目的研究二硫键对溶菌酶和肽基脯氨酰顺反异构酶(PPI)蛋白交联聚集作用的影响。方法以人总RNA为模板设计引物进行人类PPI基因的克隆,以获取的正常型表达载体为模板,利用PCR定点突变的方法破坏掉PPI基因中形成二硫键的两个半胱氨酸残基(C...目的研究二硫键对溶菌酶和肽基脯氨酰顺反异构酶(PPI)蛋白交联聚集作用的影响。方法以人总RNA为模板设计引物进行人类PPI基因的克隆,以获取的正常型表达载体为模板,利用PCR定点突变的方法破坏掉PPI基因中形成二硫键的两个半胱氨酸残基(Cys),将其构建到p Trc His C-hpp I原核表达系统,经异丙基硫代半乳糖苷(IPTG)进行诱导表达获得正常型和突变型PPI蛋白,与溶菌酶在不同温度、还原剂二硫苏糖醇(DTT)存在条件下进行蛋白聚集反应,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western印迹法分析和鉴定交联情况。结果溶菌酶和PPI在临界变性温度时有明显的二聚体和多聚体条带;反应体系中加入DTT后,几乎观察不到交联条带,两者的异源蛋白交联反应出现同样的现象;将突变型PPI置于与正常型PPI完全相同的反应条件下,均没有任何蛋白聚集现象。结论二硫键是蛋白质交联和多聚化发生的前提,破坏二硫键将导致蛋白结构和交联作用的丧失。展开更多
Aim To study the effects of binuclear copper （Ⅱ） threonine complex （Cu2 （Thr）4） as analogue of superoxide dismutase （SOD） on blood glucose, blood lipids and vessels of hearts and kidneys in diabetic mice. Met...Aim To study the effects of binuclear copper （Ⅱ） threonine complex （Cu2 （Thr）4） as analogue of superoxide dismutase （SOD） on blood glucose, blood lipids and vessels of hearts and kidneys in diabetic mice. Methods Diabetic mouse model was established by intraperitioneal injection of alloxan. Low, middle, and high doses of Cu2（Thr）4 at 0.002%, 0.02% and 0.1% were given respectively to diabetic mice following lavage. The fasting blood glucose was determined after the diabetic mice were given Cu2 （Thr）4 for 0, 30, and 45 d. The diabetic mice were killed on the 45th day. Then glycosylated hemoglobin （HbAlc） and blood lipids were assayed, and pathologic changes in hearts and kidneys stained with HE were observed. Results Compared with the control group in which the diabetic mice were given distilled water, the value of blood glucose reduced significantly in middle dose group （P 〈 0.01 ）, followed by that in low dose group （P 〈 0.05）. TC level reduced markedly and HDL level increased significantly in all three treatment groups （P 〈 0.05）. Especially in middle dose group, cardiac muscle fibers were neatly arranged, nucleus and cytoplasm well distributed, glomeruli showing normal structure, cells well distributed and staining being normal. Conclusion Cu2 （Thr）4 reduces blood glucose, regulates blood lipids, and play protective action on the vessels of hearts and kidneys in diabetic mice. The effects of it in middle dose were better than those of other doses.展开更多
A 12-week feeding trial was conducted to evaluate the effects of dietary stachyose on the growth performance, digestive enzymes activities and intestinal structures of juvenile turbot(Scophthalmus maximus L). Five iso...A 12-week feeding trial was conducted to evaluate the effects of dietary stachyose on the growth performance, digestive enzymes activities and intestinal structures of juvenile turbot(Scophthalmus maximus L). Five isonitrogenous(49.58% crude protein) and isolipidic(10.50% crude lipid) diets were formulated to contain 0(Control), 0.625%(S-0.625), 1.25%(S-1.25), 2.5%(S-2.5) and 5%(S-5) stachyose, respectively. With the increase of stachyose level, the growth performance and feed utilization of turbot, such as the specific growth rate, final mean body weight, weight gain rate and feed efficiency, increased significantly(P < 0.05) and then stabilized. The feed intake of fish fed S-5 was significantly higher(P < 0.05) than that of fish in other groups. The activities of trypsin, intestinal caseinolytic, stomach and intestinal amylase were significantly influenced by stachyose(P < 0.05). The highest values of trypsin and intestinal caseinolytic activities were observed in group S-1.25, while the highest activity of stomach amylase and the lowest activity of intestine amylase were observed in group S-5. No lesion or damage was found on the distal intestine structures of fish from all treatments, while the height of simple folds in the distal intestine was significantly increased(P< 0.05) when 1.25% or 2.5% stachyose was added in the diets. These results indicated that moderate level of stachyose(1.25%) improves the growth performance, feed utilization, digestive enzyme activities and the distal intestine structures of juvenile turbot.展开更多
Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on...Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively.Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY (P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL (P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes'expression was more sensitive to dietary PA than to SBS under the current experimental conditions.展开更多
文摘目的研究二硫键对溶菌酶和肽基脯氨酰顺反异构酶(PPI)蛋白交联聚集作用的影响。方法以人总RNA为模板设计引物进行人类PPI基因的克隆,以获取的正常型表达载体为模板,利用PCR定点突变的方法破坏掉PPI基因中形成二硫键的两个半胱氨酸残基(Cys),将其构建到p Trc His C-hpp I原核表达系统,经异丙基硫代半乳糖苷(IPTG)进行诱导表达获得正常型和突变型PPI蛋白,与溶菌酶在不同温度、还原剂二硫苏糖醇(DTT)存在条件下进行蛋白聚集反应,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western印迹法分析和鉴定交联情况。结果溶菌酶和PPI在临界变性温度时有明显的二聚体和多聚体条带;反应体系中加入DTT后,几乎观察不到交联条带,两者的异源蛋白交联反应出现同样的现象;将突变型PPI置于与正常型PPI完全相同的反应条件下,均没有任何蛋白聚集现象。结论二硫键是蛋白质交联和多聚化发生的前提,破坏二硫键将导致蛋白结构和交联作用的丧失。
