mechanism of rice defence against ShB remains unclear. Screening of transposon Ds insertion mutants identified that Calcineurin B-like protein-interacting protein kinase 31 (CIPK31) mutants were more susceptible to ShB, while CIPK31 overexpressors (OX) were less susceptible. Sequence analysis indicated two haplotypes of CIPK31: Hap_1, with significantly higher CIPK31 expression, was less sensitive to ShB than the Hap_2 lines. Further analyses showed that the NAF domain of CIPK31 interacted with the EF-hand motif of respiratory burst oxidase homologue (RBOHA) to inhibit RBOHA-induced H₂O₂ production, and RBOHA RNAi plants were more susceptible to ShB. These data suggested that the CIPK31-mediated increase in resistance is not associated with RBOHA. Interestingly, the study also found that CIPK31 interacted with catalase C (CatC); cipk31 mutants accumulated less H₂O₂ while CIPK31 OX accumulated more H₂O₂ compared to the wild-type control. Further analysis showed the interaction of the catalase domain of CatC with the NAF domain of CIPK31 by which CIPK31 inhibits CatC activity to accumulate more H₂O₂.     

Loss of DNA repair mechanisms in cardiac myocytes induce dilated cardiomyopathy

Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1^−/D mice). Ckmm-Cre^+/−;Ercc1^−/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre^+/−;Ercc1^−/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre^+/-;Ercc1^−/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre^+/−;Ercc1^−/fl and Ercc1^−/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.     

Role of pulmonary epithelial arginase-II in activation of fibroblasts and lung inflammaging

Elevated arginases including type-I (Arg-I) and type-II isoenzyme (Arg-II) are reported to play a role in aging, age-associated organ inflammaging, and fibrosis. A role of arginase in pulmonary aging and underlying mechanisms are not explored. Our present study shows increased Arg-II levels in aging lung of female mice, which is detected in bronchial ciliated epithelium, club cells, alveolar type 2 (AT2) pneumocytes, and fibroblasts (but not vascular endothelial and smooth muscle cells). Similar cellular localization of Arg-II is also observed in human lung biopsies. The age-associated increase in lung fibrosis and inflammatory cytokines, including IL-1β and TGF-β1 that are highly expressed in bronchial epithelium, AT2 cells, and fibroblasts, are ameliorated in arg-ii deficient (arg-ii^−/−) mice. The effects of arg-ii^−/− on lung inflammaging are weaker in male as compared to female animals. Conditioned medium (CM) from human Arg-II-positive bronchial and alveolar epithelial cells, but not that from arg-ii^−/− cells, activates fibroblasts to produce various cytokines including TGF-β1 and collagen, which is abolished by IL-1β receptor antagonist or TGF-β type I receptor blocker. Conversely, TGF-β1 or IL-1β also increases Arg-II expression. In the mouse models, we confirmed the age-associated increase in IL-1β and TGF-β1 in epithelial cells and activation of fibroblasts, which is inhibited in arg-ii^−/− mice. Taken together, our study demonstrates a critical role of epithelial Arg-II in activation of pulmonary fibroblasts via paracrine release of IL-1β and TGF-β1, contributing to pulmonary inflammaging and fibrosis. The results provide a novel mechanistic insight in the role of Arg-II in pulmonary aging.     

Effects of Allelochemicals on Root Growth and Pod Yield in Response to Continuous Cropping Obstacle of Peanut

Continuous cropping (CC) obstacle is a major threat in legume crops production; however, the underlying mechanisms concerning the roles allelochemicals play in CC obstacle are poorly understood. The current 2-year study was conducted to investigate the effects of different kinds and concentrations of allelochemicals, p-hydroxybenzoic acid (H), cinnamic acid (C), phthalic acid (P), and their mixtures (M) on peanut root growth and productivity in response to CC obstacle. Treatment with H, C, P, and M significantly decreased the plant height, dry weight of the leaves and stems, number of branches, and length of the lateral stem compared with control. Exogenous application of H, C, P, and M inhibited the peanut root growth as indicated by the decreased root morphological characters. The allelochemicals also induced the cell membrane oxidation even though the antioxidant enzymes activities were significantly increased in peanut roots. Meanwhile, treatment with H, C, P, and M reduced the contents of total soluble sugar and total soluble protein. Analysis of ATPase activity, nitrate reductase activity, and root system activity revealed that the inhibition effects of allelochemicals on peanut roots might be due to the decrease in activities of ATPase and NR, and the inhibition of root system. Consequently, allelochemicals significantly decreased the pod yield of peanut compared with control. Our results demonstrate that allelochemicals play a dominant role in CC obstacle-induced peanut growth inhibition and yield reduction through damaging the root antioxidant system, unbalancing the osmolytes accumulation, and decreasing the activities of root-related enzymes.

     

“DNA signaturing” database construction for Tetradesmus species identification and phylogenetic relationships of Scenedesmus-like green microalgae (Scenedesmaceae,Chlorophyta)

Species identification of Scenedesmus-like microalgae, comprising Desmodesmus, Tetradesmus, and Scenedesmus, has been challenging due to their high morphological and genetic similarity. After developing a DNA signaturing tool for Desmodesmus identification, we built a DNA signaturing database for Tetradesmus. The DNA signaturing tool contained species-specific nucleotide sequences of Tetradesmus species or strain groups with high similarity in ITS2 sequences. To construct DNA signaturing, we collected data on ITS2 sequences, aligned the sequences, organized the data by ITS2 sequence homology, and determined signature sequences according to hemi-compensatory base changes (hCBC)/CBC data from previous studies. Four Tetradesmus species and 11 strain groups had DNA signatures. The signature sequence of the genus Tetradesmus, TTA GAG GCT TAA GCA AGG ACCC, recognized 86% (157/183) of the collected Tetradesmus strains. Phylogenetic analysis of Scenedesmus-like species revealed that the Tetradesmus species were monophyletic and closely related to each other based on branch lengths. Desmodesmus was suggested to split into two subgenera due to their genetic and morphological distinction. Scenedesmus must be analyzed along with other genera of the Scenedesmaceae family to determine their genetic relationships. Importantly, DNA signaturing was integrated into a database for identifying Scenedesmus-like species through BLAST.     

色氨酸阻遏物与其操纵基因复合物的圆二色性研究

用圆二色性谱来确定色氨酸阻遏物与其操纵基因复合物在溶液中的构象变化。色氨酸阻遏物的圆二色性图谱表明它是一种富含α螺旋的蛋白质,通过比较加入Ｌ－Ｔｒｐ前和后的圆二色性谱,发现活性的与非活性的色酸阻遏物二级结构变化很小,ｔｒｐ Ｐ／Ｏ的圆二色性谱与它的单健、双链理论计算曲线对比,可近拟推测操纵基因在溶液中的存在状态。用色氨酸阻遏物对ｔｒｐ Ｐ／Ｏ进行确定,可以从中找出蛋白质与核酸结合的平衡点。从平衡时