Effect of initial substrate and biomass concentrations on anaerobic fermentative hydrogen produc
法国海岸松树皮提取物碧萝芷对长链游离脂肪酸诱导的巨噬细胞perilipin2基因表达的影响
法国海岸松树皮提取物碧萝芷对长链游离脂肪酸诱导的巨噬细胞perilipin2基因表达的影响范斌;杜强;谷剑秋;张锦【摘要】Abstract Objective To investigate the effect of Pycnogenol on oleic acid-induced perilipin2 expression in macrophages. Methods Realtime PCR and Western blot were performed to detect perilipin2 expression. Transient transfection and luciferase assay were employed to measure perilipin2 promoter activity. Results Oleic acid significantly induced perilipiti2 expression in a dose-and time-dependent manner in macrophages; oleic acid markedly enhanced perilipin2 promoter activity; Pycnogenol significantly suppressed oleic acid-induced perihpin2 expression and promoter activity. Conclusion For the first time,we demonstrated that Pycnogenol significantly suppressed oleic acid-induced perilipin2 expression and promoter activity.%目的研究碧萝芷(PYC)对油酸诱导的巨噬细胞perilipin2表达的影响及其相关分子机制.方法应用Real-time PCR 和Western blot测定油酸及PYC对perilipin2 mRNA和蛋白水平表达影响.应用荧光素酶活性分析方法检测油酸及PYC时perilipin2启动子活性的影响.结果油酸以剂量和浓度依赖方式上调perilipin2 mRNA和蛋白水平表达,并促进perilipin2启动子活性.PYC以剂量依赖方式抑制了油酸诱导的perilipin2表达及启动子活性.结论PYC抑制了巨噬细胞中油酸诱导的perilipin2的表达.PYC通过抑制perilipin2启动子活性,从而直接抑制perilipin2的表达.【期刊名称】《中国医科大学学报》【年(卷),期】2011(040)007【总页数】5页(P611-615)【关键词】长链游离脂肪酸;泡沫细胞;碧萝芷;PAT家族蛋白【作者】范斌;杜强;谷剑秋;张锦【作者单位】中国医科大学附属盛京医院神经内科,沈阳110004;中国医科大学附属盛京医院内分泌科,沈阳110004;中国医科大学附属第一医院内分泌科,沈阳110001;中国医科大学附属第一医院内分泌科,沈阳110001【正文语种】中文【中图分类】R363单核细胞通过内皮细胞的间隙移入内膜下并分化成巨噬细胞，巨噬细胞吞噬体内过剩的脂质后变成泡沫细胞，泡沫细胞聚集并形成斑块是动脉粥样硬化发生、发展的主要病理基础［1，2］。
纤维素酶的CMC酶活测定条件的研究
网络出版时间：2012-11-06 14:59网络出版地址：/kcms/detail/11.1759.TS.20121106.1459.006.html纤维素酶的CMC酶活测定条件的研究孙盈田永强赵丽坤(兰州交通大学化学与生物工程学院，甘肃兰州730070)摘要：本实验研究和分析了CMC酶活性测定法的实验条件。
通过单因素实验对酶促反应时间、酶促反应温度、pH、测定波长、底物浓度、粗酶液和底物添加量六个影响因素进行了研究。
最后结合正交实验、方差分析和多重比较,确定了纤维素酶活力测定的最佳条件组合。
结果表明，在pH6.2、粗酶液和底物添加量各为2mL的情况下，影响纤维素酶活力测定的主次因素依次为酶促反应时间、酶促反应温度、测定波长和底物浓度。
纤维素酶活力测定的最佳条件为：在酶促反应温度为40℃，pH6.2，底物浓度为10g/L，粗酶液和底物添加量各为2mL，酶促反应时间30min，测定波长为520nm。
关键词：纤维素酶，酶活力测定，条件Study on The Optimum Conditions of Determination of Cellulase ActivitySun Ying Tian Yongqiang Zhao Likun(School of Chemical and Biological Engineering, Lanzhou Jiaotong University，Lanzhou 730070,China)Abstract: The experimental conditions of CMC enzyme activity assay were researched. Six influencing factors:enzymatic reaction time, reaction-temperature, pH, detective wavelength, substrate concentration, crude enzymeand substrate addition were studied by a single factor test. Finally, orthogonal experiment, variance analysis andmultiple comparisons were married together, and the optimum conditions of determination of CMC enzymeactivity were determined. The results showed that, when the volume of crude enzyme and substrate were 2mL andpH6.2, the primary and secondary factors which affected cellulase activity were enzymatic reaction time, reactiontemperature, enzymatic determination of wavelength and substrate concentration. Experiments showed that: theenzymatic reaction temperature 40℃, pH6.2, substrate concentration 10g/L, the volume of crude enzyme andsubstrate were 2mL, enzymatic reaction time 30min, and the detective wavelength at 520nm were the optimumconditions for the determination.Key words: Cellulase; the determination of enzyme activity; condition中国分类号：TS207.3 文献标识码：A纤维素是地球上最古老、最丰富的天然高分子，也是自然界中分布最广、含量最多的一种多糖，是取之不尽用之不竭、人类最宝贵的天然可再生资源，占植物界碳含量的50%以上[1]。
大黄素治疗类风湿关节炎
大黄素治疗类风湿关节炎发布时间：2023-02-22T08:45:15.571Z 来源：《医师在线》2022年32期作者：郑若男[导读] 类风湿性关节炎（RA）是一种慢性、全身性和自身免疫性疾病，其主要病理变化是炎症细胞浸润伴有多种相关细胞因子的分泌和积累，诱发软骨和骨组织的破坏。
因此，炎症细胞和细胞因子的调节是控制RA炎症的关键治疗靶点。
本综述详细介绍了大黄素对T淋巴细胞、树突状细胞和调节性T细胞分化和成熟的影响。
郑若男黑龙江中医药大学黑龙江哈尔滨 150000摘要：类风湿性关节炎（RA）是一种慢性、全身性和自身免疫性疾病，其主要病理变化是炎症细胞浸润伴有多种相关细胞因子的分泌和积累，诱发软骨和骨组织的破坏。
因此，炎症细胞和细胞因子的调节是控制RA炎症的关键治疗靶点。
本综述详细介绍了大黄素对T淋巴细胞、树突状细胞和调节性T细胞分化和成熟的影响。
此外，系统引入大黄素直接或间接影响促炎细胞因子（TNF-α、IL-6、IL-1、IL-1β、IL-17、IL-19和M-CSF）和抗炎细胞因子（IL-4、IL-10、IL-13和TGF-β的分泌），通过多种炎性细胞因子的共调节来抑制RA炎症并促进恢复。
详细了解大黄素治疗RA的潜在机制，为大黄素今后的临床应用提供了系统的理论依据。
关键词：大黄素；类风湿关节炎；综述1 大黄素的基础生物学中药大黄和根瘤被称为“指挥官”，最早记录在经典书籍“神农草药经典”中，大约在2000年前在中国临床上使用。
本代表中药泻药可用于净化治疗凝冷、清热火、化血化瘀、降血消黄、清热、利尿[1-2].大黄素是一类最近备受关注的生物活性天然产物。
大黄素具有多种生物调节功能，如免疫抑制和抗肿瘤、抗氧化和抗炎活性.因此，大黄素在心血管系统、呼吸系统、代谢系统、神经系统和其他系统的疾病中具有治疗潜力。
1.1 大黄素的药代动力学大黄素的主要代谢途径是葡萄糖醛酸化代谢，其次是磺化代谢[3].大黄素以葡糖苷酸或硫酸酯的形式存在于血浆、肾脏和肺中，以游离形式存在于肝脏中的大黄素[4].1.2使用大黄素治疗人类疾病在口腔疾病中，大黄素降低外周血和牙龈组织中的一氧化氮（NO）水平，抑制炎症反应和牙槽骨吸收[5].在肝病中，大黄素可以通过下调丙氨酸氨基转移酶（ALT）、甘油三酯和天冬氨酸氨基转移酶的水平来改善乙醇介导的肝脂肪变性并治疗酒精性肝病[6].关于氧化应激损伤，大黄素发挥潜在的抗氧化作用，例如调节自由基和活性氧（ROS）水平并影响氧化应激诱导的损伤[7].在心血管疾病中，大黄素通过抑制醛糖还原酶活性和改善视网膜血管生成对糖尿病视网膜病变发挥潜在的治疗作用[8].1.3免疫细胞中大黄素的调节大黄素的免疫调节功能主要影响T淋巴细胞、树突状细胞（DC）和调节性T细胞（Tregs）的分化和成熟以及多种促炎和抗炎细胞因子的分泌，以达到免疫调节作用[9].2 大黄素通过调节炎性细胞因子影响 RA 进程大黄素的免疫调节作用可能部分归因于其对淋巴细胞的抗增殖作用及其对 TH1/TH2 和 TH17/Treg 平衡的调节 [10].为了抑制炎症，大黄素降低血浆中TNF-ɑ和IL-6的水平，并抑制PGE（2）的产生，PGE（2）是COX-2在滑膜组织中的蛋白质表达[11]以及抗炎细胞因子IL-4，IL-10，IL-13，IL-11和TGF-β在细胞内外的协同作用。
牛蒡子提取物对阿尔兹海默病模型小鼠学习记忆障碍的改善作用
・基础论著・ 牛蒡子提取物对阿尔兹海默病模型小鼠学习记忆障碍的改善作用肖五一1王月1邵天萌1郑涵1张晓宇1王双2【摘要】 目的 研究牛蒡子提取物（FAE）对阿尔兹海默病（AD）模型小鼠学习记忆的影响及机制。
方法50只小鼠随机分为5组，分别为阴性对照组、模型对照组、阳性药奥拉西坦（OC）组（每天800 mg/kg）、FAE高低剂量组（每天800 mg/kg、400 mg/kg），除阴性对照组外各组小鼠皆建立D-半乳糖-氯化铝复合AD模型，每天腹腔注射D-半乳糖100 mg/kg灌胃氯化铝10 mg/kg，实验组在建模第15天开始灌胃给药。
建模60 d后对小鼠进行跳台实验、水迷宫实验等行为学检测并测定脑组织中超氧化物歧化酶（SOD）、总氧化能力（T-AOC）、丙二醛（MDA）、钙离子浓度（[Ca2+]）、一氧化氮（NO）和一氧化氮合酶（NOS）含量，数据采用SPSS软件进行单因素方差分析。
结果与模型组比较，FAE组小鼠跳台实验潜伏期延长、游出迷宫时间缩短、水迷宫及跳台实验中错误次数减少（F=11.07，P=0.004；F=8.13，P=0.011；F=6.25，P=0.02），生化指标显示脑组织中MDA、NO、NOS及[Ca2＋]含量与模型组比较明显降低（F=5.12，P=0.04，F=4.49，P=0.05，F=4.55，P=0.05，F=4.62，P=0.05）；而SOD、T-AOC的活性显著提高（F=6.49，P=0.02；F=4.76，P=0.05）。
结论FAE对AD模型小鼠学习记忆障碍具有改善作用。
其机制与提高抗氧化酶活性，减少自由基的产生及NO的神经损伤有关。