文摘Aim To study the effects of binuclear copper （Ⅱ） threonine complex （Cu2 （Thr）4） as analogue of superoxide dismutase （SOD） on blood glucose, blood lipids and vessels of hearts and kidneys in diabetic mice. Methods Diabetic mouse model was established by intraperitioneal injection of alloxan. Low, middle, and high doses of Cu2（Thr）4 at 0.002%, 0.02% and 0.1% were given respectively to diabetic mice following lavage. The fasting blood glucose was determined after the diabetic mice were given Cu2 （Thr）4 for 0, 30, and 45 d. The diabetic mice were killed on the 45th day. Then glycosylated hemoglobin （HbAlc） and blood lipids were assayed, and pathologic changes in hearts and kidneys stained with HE were observed. Results Compared with the control group in which the diabetic mice were given distilled water, the value of blood glucose reduced significantly in middle dose group （P 〈 0.01 ）, followed by that in low dose group （P 〈 0.05）. TC level reduced markedly and HDL level increased significantly in all three treatment groups （P 〈 0.05）. Especially in middle dose group, cardiac muscle fibers were neatly arranged, nucleus and cytoplasm well distributed, glomeruli showing normal structure, cells well distributed and staining being normal. Conclusion Cu2 （Thr）4 reduces blood glucose, regulates blood lipids, and play protective action on the vessels of hearts and kidneys in diabetic mice. The effects of it in middle dose were better than those of other doses.
基金supported by the Research Fund for the Doctoral Program of Higher Education of China(No.20120132120025)National Program on Key Basic Re-search Project(973 Program,2014CB138600)+1 种基金the Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province(No.BS2013 SW007)Modern Agro-Industry Technology Research System(No.nycytx-50-G07)
文摘A 12-week feeding trial was conducted to evaluate the effects of dietary stachyose on the growth performance, digestive enzymes activities and intestinal structures of juvenile turbot(Scophthalmus maximus L). Five isonitrogenous(49.58% crude protein) and isolipidic(10.50% crude lipid) diets were formulated to contain 0(Control), 0.625%(S-0.625), 1.25%(S-1.25), 2.5%(S-2.5) and 5%(S-5) stachyose, respectively. With the increase of stachyose level, the growth performance and feed utilization of turbot, such as the specific growth rate, final mean body weight, weight gain rate and feed efficiency, increased significantly(P < 0.05) and then stabilized. The feed intake of fish fed S-5 was significantly higher(P < 0.05) than that of fish in other groups. The activities of trypsin, intestinal caseinolytic, stomach and intestinal amylase were significantly influenced by stachyose(P < 0.05). The highest values of trypsin and intestinal caseinolytic activities were observed in group S-1.25, while the highest activity of stomach amylase and the lowest activity of intestine amylase were observed in group S-5. No lesion or damage was found on the distal intestine structures of fish from all treatments, while the height of simple folds in the distal intestine was significantly increased(P< 0.05) when 1.25% or 2.5% stachyose was added in the diets. These results indicated that moderate level of stachyose(1.25%) improves the growth performance, feed utilization, digestive enzyme activities and the distal intestine structures of juvenile turbot.
基金financially supported by grant from the National Natural Science Foundation of China(No.31072219)
文摘Soybean stachyose (SBS) and phytic acid (PA) are anti-nutritional factors (ANF) which have deleterious effects on the growth and digestibility in fish. The present research studied the effects of dietary SBS and PA on the expression of three serine protease genes in the liver of Japanese flounder (Paralichthys olivaceus). These genes are trypsinogen 1 (poTRY), elastase 1 (poEL) and chymotrypsinogen 1 (poCTRY). Eight artificial diets with graded levels of supplemented ANFs were formulated to 4 levels of SBS (0.00, 0.40, 0.80 and 1.50%), 4 levels of PA (0.00, 0.20, 0.40 and 0.80), respectively.Japanese flounder (initial weight 2.45 g ± 0.01 g) were fed with these diets for 10 weeks with three replications per treatment. At the end of 10 weeks, supplementation of 0.40% of dietary SBS or PA significantly increased the gene expression of poTRY and poCTRY (P<0.05). The same level of dietary SBS significantly decreased the gene expression of poEL. In comparison with the control group (ANF-free),dietary PA (0.2% and 0.8%) significantly decreased the gene expression of poTRY, poCTRY and poEL (P<0.05). However, excessive supplement of dietary SBS (1.5%) has no significant effects on these gene expressions (P>0.05). These results suggested that dietary SBS and dietary PA could directly affect the serine protease genes at the transcriptional level in Japanese flounder, and these genes'expression was more sensitive to dietary PA than to SBS under the current experimental conditions.
