TROSY and NMR of Large Proteins
Impact of TROSY on Solution NMR
Fernandex and Wider, Current Opinion in Structural Biology 2003, 13:570-580
Application of TROSY NMR on Biological Systems
Figure from K. Pervushin in EMBO Practical NMR Course 2003 http://www.embl-heidelberg.de/nmr/sattler/embo/coursenotes.html
Transverse Relaxation-Optimized Spectroscopy (TROSY)
(A) At High Magnetic Field (TROSY line-narrowing effect)
DD + CSA
(large) (large)
1H
(large) (large)
DD – CSA
DD
(large)
ห้องสมุดไป่ตู้
+ CSA
(small)
DD
(large)
– CSA
(small)
(B) At Low Magnetic Field (almost no TROSY line-narrowing effect)
Pushing the Size Limit by TROSY ,Deuteration, and Selective Isotope Labeling 723 a.a., 81.4 kDa
TROSY-HSQC of U-[2H,15N]-MSG at 800 MHz
MSG: Malate synthase G
Non-decoupling HSQC
TROSY-HSQC
Pervushin et al. PNAS USA, v94, p12366 (1997)
The Sensitivity and Resolution Gain by TROSY and Deuteration
u-2H,15N-Gyrase-45 (45 kDa), 750 MHz
Wider and Wuthrich, Current Opinion in Structural Biology, 1999, 9:594-601
TROSY Effect is Field Dependent and Motion Dependent
800 (kDa)
Linewidth
150 kDa 50 kDa
TROSY, 40 kDa, 750 MHz
Decoupled HSQC (during t1 &t2)
Linewidrh: 60% reduction in 1H, 40%reduction in 15N If perdeuterated: Expected reduction 40-fold for 1H & 10-fold for 15N
TROSY Application
• The implication of TROSY technique is revolutionary and wide spreading for large molecules (>20 KDa). Virtually all 1H,15N-HSQCbased double resonance and triple resonance experiments gain sensitivity and spectral resolution via TROSY. • Salzmann et al., “TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins” PNAS USA, v95, 13585-13590 (1998). • D. Yang and L.E. Kay, JACS, “TROSY Triple-Resonance FourDimensional NMR Spectroscopy of a 46 ns Tumbling Protein “ • Salzmann et al., “NMR Assignment and Secondary Structure Determination of an Octameric 110 kDa Protein Using TROSY in Triple Resonance Experiments”, JACS, 122, 7543-7548 (2000).
•DD relaxation is field-independent. However, CSA∝ B02, therefore at high magnetic fields, CSA relaxation can be comparable to DD relaxation, and the interference effect on relaxation can be observed.
(a). None-decoupled HSQC (b). Decoupled HSQC (c). TROSY-HSQC
15N
1. Main relaxation source for 1H and 15N: dipole-dipole (DD) coupling and, at high magnetic fields, chemical shift anisotropy (CSA). 2. Different relaxation rates (line width) for each of the four components of 15N-1H correlation. 3. The narrowest peak (the blue peak) is due to the constructive canceling of transverse relaxation caused by chemical shift anisotropy (CSA) and by dipole-dipole coupling at high magnetic field. 4. TROSY selectively detect only the narrowest component (1 out of 4).
Linewidth ∆υ1/2= 1/(πT2)
1D-HSQC
Figures from M. Sattler’s website
Pictures from M. Sattler, http://www.embl-heidelberg.de/nmr/sattler/teaching/
Current Techniques Designed to Attack the Size Limit
NMR hardware, new NMR methods, advanced in molecular biology • • • • • • • • • • Isotope labeling (15N, 13C), 3D, 4D triple resonance experiments: overlap NMR hardware: bigger magnets (overlap, s/n) Cryogenic probe: s/n (optimal 4-fold increase) TROSY, CRINPT: overlap, s/n Deuteration: overlap, s/n Selective isotope labeling: overlap Line-narrowing by low viscosity solvent: overlap, s/n Segmental Labeling: overlap Residual dipolar coupling: extra angle and long distance information Cross saturation: identify binding surface
>95% 1H, 15N, 13Cα, 13CO, 13Cβ assigned !!!
Tugarinov et al, JACS, 2002, 124, 10025-10035
3D NOESY-[1H, 15N, 1H]-ZQ-TROSY
NOESY[1H, 15N, 1H]ZQ-TROSY
110 kDa, [70% 2H, U-15N]-DHNA Diagonal peaks in NOESY are suppressed !
P. Gunter, Q. Rev. Biophys, 1998, v31, p145
Intrinsic Problems of Solution NMR of Large Molecules
• • Many more signals Slow tumbling fast decay of signal) spectral overlap fast transverse relaxation rate (short T2 , poor signal to noise (s/n)
1H
TROSY-HSQC and Conventional HSQC
TROSY-HSQC TROSY-HSQC (1) No 1H decoupling during 15N evolution. (2) No 15N decoupling during 1H acquisition. (3) Use the TROSY-HSQC pulse sequence to selectively observe the most slowly relaxing component.