【关键词】 牛蒡子提取物； 阿尔茨海默病； 学习记忆； 模型Effect of Fructus arctii extract on learning and memory deficit in Alzheimer disease model of miceXiao Wuyi1, Wang Yue1, Shao Tianmeng1, Zheng Han1, Zhang Xiaoyu1, Wang Shuang2. 1College ofEnvironment & Chemical Engineering, Yanshan University, Qinhuangdao 066004, China; 2School of LifeScience and Bio-engineering, Beijing University of Technology, Beijing 100124, ChinaCorresponding author: Zhang Xiaoyu, Email: xyzhang05@; W ang Shuang, Email: wangshuang01@【Abstract】 Objective To investigate the effect of Fructus arctii extract on Alzheimer disease(AD) model of mice and its mechanism. Methods Fifty healthy male Kunming mice were randomlydivided into 5 groups, respectively, the negative control group, AD model group, Oxiracetam Capsules(400 mg/kg) and Fructus arctii extract (FAE 800 mg/kg, 400 mg/kg). The AD model was intraperitoneallyinjected with D-galactose 100 mg/kg and aluminum trichloride 10 mg/kg by intragastric administrationevery day. Since the 15th day of AD model developed, the mice in experimental groups wereintragastric administrated with the corresponding dosage each day for 45 days. Behavioral testing inexposed and control mice were developed using step-down assays and water maze test, and the chemicalparameters SOD, T-AOC, MDA, [Ca2+], NO and NOS of brain tissue were measured. Differences amonggroups were compared by SPSS statistics software using ANOV A. Results Compared with model group,the latency period in step-down assays was statistically lengthened and the whole swimming time in watermaze test was reduced in the FAE groups, and the rate of errors was decreased in step-down tests(F=11.07, P=0.004. F=8.13, P=0.011, F=6.25, P=0.02). The chemical parameter showed that the levels ofMDA, NO, NOS and [Ca2＋] in brain tissue were significantly decreased in the FAE group (F=5.12, P=0.04F=4.49, P=0.05, F=4.55, P=0.05, F=4.62, P=0.05) and the activity of the SOD and T-AOC (F=6.49,P=0.02. F=4.76, P=0.05) increased greatly. Conclusions The FAE can protect the deficit of learning andmemory of the AD model mice. The mechanisms of effects may be related to the improvement of enzymes,reduce the formation of free radical; resulting in prevention of peroxide generation and reduction ofDOI:10.3877/cma.j.issn.1674-0785.2016.23.017作者单位：066004河北秦皇岛，燕山大学环境与化学工程学院1；100124北京工业大学生命科学与生物工程学院2通讯作者：张晓宇，Email: xyzhang05@；王双，Email: wangshuang01@NO-related neurological damage.【Key words】Fructus arctii;Alzheimer disease;Learning and memory;Model阿尔兹海默病（Alzheimer disease，AD）亦称老年痴呆症，是以进行性认知障碍和记忆能力损害为主要临床表现的中枢神经系统退行性疾病。
杏鲍菇废弃菌渣中D-氨基葡萄糖盐酸盐的制备工艺及生物学活性分析
张倩如，吴启赐，薛钰，等. 杏鲍菇废弃菌渣中D-氨基葡萄糖盐酸盐的制备工艺及生物学活性分析[J]. 食品工业科技，2023，44（17）：263−271. doi: 10.13386/j.issn1002-0306.2022110139ZHANG Qianru, WU Qici, XUE Yu, et al. Preparation and Biological Activity of D-Glucosamine Hydrochloride from the Waste Residues of Pleurotus eryngii [J]. Science and Technology of Food Industry, 2023, 44(17): 263−271. (in Chinese with English abstract).doi: 10.13386/j.issn1002-0306.2022110139· 工艺技术 ·杏鲍菇废弃菌渣中D-氨基葡萄糖盐酸盐的制备工艺及生物学活性分析张倩如1，吴启赐1, *，薛 钰1，林志超1，黄家福1，吕昊坤1，彭 伟1，潘裕添1，林进妹2,*（1.闽南师范大学菌物产业福建省高校工程研究中心，福建漳州 363000；2.闽南师范大学化学化工与环境学院，福建漳州 363000）摘 要：本文以杏鲍菇废弃菌渣为原料，探究了D-氨基葡萄糖盐酸盐（D-glucosamine hydrochloride ，GAH ）的制备工艺、液相-质谱（HPLC-MS ）、红外光谱、理化指标及其对斑马鱼胚胎发育的影响。
采用单因素和响应面优化试验，获得盐酸水解制备GAH 的最佳条件：盐酸浓度31%，水解时间4 h ，水解温度82 ℃，液固比5 mL/g ，此时GAH 得率可达23.61%。
液相-质谱、红外光谱和理化指标分析显示，GAH 纯化样品纯度是标准品的101.9%，质谱和红外光谱图与标准品一致，各项指标均符合甚至优于美国药典43-国家处方集38（USP43-NF38）的质量标准，砷含量仅0.21 μg/g 。
检验专业英语试题及答案
检验专业英语试题及答案一、选择题（每题2分，共20分）1. Which of the following is not a routine test in clinical laboratory?A. Blood countB. Urine analysisC. Liver function testD. DNA sequencing2. The term "hemoglobin" refers to:A. A type of proteinB. A type of enzymeC. A type of hormoneD. A type of lipid3. What is the primary function of the enzyme amylase?A. To break down proteinsB. To break down carbohydratesC. To break down fatsD. To break down nucleic acids4. The process of identifying the presence of a specific microorganism in a sample is known as:A. CulturingB. IsolationC. IdentificationD. Quantification5. Which of the following is a common method for measuring the concentration of glucose in blood?A. SpectrophotometryB. ChromatographyC. ElectrophoresisD. Enzymatic assay6. The term "ELISA" stands for:A. Enzyme-Linked Immunosorbent AssayB. Electrophoresis-Linked Immunosorbent AssayC. Enzyme-Linked Immunofluorescence AssayD. Electrophoresis-Linked Immunofluorescence Assay7. In medical diagnostics, what does "PCR" refer to?A. Polymerase Chain ReactionB. Protein Chain ReactionC. Particle Count ReactionD. Pathogen Characterization Reaction8. The process of measuring the amount of a specific substance in a sample is known as:A. TitrationB. CalibrationC. QuantificationD. Qualification9. Which of the following is a common type of clinical specimen?A. BloodB. SoilC. HairD. Water10. The term "antibodies" refers to:A. Proteins that recognize and bind to specific antigensB. Substances that neutralize toxinsC. Hormones that regulate immune responseD. Cells that produce immune responses二、填空题（每空1分，共10分）1. The process of separating molecules based on their size is known as __________.2. In clinical chemistry, the term "assay" refers to a__________ method.3. The unit of measurement for pH is __________.4. A common method for detecting the presence of antibodies in a sample is the __________ test.5. The process of identifying the type of bacteria in a sample is known as __________.6. The process of separating DNA fragments based on their size is known as __________.7. The term "ELISA" is used in __________ to detect the presence of specific antibodies or antigens.8. The process of identifying the genetic makeup of an organism is known as __________.9. The process of measuring the amount of a substance in a sample using a specific wavelength of light is called__________.10. The process of identifying the presence of specific microorganisms in a sample is known as __________.三、简答题（每题5分，共20分）1. Describe the principle of the Enzyme-Linked Immunosorbent Assay (ELISA).2. Explain the importance of maintaining aseptic technique ina clinical laboratory.3. What are the steps involved in performing a blood count?4. Discuss the role of antibodies in the immune response.四、论述题（每题15分，共30分）1. Compare and contrast the methods of Chromatography and Electrophoresis in terms of their applications in clinical diagnostics.2. Discuss the ethical considerations in the use of genetic testing for medical purposes.五、翻译题（每题5分，共10分）1. 将以下句子从中文翻译成英文：在临床实验室中，酶联免疫吸附测定法是一种常用的检测特定抗体或抗原的方法。
亚砷酸钠诱导酵母细胞凋亡中SOD1和SOD2基因的作用
亚砷酸钠诱导酵母细胞凋亡中SOD1和SOD2基因的作用细胞生物学论文第三篇：亚砷酸钠诱导酵母细胞凋亡中SOD1和SOD2基因的作用摘要：为了探讨SOD1和SOD2基因在亚砷酸钠诱导酵母细胞凋亡中的作用，本实验以酿酒酵母野生株BY4741（WT）及其突变体Δsod1和Δsod2为材料，研究了亚砷酸钠对酵母细胞生长和相对存活率的影响，以及酵母细胞在亚砷酸钠胁迫下活性氧（Reactive oxygen species, ROS）水平、线粒体膜电位和细胞凋亡率的变化。
结果显示，亚砷酸钠可抑制酵母细胞生长，诱导胞内ROS水平和细胞凋亡率升高。
在相同砷处理组中，Δsod1相对存活率、细胞胞内ROS水平和细胞凋亡率显著高于野生株，线粒体膜电位显著低于野生株；Δsod2细胞凋亡率显著高于野生株。
结果表明，亚砷酸钠诱导的酵母细胞凋亡与胞内ROS水平的升高有关，而超氧化物歧化酶基因与亚砷酸钠引起的细胞凋亡密切相关。
关键词：亚砷酸钠; 酵母; 凋亡; 超氧化物歧化酶; 活性氧;Effects of SOD1 and SOD2 Gene Deletions on Arsenic-induced Apoptosis in Yeast CellsWU Lihua YI Huilan CHEN Yanfei QIAO Hongping ZHAO WenjingDepartment of Biology, Taiyuan Normal University School of Life Science, Shanxi UniversityAbstract：To explore the role of SOD1 and SOD2 in sodium arsenite-induced apoptosis in yeast cells, yeast wild-type (BY4741), SOD1 mutant (Δsod1) and SOD2 (Δsod2) mutant strains were used to study the effects of sodium arsenite on the growth and relative survival rate in the yeast cells. In addition, intracellular reactive oxygen species (ROS) level, mitochondrial membrane potential and apoptotic rate of the yeast cells under sodium arsenite-induced stress were determined in this study. The results showed that sodium arsenite induces growth inhibition and cell apoptosis in yeast cells with increased intracellular ROS levels. In the same treatment group, relative growth and clonogenic survival rate were lower, meanwhile, apoptosis rate and intracellular ROS levels were higher significantly in the yeast strain lacking SOD1 (Δsod1) compared to the WT strain, but no significant difference except for apoptosis rate was observed between in Δsod2 and WT strains. These results indicated that sodium arsenite-induced yeast apoptosis was associated with increased intracellular ROS level, and superoxide dismutase is essential in sodium arsenite- induced yeast apoptosis.0 引言凋亡，是一种由基因严格控制的程序性细胞死亡方式，在调控机体生长发育和对外界刺激的反应等生物学过程中起重要作用。
非等位基因
非等位基因概述非等位基因是指同一基因座上的不同等位基因。
等位基因是指在某个给定的基因座上，可以存在多种不同的变体。
每个个体继承了一对等位基因，一对等位基因可能会导致不同的表型表达。
非等位基因的存在使得遗传学研究更加复杂，因为不同的等位基因会对个体的表型产生不同的影响。
背景在生物学中，基因座是指染色体上一个特定的位置，该位置上的基因决定了某个特征的表达方式。
每个基因座上可以有多种不同的等位基因。
等位基因是指在某个特定基因座上的不同基因变体。
每个个体都会继承一对等位基因，通过这对等位基因的不同组合，决定了个体的表型。
然而，并非所有基因座上的等位基因都具有相同的表现型。
非等位基因的影响非等位基因的存在导致不同等位基因会对个体表型产生不同的影响。
有些非等位基因会表现出显性效应，也就是说，当个体继承了一个突变的等位基因时，即使同时继承了一个正常的等位基因，但显性效应会使得突变的等位基因的表型表达得到体现。
相反，有些非等位基因会表现出隐性效应，当个体继承了两个突变的等位基因时，才会表现出突变的表型。
除了显性和隐性效应之外，非等位基因还可能发生两种其他类型的表型效应。
一种是共显效应，当个体继承了两个不同的突变等位基因时，在表型表达上会表现出一种新的特征，这个特征并不是单个突变等位基因所能导致的。
另一种是部分显性效应，当个体继承了两个不同的突变等位基因时，表型表达将介于两个单独突变等位基因的表型之间。
重组和非等位基因重组是指两个不同的染色体交换部分基因序列的过程。
在重组的过程中，非等位基因可能会发生改变，导致新的等位基因组合形成。
这一过程使得非等位基因的表型效应更加复杂，因为新的等位基因可能将不同基因座的效应组合起来。
非等位基因的重要性非等位基因对生物的适应性和多样性起着重要作用。
通过对等位基因的各种组合的研究，人们可以更好地理解基因与表型之间的关系，并揭示遗传变异对物种适应环境的重要性。
总结非等位基因是指同一基因座上的不同等位基因。
代谢工程改造微生物合成生物基单体的进展与挑战
化工进展Chemical Industry and Engineering Progress2023 年第 42 卷第 8 期代谢工程改造微生物合成生物基单体的进展与挑战高聪，陈城虎，陈修来，刘立明（江南大学食品科学与技术国家重点实验室，江苏 无锡 214122）摘要：单体是合成聚合物所用的小分子基础原料，目前主要来源于化石燃料。
利用微生物制备生物基单体具有生产条件温和、环境友好、可持续的优势，是实现高分子材料行业绿色制造的重要途径。
借助代谢工程和合成生物学元件，目前已经实现了多种单体的微生物制造，然而与石油基生产工艺相比，这些单体微生物细胞工厂的生产性能普遍较低。
围绕代谢工程改造微生物合成生物基单体过程中存在的瓶颈问题，本文基于具体案例分析，从廉价底物的高效利用、提高生物基单体合成效率、强化细胞环境耐受性三个方面，总结了改造微生物合成单体的最新研究进展。
同时，讨论了单体微生物细胞工厂目前存在的挑战和未来发展方向。
关键词：微生物细胞工厂；塑料单体；底物利用；调控策略；环境耐受性中图分类号：Q815; TQ92 文献标志码：A 文章编号：1000-6613（2023）08-4123-13Progress and challenges of engineering microorganisms to producebiobased monomersGAO Cong ，CHEN Chenghu ，CHEN Xiulai ，LIU Liming(State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China)Abstract: Monomers are the basic raw materials used in the synthesis of polymers, which mainly come from fossil fuels. Engineering microorganisms to synthesize monomers has the advantages of mild production conditions, environmental friendliness, and sustainability, which is an important way to achieve green manufacturing in the material industry. With the help of metabolic engineering and synthetic biology parts, microbial manufacturing of various monomers has been realized at present. However, compared with petroleum-based production processes, the production performance of these microbial cell factories is limited. Focusing on the bottleneck problems in engineering microorganisms to synthesize bioplastic monomers, this review summarizes the latest research progress in the metabolic engineering of microorganisms to produce monomers from three aspects: efficient utilization of cheap substrates, improvement of monomer synthesis efficiency, and enhancement of cell environment tolerance,based on specific case studies. At the same time, the current challenges and future direction of the microbial monomer cell factory are discussed.Keywords: microbial cell factories; bioplastic monomer; substrate utilization; regulation strategy;environmental tolerance特约评述DOI ：10.16085/j.issn.1000-6613.2023-0289收稿日期：2023-02-28；修改稿日期：2023-04-08。
Oxygen transfer effects in serine
Enzyme and Microbial Technology 23:451– 461, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
bioreactor performance in relation with the physiology of the microorganism. Synthesis of extracellular proteases by Bacillus strains is subject to carbon repression2–4 and nitrogen repression;3,4 nevertheless, many of the investigations performed on extracellular enzyme production suffer systematic study. Table 1 summarizes the literature on protease research giving the investigated effects and the related measurements; furthermore, Table 2 emphasizes the oxygen transfer and other bioreactor operation conditions used in the literature.
类风湿因子引起免疫比浊法测定肌钙蛋白I假阳性的分析与对策
2013年10月结果。
该种蛋白丢失现象还从对附睾液新蛋白组分的测定而得到证实。
这种现象可能归因于精子在附睾成熟过程中精子膜经历了蛋白质裂解过程,裂解作用主要是存在于精子膜表面、内部或周围附睾液中蛋白水解酶作用的结果,其中附睾液作用起决定影响。
精子膜表面新蛋白的形成是精子表面膜蛋白改变的另一个重要特征。
精子在附睾转运中大多数膜蛋白新组分是以低分子量和高度糖基化为特征,这种低分子量和高度糖基化蛋白的形成和精子所处的周围附睾液密切相关。
但对新蛋白的形成机制却有争论,有人认为附睾分泌的糖蛋白在精子成熟过程中是逐渐沉积在精子膜上,也有人认为,精子表面新蛋白组分的加入主要是由于附睾液与精子表面相互作用的结果。
目前大多认同第2种机制,其理由是精子本身不具备生物合成能力,而附睾液中富含氨基酸和多种有关蛋白质合成、转移、糖基化修饰的酶及转移载体长萜醇。
脂质作为精子膜主要组成部分其成分主要有磷脂、中性脂和糖脂,它们的变化与精子成熟程度密切相关。
Awano等(1989)发现仓鼠精子在附睾转运过程中脂肪酸和固醇总量没有改变,但24-脱氢胆固醇含量升高,7、24-二烯胆固烷醇含量升高,而胆固醇含量下降。
Rana等(1991)研究结果发现山羊精子膜脂质在附睾转运和成熟过程中总脂、磷脂和糖脂含量显著下降,而中性脂含量增加。
这些脂质的改变导致精子表面净负电荷的增加,并使膜成分构象发生变化,改变膜的稳定性,同时负电荷增加有利于精子在附睾尾处于静息状态和非凝集状态。
总之,精子表面成分的改变其目的一是为受精作好准备,二是暂时抑制其受精功能。
2.3代谢改变 精子在附睾中成熟过程经历一系列代谢改变,其中钙离子的跨膜转运具有重要生理意义。
据报道,精子在附睾转运过程中钙离子积累能力有很大的变化,附睾头精子钙离子浓度大概是尾部的2倍,附睾头精子从外部积累钙离子的速度比尾部大概快2～4倍。
另外,精子在附睾成熟过程中,由附睾头无规则运动转变成附睾尾精子直线运动的变化过程与钙离子依赖性机制的代谢改变有关。
敲减Wingless_Wnt1基因表达对赤拟谷盗发育中的影响_英文_
ISSN 1007-7626CN 11-3870/Q中国生物化学与分子生物学报http ：//cjbmb．bjmu．edu．cnChinese Journal of Biochemistry and Molecular Biology2012年8月28（8）：733 738Received ：March 26，2012；Accepted ：June 6，2012Supported by National Natural Science Foundation of China （No．31040018and No．31172146），Shanxi Scholarship Council of China （2010-2012）and International Cooperation Projects of Shanxi Province*Corresponding author Tel ：86-25-85891763；E-mail ：libin@njnu．edu．cn ；Tel ：0086-351-7018268，E-mail ：lzy@sxu．edu．cn收稿日期：2012-03-26；接受日期：2012-06-06国家自然科学基金（（No．31040018；No．31172146）和山西省国际合作项目资助*联系人Tel ：86-25-85891763；E-mail ：libin@njnu．edu．cn ；Tel ：0086-351-7018268，E-mail ：lzy@sxu．edu．cnKnocking-down of Wingless /Wnt1Influences the Development of Tribolium castaneumPENG Ya-Nan 1），LI Cheng-Jun 2），Li Bin 2）*，LI Zhuo-Yu 1）*（1）Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education ，Institute of Biotechnology ，Shanxi University ，Taiyuan030006，China ；2）Jiangsu Key Laboratory for Biodiversity and Biotechnology ，College of Life Sciences ，Nanjing Normal University ，Nanjing210046，China ）Abstract Known as highly conserved during evolution ，the Wnt signaling pathway plays a vital role in regulating animal embryonic axis ，embryonic differentiation ，and deciding cell polarity and maintaining adult dynamic equilibrium．Mutations or deregulations of its components might cause the occurrence of carcinoma．We studied the role of Wingless /Wnt1during larva-adult development of the red flour beetle ，Tribolium castaneum ，with dsRNA-mediated Wingless （Wg ）/Wnt 1gene knocked down．The treated late larvae metamorphosed into pupae with drastically increased wing interval and decreased wing width （P ＜0.01）．The pursuant pupa-adult eclosion was also severely affected and most of pupae died during this period．The qPCR result showed that the mRNA level of Cadherin-like and Smoothened （Smo ）geneswere up-regulated greatly ，and that of armadillo-2was slightly higher ，after Wingless /Wnt 1gene was knocked down．We drew the conclusion that Wnt-1signaling pathway is closely related to the proper wingdevelopment and adult metamorphosis of Tribolium ．In addition ，the elevated expression of Cadherin-like and Armadillo-2may be accountable for the reduced wing width and enlarged wing interval caused by wggene silencing ，because those alterations can either enhance cell adhesion or change cell morphology．Importantly ，the up-regulation of smo gene indicates that Hedgehog signaling pathway may be affected by the RNAi of Wg and involved in the abnormal or lethal phenotypes observed in our experiment．Key words Tribolium castaneum ；Wg RNAi ；Wingless /Wnt1protein ；Wnt signaling pathway ；eclosion敲减Wingless /Wnt1基因表达对赤拟谷盗发育中的影响彭亚男1），李承军2），李斌2）*，李卓玉1）*（1）化学生物学与分子工程教育部重点实验室山西大学生物技术研究所，太原030006；2）南京师范大学生命科学学院，南京210046）摘要Wnt 信号通路是进化中高度保守的一条信号转导途径，在调控动物的胚胎轴向正常发育、胚胎分化、决定细胞极性、维持成体动态平衡等方面发挥重要作用．该信号通路的异常激活还与肿瘤的发生密切相关．本实验将体外人工合成的Wingless （Wg ）/Wnt 1基因dsRNA 显微注射入赤拟谷盗晚期幼虫体内，研究Wingless /Wnt1蛋白在赤拟谷盗发育过程中发挥的作用．实验结果显示，注射Wingless （wg ）/Wnt 1基因dsRNA 后，赤拟谷盗发育形成的蛹，翅膀宽度减小，翅间距明显增大，且羽化过程也受到严重影响．此外，qPCR 结果表明，赤拟谷盗Wingless （Wg ）/Wnt 1基因被沉默后，Cadherin-like 和Smoothened （Smo ）基因的表达显著上调，A rmadillo -2基因略上调．这些结果揭示，Chinese Journal of Biochemistry and Molecular Biology Vol．28Wnt-1信号通路和赤拟谷盗翅膀发育以及成虫羽化过程密切相关．蛹翅宽减小，翅间距增大，可能是由于调控细胞粘连及细胞形态的Cadherin-like和Armadillo-2基因的上调所引起．更重要的是，Smo基因的上调，表明了Wnt信号通路和Hedgehog信号通路在赤拟谷盗发育过程中有交互作用．关键词赤拟谷盗；Wg RNAi；Wingless/Wnt1蛋白；Wnt信号通路；羽化中图分类号Q966Wnt gene was first reported by Nusse and his colleagues in1982，and named as int-1．When this gene was abnormally activated，it would induce tumor［1］．The subsequent name‘wnt’was derived from a combination of int-1and wingless（a developmental patterning gene in Drosophila），because these two genes were shown to be homologous［2］．Later，the family of wnt genes was confirmed to exist in many species from nematodes to vertebrates：nineteen in human［3］，seven in Drosophila，seven in Apis，six in Anopheles，and nine in Tribolium castaneum［4，5］．Wnt genes encode a large family of secreted，cysteine-rich proteins that play a key role in animal development，as intercellular signaling molecules．Most Wnt proteins are comprised of350to380amino acids，with more than100conserved residues scattering across the entire sequence．The initiation of these proteins is a sequence of hydrophobic signal followed by a site that can be recognized by signal peptides．Each protein has one or more sites for N-linked glycosylation and up to 24conservative cysteine residues that form disulfide bonds［6］．Wnt ligands（Wnts）bind to various transmembrane receptors by autocrine or paracrine and drive the Wnt signaling pathway，thereby triggering intercellular cascades to regulate transcription in target genes．Wnt signaling pathway activated by Wnt proteins is involved in embryonic development，as well as cell proliferation and differentiation in adult development．It is required in the processes of regulating the establishment of head-to-tail axis，the differentiation of neural crest，the correct formation of brain and heart，kidney morphogenesis and sex determination［7］．The disruption of this precise system will induce developmental disabilities．A total of nine Wnt genes have been reported in Tribolium genome，including Wnt A，Wnt8，orthologs of the vertebrate Wnt5-7and Wnt9-11genes，in addition to Wg or Wnt1［5］．The expression patterns of these nine Wnt genes in embryogenesis have been discussed at length in existing studies，especially their segment polarity function of the canonical Wg/Wnt1 gene．However，less is known about the role of Wnt signaling pathway in postembroyonic developmental process of Tribolium．An investigation had been carried out about the role of Wg/Wnt1protein during the larval-adult development of Tribolium．1Materials and Methods1.1Beetle strain and maintenanceThe Georgia-1（GA-1）strain of Tribolium castaneum used in this study was reared at30ħand all experiments were performed at room temperature 25ħ［8］．1.2Analysis of Wg gene transcript levels in different developmental stages by RT-PCREarly and late eggs，larvae，pupae and adults were collected for RNA preparation，followed byRT-PCR．The first day of the embryonic period was taken for early egg stage，the initial1-7days of larval stage for early larval stage，and the starting1-3days of pupa/adult stage for early pupa/adult stage．Sequence of designed primers was Wg-F1：5'-GTGCCAATA ATGCGATTCAC-3'，Wg-R1：5'-TTCCTTTGTAGT GCGTTTCG-3'．Amplification conditions of RT-PCR were：94ħ5min，94ħ30s，60ħ30s，72ħ30s，35cycles，25ħ10min．The housekeeping gene Rps3（Tribolium ribosomal protein3）was used as an internal control．Amplification conditions of RT-PCR were：94ħ5min，94ħ30s，60ħ30s，72ħ30s，28cycles，25ħ10min．1.3RNA interferenceDouble-stranded RNA was produced from a714 bp fragment of the T．castaneum Wingless gene （fragment positions493 1206）．Template for dsRNA synthesis was amplified by using gene-specific primers that have T7promoter sequences at the end．The sequences of primers were as follows：Wg-F2：5'-TAATACGACTCACTATAGGGATAG ATACGTGCAAC TGCGA-3'，Wg-R2：5'-TAATACGACTCACTATAGG GCTCGAATACGACGACTTCCT-3'．Amplification conditions of RT-PCR were：94ħ5min，94ħ30s，60ħ30s，72ħ1min，35cycles，25ħ10min．Double-stranded RNA was synthesized and purified using the Transcript Aid TM T7High Yield Transcription kit．dsRNA was diluted to1μg/μL with1ˑinjection buffer（5mmol/L KCl，0.1mmol/L K3PO4，pH6.8）prior to injection．For injection，T．castaneum late larvae were affixed to microscope slides with tweezers at their posterior abdomen．Approximately0.2μL of dsRNA solution was injected into each pupa through a micromanipulator set-up，at a ventrolateral position between abdominal segments three and four．Untreated late larvae of the same number were selected as437No．8PENG Ya-Nan et al：Knocking-down of Wingless/Wnt1Influences the Development of Tribolium castaneum controls．Beetles of group IB were injected with0.2μL of injection buffer（1ʒ10）．Larva RNAi wasperformed with WPI Nanoliter microscopic injectionsystem．After，these beetles were reared in the sameconditions mentioned above．Their development statuswas observed every24hours．Five days after injection，three insects from each treatment were collected forRT-PCR to confirm Wg gene was silenced．Anotherreaction with a pair of primers for rps3gene was usedas the internal loading control．Sequences of primersfor Rps3g ene was：Rps3-F5'-TCAAATTGATCGGAGGTTTG-3'，Rps3-R5'-GTCCCACGGCAACATAATCT-3'，and Wg-F1and Wg-F2were used for Wggene．1.4Detection of mRNA level of other relatedgenes by qPCRTc-Armadillo-2，NCBI mRNA accession numberXM_966892，is located on LG9；smo，XM_966834，is on LG8；cadherin-like，XM_966295．Primers ofqPCR were designed and synthesized by TakaraBiotechnology CO．（Dalian，China）．The detailedinformation of primer sequences was described in Table1．Three cDNA templates were prepared with threepupae from each group．Technical triplicates of eachreaction mix were prepared in20μL final volumecontaining：10μL of2ˑSYBR Green PCR Mix，0.4μL of Rox，3μL of water-diluted cDNA（1︰20），which corresponds to1μg of total RNA，0.8μL ofeach primer（10pmol）．Finally，5μL DEPC H2Owas introduced to raise the system to20μL．Thefollowing cycling protocol was used：40cycles of30sat95ħ，5s at95ħ，34s at60ħ．To verify theapplicant’s consistency，the product was tested in amelting point analysis conducted directly afteramplification．Relative expression level of each genewas determined versus a constitutively expressed generps3．The results were displayed by meansʃstandarderrors with three independent experiments．Table1Details of primers used for the three evaluatedgenesGene Sequences（5'→3'）Product length（bp）Armadillo-2For：caatcacggtagtcagccttttc81Rev：tgtgtgccaatctccagtccSmo For：atcggttactgcgtcctggt95Rev：aaggcggggtatttgttggCadherin-like For：gacttcaaagatgctcagtcgaaa118Rev：taacaactaaaacggcaaccacac2Results2.1Knockdown of Wg gene in Tribolium late larvaeWe first examined expression pattern of Wg gene by reverse transcription-PCR（RT-PCR）．The results indicated that wg gene showed a high expression from late larval stage to late pupa stage，and the expression was declined in adult stage（Fig．1A）．It is likely that Wg gene plays a crucial role in larva-pupa-adult transitions．To study the role of Wg/Wnt1protein during these transitions，we injected Wg-dsRNA into late larvae．Five days later，we verified the mRNA level of Wg gene by using RT-PCR．The results showed that Wg gene of beetles was silenced as expected （Fig．1B）．Fig．1Analysis of wg gene expression by reversetranscription-PCR（A）The mRNA level of Wg ineight developmental stages．1：early egg；2：late egg；3：early larva；4：late larva；5：early pupa；6：late pupa；7：early adult；8：late adult．（B）Knocking-down of Wgtranscript by RNAi．Control：no injecting；IB：injectinginjection buffer；RNAi：injecting Wg-dsRNA．PCRanalysis of Rps3with the same cDNA template served asinternal control2.2Wnt1protein is required for wing develop-ment of Tribolium pupaeBecause a fraction of late larvae died as a result of injury caused by dsRNA injections，we observed phenotypes of the remaining bettles：n（group RNAi）=27；n（group IB）=25；n（group Control）=30．All of the remaining larvae survived on the pupa stage，but the wing interval of pupae from group RNAi was bigger than that of control beetles（Fig．2A）．Furthermore，body size，the length，width and interval of each pupa wing were measured using OLYMPUS microscope．The experiments showed that the wing width of Wg RNAi pupae decreased，and the wing interval expanded distinctly （Fig．2B）．However，the body size and wing length did not appear to be affected in comparison to the two control groups（Fig．2B）．2.3Wnt1protein contributes to pupa-adult moltingAlthough the wg RNAi larvae had metamorphosed into pupae，three defective phenotypes were revealed at the ensuing adult eclosion（Fig．3）．Ultimately，these abnormal insects died during the eclosion process．The proportion of three cases in each group was calculated：n（group RNAi）=27；n（group IB）=25；n（group Control）=30．And the result was indicated in Table 2．Most insects in group Control and IB showed normal phenotype．However，the majority of insects in group537Chinese Journal of Biochemistry and Molecular Biology Vol．28Fig．2RNAi results of wg on Tribolium pupae（A）Phenotype deficiency of pupae by RNAi．（a）Pupae in group of control；（b）Pupae in group of IB；（c）and（d）RNAi treated pupae．Arrow indicates the wing interval．These indexes were measured under OLYMPUS microscope．The photo was magnified by2.5times．（B）The statistical results of body size，wing length，wing width and wing interval of Tribolium pupae in group of control，IB and RNAi．Data processing was made usingsoftware SPSS．Star markers（＊＊）indicates a highly significant difference between control and treated insects（P＜0.01）Fig．3RNAi effects of wg on Tribolium adult eclosion（A）and（E）：normal adult；（B）and（F）：deficiency adult stopped at the prophase of emergence；（C）and（G）：deficiency adult emerged incompletely；（D）and（H）：defective adult exhibiting abnormal phenotypes（dorsal is on top row；ventral is on bottom row）637No．8PENG Ya-Nan et al ：Knocking-down of Wingless /Wnt1Influences the Development of Tribolium castaneum Table 2Statistical results of three kinds of emergence phenotypes in each groupGroup Normal eclosion Stop at beginning eclosion Incomplete eclosion Finish eclosion yet with phenotype defects Control 94%0%0%6%IB 97%0%0%3%RNAi7%41%22%30%The percentage in theTable 2is the ratio of Tribolium with certain emergence phenotype in corresponding groupRNAi did not finish the adult eclosion completely．Some of the pupae stopped at the beginning eclosion ；some went further ；and others metamorphosed into pharate adults with phenotype defects．2.4Knock-down of Wg has effects on a few genesat transcript levelIn order to study the molecular mechanism of Wnt1/Wingless protein in regulating wing development and pupa-adult molting of Tribolium ，the isolated total RNA of pupae from each group was screened to test whether the observed effects of Wg gene knock-down were caused by those factors ：Armadillo -2，Smo a ndCadherin-like ，which have been demonstrated to play some roles in the wing development of Drosophila ［9-11］．The result of qPCR indicated that the expression of Cadherin-like and Smo were up-regulated markedly ，the level of Armadillo-2with a slightincrease．Fig．4The mRNA level of Cadherin-like ，Smo ，andArmadillo-2affected by the RNAi of Wg The Y-axis denoted mRNA relative expression levels（normalized by Tcrps 3）．Mean ʃSE of mRNA relative expression levels of three genes mentioned above were showed．Star markers （*）indicated a significant difference between control and treated insects （P ＜0.05）．Double star markers （＊＊）indicated a highly significant difference between control and treated insects （P ＜0.01）3DiscussionStudies have suggested that Wnts can signal through three different pathways ：the canonical β-catenin pathway ，the non-canonical planar cell polarity （PCP ）and Wnt /Ca 2+pathways．The β-catenin pathway is required for growth and cell fate specification ［12］，whereas the two non-canonical pathways are implicated in cell polarity and cellmovement ［13］．The classic Wingles s /Wnt signaling pathway has been demonstrated to play a key role in the wing development of Drosophila ．The functional mechanism is also clear．The pathway is involved in the growth ，specification ，and morphogenesis of the wing pouch of Drosophila ［11］，one region of the wing disc that will be transformed to the adult wing blade during pupa development ［14］．Further studies suggested that Wingless promoted the wing disc pouch growth mainly through the inhibition of apoptosis ［15］．Meanwhile ，the regulation of wingless for vestigial ，one of its targets which defines the wing primordium and is required for its growth ，contributes partly to the increase of thewing pouch size ［16，17］．Wingless /Wnt signaling also participates in the cell specification of the wing disc．The depletion of wingless gene during the development of Drosophila early larvae will lead to the result of losing normal wing structures．Meanwhile ，wingless can induce the expression of genes along dorsal-ventral boundary during late larval development ，including senseless regulating cell fate specification at the wing margin ［18］．The latest studies have revealed the role of wingless in controlling the cell shape of late larval wing disc ［11］．In addition ，Wingless /Wnt signaling is implicated in cell adhesion by directing the graded expression of Shotgun ，which encodes E-cadherin in Drosophila ．For cells along the proximodistal axis of the developing wing epithelium ，those further away from the source of Wg signaling in the wing imaginal discs display lower levels of DE-Cadherin expression when compared with those receiving high threshold of Wg signaling ［19］．The model insect Tribolium used in our experiment is one of the well-known holometabolous pests for stored products ，mainly in tropical or warmer regions around the world ［20］．It belongs to Tenebrionidae ，Coleopteran ，and the genome sequence of the Tribolium is now available ［21］．Although Tribolium belongs to holometabolous taxa as well as Drosophila ，its developmental pattern differs from that of Drosophila ．In Tribolium ，primordia of anterior segments appears first ，then primordia of more posterior segments are generated from a posterior growth zone．However ，all segments in Drosophila occur nearly simultaneously ［22］．Our data therefore complement the Drosophila on functional genetic737Chinese Journal of Biochemistry and Molecular Biology Vol．28analysis of basic biological questions．In this study，the results showed that the disruption of canonical Wingless/Wnt signaling pathway in Tribolium also resulted in abnormal wing phenotype，along with severely affected emergence process，indicating that Wnt1protein is necessary for the wing development of Tribolium．This result also shows that Tribolium is an excellent model for studying genetic regulation．Furthermore，the qPCR result shows that Cadherin-like，Armadillo-2and Smo genes of T．castaneum pupae with abnormal wings were upregulated．Because E-cadherin and Armadillo-2play some roles in wing cell adhesion and shape［23］，we believe that Cadherin-like and Armadillo-2may influence the wing growth of Tribolium by strengthening cell adhesion or changing cell shapes．In addition，the abnormal activation of Smo implies that smo gene is possibly related to the effects of wing development and adult emergence．This finding points to a conclusion that Wnt1protein may play an additional role independent of Wnt signaling pathway in postembryonic development of Tribolium．Importantly，it may mediate the cross-talk of Wnt signaling pathway and Hedgehog pathway．Further studies need to be completed in the future．参考文献（References）［1］Nusse R，Varmus H E．Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the sameregion of the host genome［J］．Cell，1982，31（1）：99-109［2］Rijsewijk F，Schuermann M，Wagenaar E，et al．The Drosophila homolog of the mouse mammary oncogene int-1is identical to thesegment polarity gene wingless［J］．Cell，1987，50（4）：649-657［3］Garriock R J，Warkman A S，Meadows S M，et al．Census of vertebrate Wnt genes：isolation and developmental expression ofXenopus Wnt2，Wnt3，Wnt9a，Wnt9b，Wnt10a，and Wnt16［J］．Dev Dyn，2007，236（5）：1249-1258［4］Murat S，Hopfen C，McGregor A P．The function and evolution of Wnt genes in arthropods［J］．Arthropod Struct Dev，2010，39（6）：446-452［5］Bolognesi R，Beermann A，Farzana L，et al．Tribolium Wnts：evidence for a larger repertoire in insects with overlappingexpression patterns that suggest multiple redundant functions inembryogenesis［J］．Dev Genes Evol，2008，218（3-4）：193-202［6］Cho SJ，Valles Y，Giani V C，et al．Evolutionary dynamics of the Wnt gene family：a lophotrochozoan perspective［J］．MolBiol Evol，2010，27（7）：1645-1658［7］Agholme F，Aspenberg P．Wnt signaling and orthopedics，anoverview［J］．Acta Orthop，2011，82（2）：125-130［8］Haliscak J P，Beeman R W．Status of Malathion Resistance in5 Genera of Beetles Infesting Farm-Stored Corn，Wheat，and Oatsin the United-States［J］．J Econ Entomol，1983，76（6）：717-722［9］Terriente-Félix A，López-Varea A，de Celis J F．Identification of genes affecting wing patterning through a loss-of-functionmutagenesis screen and characterization of med15function duringwing development［J］．Genetics，2010，185（2）：671-684［10］Blair S S，Ralston A．Smoothened-mediated Hedgehog signaling is required for the maintenance of the anterior-posterior lineagerestriction in the developing wing of Drosophila［J］．Development，1997，124（20）：4053-4063［11］Logan C Y，Nusse R．The Wnt signaling pathway in development and disease［J］．Annu Rev Cell Dev Biol，2004，20：781-810［12］Kohn A D，Moon R T．Wnt and calcium signaling：beta-catenin-independent pathways［J］．Cell Calcium，2005，38（3-4）：439-446［13］Widmann T J，Dahmann C．Wingless signaling and the control of cell shape in Drosophila wing imaginal discs［J］．Dev Biol，2009，334（1）：161-173［14］Gonsalves F C，DasGupta R．Function of the wingless signaling pathway in Drosophila［J］．Methods Mol Biol，2008，469：115-125［15］Johnston L A，Sanders A L．Wingless promotes cell survival but constrains growth during Drosophila wing development［J］．NatCell Biol，2003，5（9）：827-833［16］Kim J，Sebring A，Esch JJ，et al．Integration of positional signals and regulation of wing formation and identity byDrosophila vestigial gene［J］．Nature，1996，382（6587）：133-138［17］Zecca M，Struhl G．Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial auto regulation［J］．Development，2007，134（16）：3001-3010［18］Jafar-Nejad H，Tien A C，Acar M，et al．Senseless and Daughterless confer neuronal identity to epithelial cells in theDrosophila wing margin［J］．Development，2006，133（9）：1683-1692［19］Jaiswal M，Agrawal N，Sinha P．Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wingdevelopment in Drosophila［J］．Development，2006，133（5）：925-935［20］Brown S J，Shippy T D，Miller S，et al．The red flour beetle，Tribolium castaneum（Coleoptera）：a model for studies ofdevelopment and pest biology［J］．Cold Spring Harb Protoc，2009，2009（8）：pdb．emo126［21］Tribolium Genome Sequencing Consortium，Richards S，Gibbs R A，et al．The genome of the model beetle and pest Triboliumcastaneum［J］．Nature，2008，452（7190）：949-955［22］Ober K A，Jockusch E L．The roles of wingless and decapentaplegic in axis and appendage development in the redflour beetle，Tribolium castaneum［J］．Dev Biol，2006，294（2）：391-405［23］Menzel N，Melzer J，Waschke J，et al．The Drosophila p21-activated kinase Mbt modulates E-cadherin-mediated celladhesion by phosphorylation of Armadillo［J］．Biochem J，2008，416（2）：231-241837。
底物特异性的生物催化与酶设计改造
化工进展CHEMICAL INDUSTRY AND ENGINEERING PROGRESS2019年第38卷第1期底物特异性的生物催化与酶设计改造姜恬1，冯旭东2，李岩1，李春1，2（1北京理工大学生命学院，北京100081；2北京理工大学化学与化工学院，北京100081）摘要：随着生物产业的发展，生物酶催化发挥着越来越重要的作用。
然而，部分酶在应用过程中仍然存在诸多问题，影响了生物催化的进一步发展。
本文以酶的底物特异性为切入点，回顾了酶的专一性、高效性和环保性；介绍了酶在药物合成和天然产物改性领域的应用以及所遇到的问题；综述了酶的底物特异性改造过程中各种方法的应用，包括化学修饰、非理性和理性设计。
化学修饰作为一种直观的修饰方法，通过化学反应对酶分子进行改造；非理性设计是利用易错PCR 和DNA Shuffling 等手段获得底物特异性提高的突变体；理性设计是基于序列和结构信息对酶分子进行改造。
本文从重塑活性口袋提高酶的底物特异性和重塑活性口袋改变酶促反应类型两个方面出发，详述了理性设计改变酶的底物特异性的方法，为酶的特异性改造提供借鉴。
关键词：生物催化；酶设计；底物特异性；分子改造中图分类号：Q814文献标志码：A文章编号：1000-6613（2019）01-0606-09The biocatalysis and enzyme modification of substrate specificityJIANG Tian 1，FENG Xudong 2，LI Yan 1，LI Chu 1，2(1School of Life Science,Beijing Institute of Technology,Beijing 100081,China;2School of Chemistry and ChemicalEngineering,Beijing Institute of Technology,Beijing 100081,China)Abstract:Enzymatic catalysis plays an increasing role in the development of bio-industry.However,there are still many problems in the application of some enzymes,which hinder the development of biocatalysis.This paper focused on the enzyme substrate specificity and reviewed the advantages of enzymes,such as high specificity,increased efficiency,and environmentally-friendly process.The applications of enzymes in fine chemistry and pharmaceutical synthesis were introduced.The paper also summarized the current methods used in engineering of enzyme substrate specificity,including chemical modification,irrational and rational design.Chemical reaction was a direct-viewing method applied in enzyme modification.Irrational design was often employed to obtain the better mutants with increased substrate specificity through error-prone PCR and DNA shuffling.In the rational design,enzyme engineering was based on their sequences and structures.Starting from reshaping the active pockets for increasing the substrate specificity and changing the enzymatic reaction type,this paper elaborated the methods of rational design to enhance the substrate specificity and provided a reference for future substrate specificity engineering.Keywords:biocatalysis;design of enzyme;substrate specificity;molecular modification特约评述DOI ：10.16085/j.issn.1000-6613.2018-1136收稿日期：2018-05-31；修改稿日期：2018-08-23。
大学生物化学课件Factors affect enzyme activity
Introduction
V
Vm * [S]
V=
Km + [S]
Km
[S]
Introduction
1/V
Km/Vm
-1/Km
1/Vm
1/V=Km/Vm * 1/[S] + 1/Vm
1/[S]
P (mol) V=
V(L)*t (min)
Introduction
AKP
-OPO3Na2 + H2O
Na2HPO4 +
Result
Km Vm
Notes
The amount of substrate and enzyme must be very accurate.
The time of every tube must the same.
E+
K1
S
ES
K3
E
+
P
K2
Here K1,K2,K3 represent the velocity constants for the respective reactions.
V = P / time
Km , the Michaelis-Menten constant is given by the formula
Let us assume that the measured velocity (v) is equal to ½ Vmax , then the above equation may be substituted as follows:
½ Vmax = Vmax [s] / (Km + [s])
phenylphosphate
the hallmarks of cancer(大牛的经典肿瘤学综述PPT).
• Downregulating proapoptotic factors (Bax, Bim, Puma), or by short-circuiting the extrinsic ligandinduced death pathway
Autophagy
• Can be strongly induced in certain states of cellular stress, the most obvious of which is nutrient deficiency
Resisting Cell Death
• Apoptosis • Autophagy • Necrosis
Apoptosis
• The concept that apoptosis serves as a natural barrier to cancer development has been established over the last two decades
“contact inhibition” is abolished in various types of cancer cells in culture, suggesting that contact inhibition is an in vitro surrogate of a mechanism that operates in vivo to ensure normal tissue homeostasis
• Cancer cells, by deregulating these signals, become masters of their own destinies.
植物生理学：第三节 水分(亏缺)胁迫及植物的抗旱性
Aerenchyma: continuous, columnar intracellular
spaces formed in root cortical tissues
Aerenchyma development in root cortex of maize after oxygen deprivation. (A) under aerobic conditions, (B) under 72h of hypoxia
• Effect of manipulating ABA concentration in xylem sap on stomatal conductance of leaves (abaxial
epidermis) in maize.
透调节物质(osmolyte) :
高度水溶性、不干预细胞代谢的有机化合物。
• (B) Photographs comparing internode elongation in aerobic ( l e f t ) and submerged (right) plants. Arrows indicate positions of nodes.
During short-term acclimation to anoxic conditions, plants generate ATP through glycolysis and fermentation
Ethylene promotes long-term acclimative
responses:
including formation of aerenchyma and stem
elongation
Treatment
Action
生物正交化学英文
生物正交化学英文In recent years, the concept of "bio-orthogonal chemistry" has gained significant attention in the field of chemical biology. It refers to chemical reactions that can occur in living systems without interfering with the biological processes. These reactions are crucial for studies of cellular processes, imaging, drug delivery, and biomaterials development.Step 1: Definition of Bio-orthogonal chemistryBio-orthogonal chemistry refers to a set of chemical reactions that can co-exist with biological molecules without interfering with their normal functions. It enables researchers to study biological processes in live cells and tissues with minimal disruption. Scientists can label and track specific molecules, proteins, and other biomolecules with fluorescent dyes or probes using these reactions, without affecting their function. Bio-orthogonal reactions are typically fast, selective, and can be performed under physiological conditions.Step 2: Importance of Bio-orthogonal chemistryBio-orthogonal chemistry has become a powerful tool in the field of chemical biology. It has enabled researchers to gain insights into cellular processes such as protein trafficking, signal transduction, and gene expression without disturbing the natural environment inside the cell. This technique has also been instrumental in the development of new imaging techniques, such as super-resolution microscopy, which has enabled scientists to visualize cellular structuresat a higher resolution than ever before.Bio-orthogonal chemistry has also played an essential role in the development of drug delivery systems. Byutilizing bio-orthogonal reactions, scientists canselectively target specific cells or tissues, reducing the chance of side effects. Finally, bio-orthogonal chemistry has also enabled the development of new biomaterials with a wide range of applications in tissue engineering, drug delivery, and regenerative medicine.Step 3: Examples of Bio-orthogonal reactionsThere are several examples of bio-orthogonal reactions, including the Strain-promoted Azide-alkyne Cycloaddition (SPAAC), Copper-catalyzed Azide-alkyne Cycloaddition (CuAAC), Tetrazine-click chemistry, and Photo-induced Tetrazine-alkene Cycloaddition (PTA).SPAAC involves the reaction of an azide and an alkyne in the presence of a strained-ring cyclooctyne. It is a copper-free reaction that proceeds rapidly under physiological conditions. CuAAC is a similar reaction that uses a copper catalyst to facilitate the reaction. It is also fast and can be performed in water.Tetrazine-click chemistry involves the reaction of a tetrazine and a trans-cyclooctene (TCO) group attached to a molecule of interest. This reaction is also fast and isuseful in imaging applications. Finally, PTA involves the photo-activated reaction of tetrazine and alkenes. This reaction is noteworthy because it is activated only when a specific wavelength of light is present, allowing for precise control over the reaction.In conclusion, Bio-orthogonal chemistry has become a useful technique in chemical biology. Through the use ofthese reactions, researchers can study biological processes with unprecedented accuracy without interfering with their function. The continued development of bio-orthogonal chemistry will lead to new insights into the workings of living organisms and the development of new treatments for diseases.。
211091445_牡丹籽粕蛋白提取工艺优化和功能性质分析
李若敏，张焕新，盘赛昆，等. 牡丹籽粕蛋白提取工艺优化和功能性质分析[J]. 食品工业科技，2023，44（8）：197−204. doi:10.13386/j.issn1002-0306.2022050251LI Ruomin, ZHANG Huanxin, PAN Saikun, et al. Process Optimization and Functional Properties of Peony Seeds Protein[J]. Science and Technology of Food Industry, 2023, 44(8): 197−204. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022050251· 工艺技术 ·牡丹籽粕蛋白提取工艺优化和功能性质分析李若敏1,2，张焕新1, *，盘赛昆2，叶静静1（1.江苏农牧科技职业学院，江苏泰州 225300；2.江苏海洋大学食品科学与工程学院，江苏连云港 222005）摘 要：以酶水解-超声辅助碱溶酸沉法提取蛋白工艺为基础，初步对牡丹籽中粗蛋白进行分离提取。
通过单因素实验和响应面试验，考察料液比、超声温度、酶用剂量、超声时间四个因素对牡丹籽粕蛋白提取率的影响，确定最佳提取工艺，并测定其功能特性。
结果表明，酶水解-超声辅助碱溶酸沉法提取牡丹籽粕蛋白最优工艺条件为：料液比为1:9.8（w/v ），超声温度为49.5 ℃，酶用剂量为1.9%，超声时间为119 min 。
在此条件下，蛋白质提取率达到90.95%。
此时所得蛋白与常规法提取蛋白相比，氨基酸种类齐全、必需氨基酸含量均有所提高，功能特性如持水性、吸油性、乳化性皆优于常规法提取蛋白的功能特性，且乳化的稳定性更优，由此推测可作为食品加工乳化剂。
因此酶水解-超声辅助碱溶酸沉法提取的牡丹籽粕蛋白具有更高的营养价值和更好的功能特性。
断裂内含肽在含有抗体铰链区氨基酸序列的非天然外显肽中的剪接
ＤＯＵＴｏｎｇｌｕ牞ＣＨＥＮＨａｏ牞ＣＡＯＪｉｎ牞ＣＨＥＮＪｕｎｓｈｅｎｇ牞ＺＨＵＪｉａｎｗｅｉ
摘 要 内含肽是一种能够介导蛋白质分子从前体分子中自我切除，同时将其双侧的外显肽蛋白通过肽键连接起来的功 能性蛋白质，其中断裂型内含肽在抗体连接领域有着广泛应用。但由于天然断裂型内含肽 Ｃ端可特异性识别外显肽的前 ３ 位氨基酸序列———半胱氨酸、苯丙氨酸和天冬酰胺（ＣＦＮ），因此在蛋白剪接反应后不可避免的会引入外源氨基酸。由于本 课题组前期开发出的断裂内含肽介导的双特异性抗体药物装配平台也存在该缺陷，因此本研究尝试采用抗体铰链区的半 胱氨酸、天冬氨酸和赖氨酸（ＣＤＫ）取代“ＣＦＮ”作为内含肽的识别位点，并构建突变型断裂内含肽 ＮｐｕＧＥＰ ＤｎａＥ。结果表 明，突变体可以识别“ＣＤＫ”并成功发生了内含肽剪接反应。进而对影响该内含肽剪接反应平台的各因素如 ＮａＣｌ浓度、ＤＴＴ 浓度、ｐＨ和温度等进行了考察，结果表明在所考察的范围内断裂内含肽的剪接反应均可很好的进行。该内含肽剪接反应 平台的建立将有助于减少外源氨基酸的引入，进一步拓展了内含肽底物宽泛性，为内含肽应用于抗体药物特别是双特异性 抗体药物的装配提供了技术支持。 关键词 断裂内含肽；外显肽；双特异性抗体药物；蛋白质剪接；外源氨基酸 中图分类号 Ｑ８１６；Ｑ５１１ 文献标志码 Ａ 文章编号 １０００－５０４８（２０１９）０５－０６０６－０８
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何首乌二苯乙烯苷对2型糖尿病大鼠骨骼肌胰岛素抵抗的影响
何首乌二苯乙烯苷对2型糖尿病大鼠骨骼肌胰岛素抵抗的影响王婷;范益【摘要】目的考察何首乌二苯乙烯苷(TSG)对高脂饲料联合链脲菌素(STZ)诱导的2型糖尿病大鼠糖脂代谢紊乱及骨骼肌胰岛素抵抗的改善作用.方法雄性SD大鼠高脂饲料喂养6周后,腹腔注射STZ 30 mg/kg建立2型糖尿病模型.将造模动物分为4组:模型组(DM组)、罗格列酮组(RGLT组)、TSG高剂量(100 mg/kg)组(TSG-H组)、TSG低剂量(50 mg/kg)组(TSG-L组),连续灌胃给药4周.另取10只作为正常对照组.改善作用的评价指标包括一般状态、体重、血糖、糖耐量试验、血清胰岛素水平、胰岛素敏感/抵抗指数、骨骼肌脂质含量、脂质过氧化物水平和抗氧化酶活力等.结果给药期间,TSG对大鼠一般状态和体重无明显影响.模型大鼠出现明显的高血糖、糖耐量异常和胰岛素抵抗,同时骨骼肌组织中脂质水平升高,出现氧化应激状态.与DM组比较,TSG-H组、TSG-L组给药4周血糖水平明显降低(P＜0.05),模型大鼠的葡萄糖耐量明显改善.与DM组比较,TSG-H组、TSG-L组大鼠的胰岛素敏感指数显著升高(P＜0.01),同时胰岛素抵抗指数显著降低(P＜0.01).TSG可剂量依赖性地显著降低骨骼肌中三酰甘油和游离脂肪酸水平(P＜0.05),对于骨骼肌组织中的氧化应激状态也有明显的改善作用,降低丙二醛水平(P＜0.01),显著升高超氧化物歧化酶和过氧化氢酶活力水平(P＜ 0.01、P＜0.05).结论 TSG对于高脂饲料联合STZ诱导的2型糖尿病大鼠骨骼肌脂质蓄积及氧化应激具有明显的抑制作用,进而改善胰岛素抵抗及糖脂代谢紊乱.【期刊名称】《中国医药导报》【年(卷),期】2016(013)014【总页数】5页(P25-28,56)【关键词】何首乌;二苯乙烯苷;糖尿病;胰岛素抵抗;骨骼肌【作者】王婷;范益【作者单位】南京医科大学基础医学院,江苏南京210029;南京医科大学基础医学院,江苏南京210029【正文语种】中文【中图分类】R587［Abstract］Objective To investigate the improvement of tetrahydroxy stilbene glucoside（TSG）from Polygoni Multiflori Radix on glucose and lipid metabolism disorder and insulin resistance in skeletal muscle of type2 diabetes rats induced by high fat diet and streptozotocin（STZ）.Methods Male SD rats were administrated intraperitoneally 30mg/kg of STZ after 6 months of high fat diet fed.Model rats were dividedin 4 groups，model group（DM group），Rosiglitazone （RGLT group），TSG high dose（100 mg/kg）group（TSG-H group），TSG low dose（50 mg/kg）group（TSG-L group）.Therapeutic drugs were administrated intragastrically for 4 consecutive weeks.Another 10 rats were selected as normal control group.The improvement of TSG were evaluated by a range of indicators consisted of general state，body weight，blood glucose，glucose tolerance，serum insulin，insulin sensitivity/resistance index，the levels of lipids，lipid peroxide and antioxidant enzyme activities in skeletal muscle.Results During the period of drug delivery，TSG had no obviouseffect on general state and body weight.There were significantly changesin model rats，such as hyperglycemia，impaired glucose tolerance，insulin resistance，lipid deposition and oxidative stress in skeletal pared with DM group，TSG-H group and TSG-L group could significantly reduce blood glucose（P＜0.05）and ameliorate glucose pared with DM group，the insulin sensitivity index of TSG-H group and TSG-L group was increased（P＜0.01），while the insulin resistance was decreased significantly （P＜0.01）.TSG could dose-dependently reduce the levels of triglyceride and free fatty acids in skeletal muscle（P＜0.05），as well as inhibit oxidative stress involving of decrease of MDA （P＜0.01），raise the activities of superoxide dismutase and catalase （P＜0.01，P＜0.05）.Conclusion TSG can alleviate lipids accumulation and oxidative stress in skeletal muscle of type 2 diabetes rats induced by high fat diet and STZ，and then improve insulin resistance and glucose and lipid metabolism disorder.［Key words］Polygoni Multiflori Radix；Tetrahydroxy stilbene glucoside；Diabetes mellitus；Insulin resistance；Skeletal muscle近年来，肥胖、高血脂、高血糖等代谢性疾病发病率迅速增高，而胰岛素抵抗（insulin resistance，IR）是这些代谢性疾病的共同病理机制之一［1-2］。