Course Topic: TROSY and NMR of Large Proteins
紫茎泽兰花粉变应原的蛋白组分
１ ０ ，ａｄ ７０ ０ ｈｗ ｎ ｔｅＳ ＳＰ Ｇ ｆＥ ｐ ｔ ｉｍ ａｅｏ ｈ ｒｍ ｐｏｅ ｘ ａｔａｄ ｔ ｒ ａ ４０ ０ ｎ ０ ）ｓｏ ｎ ｉ ｈ Ｄ —Ａ Ｅ ｏ ｕ ａ ｒ ｄｎｐ ｏｕ ｒｔ ｎ ｅｔ ｃ ｎ ｈ ｅｗ ｓ ｏｕ ｉ ｒ ｅ
（ 明 医学 院 昆 第 一 附 属 医 院 耳鼻 喉科 ，昆 明 ６ ０ ３ ） ５０ ２
摘 要 ：目的 明确紫茎泽 兰花粉变应原 的蛋 白组分 ，探 索紫茎泽兰花粉 是否为云南 主要的春季致 敏花 粉之
一。ຫໍສະໝຸດ 方 法 适 时 采 集 紫 茎 泽 兰 花 粉 制 成 变 应 原 浸 液 ，提 取 蛋 白 ，通 过 十 二 烷 基 硫 酸 钠 ～ 丙 烯 酰 胺 凝 胶 电 泳 聚 Ｓ ＳＰ Ｇ 电 泳 检 测 发 现 ，青 蒿 Ｄ －Ａ Ｅ
ｆ Ｄ －Ａ Ｅ 检 测 其 蛋 白组 分 大 小 并 比 较其 与 青 蒿 花 粉 蛋 白组 分 的 相 似 性 。 结 果 Ｓ ＳＰ Ｇ ）
花粉 有 ６条 蛋 白条 带 ，相 对 分 子 质 量 分 别 为 ６ ０ ４００、３ ０ 、２ ０ 、１ ０ 、１ ０ ４００ ２００ ７００ ４０ ０和 ７００ ０ ；紫 茎 泽 兰 有 ４条 蛋 白条 带 ．相 对 分 子 质 量 分 别 为 ２ ０ 、１ ０ 、 １ ０ ２ ０ ７００ ４００和 ７ ０ 。青 蒿 和 紫茎 泽 兰 蛋 白条 带 平 均 灰 度 值 基 本 相 同 （ ０ ０ ０ Ｐ ＞． ） ００ 。结 论 紫茎 泽 兰 和 青 蒿 花粉 有 ４条 相 对 分子 质量 相 近 的蛋 白条 带 ，提 示两 种 花粉 可 能有 相 似 的变 应 原 致 敏 ５
（ ｅ ａｔ ｅ ｔｏ ｔ ａｙ ｇｌ ｙ ｉ ｔ ｆ ｌ ｔｄ Ｈ ｓ ｉ ｌ ｆ ｕ ｍ ｎ ｄｃｌＵ ｉｅ ｉ ，Ｋ ｎ ｉｇ６ ０ ３ ，Ｃ ｉａ Ｄ ｐ ｒ ｎ ｆ ａ ｒｎ ｏ ｇ ，Ｆｒ ｆｉ ｅ ｏｐｔ ｎ ｉ Ｍｅ ｉ ｎ ｒ ｔ ｍ Ｏｌ ｏ ｓ Ａ ｉａ ａｏ Ｋ ｇ ａ ｖ ｓｙ ｕ ｍ ｎ ５ ０ ２ ｈｎ ）
穹窿海马伞切断大鼠不同脑区酪氨酸激酶A表达的变化
穹窿海马伞切断大鼠不同脑区酪氨酸激酶A表达的变化郭宗君;金丽英;杜芳【期刊名称】《中国组织工程研究》【年(卷),期】2005(009)045【摘要】BACKGROUND: Cholinergic nervous system has a site which is affected by both blineurine and nerve growth factor receptor tyrosine kinase A (NGF-RTK-A). Whether cholinergic injury can affect the changes of tyrosine kinase A (TrKA) is significant to the application of nerve growth factor (NGF) to interfere cognitive disorder disease.OBJECTIVE: To observe the expressive variety of TrKA in different cerebral regions of rats with bilateral fimbria/fornix transection, and comprehend the effect offimbria/fornix transection on nerve growth factor receptor system.DESIGN: A completely randomized and controlled study on the basis of animals.SETTING: Institute of Cerebrovascular Disease, Affiliated Hospital of Qingdao University Medical College.MATERIALS: The experiment was completed in the Institute of Cerebrovascular Disease, Affiliated Hospital of Qingdao University Medical College from March to December 2003. Totally 14 male Wistar rats were randomly divided into control group and model group with 7 in each group.METHODS: The bilateral fimbria/fornix transection was performed on rats in the model group, but rats in the control group were treated with the same methods except fimbria/fornix transection. Twenty eight days after operation, rats in the two groups werekilled under anesthesia to take out brain tissues to stain with immunohistochemical staining.MAIN OUTCOME MEASURES: The number of positive TrKA cells in the hippocammpal CA1, cortex, amygdala and Meynert of the basal forebrain of rats in both groups.RESULTS: Totally 14 rats entered the final analysis. The numbers of positive TrKA cells in the hippocammpal CA1, cortex, amygdala and Meynert of the basal forebrain of rats in the model group were obviously smaller than those in the control group [(18.91 ±6.27), (15.17±5.23),(18.71±9.05), (8.03±2.33); (54.77±11.84), (59.69±10.40), (49.23±15.84),(21.49±15.54), t=4.17-10.00; P ＜0.01].CONCLUSION: The expression of TrkA positive neurons is decreased in many sections of the brain in the rats with fimbria/fornix transection.Possibly it is the cause leading to the cognitive disorder and senile dementia.%背景:胆碱能神经系统存在胆碱与神经生长因子受体酪氨酸激酶A共同作用的位点,胆碱能损伤是否影响到酪氨酸激酶A的变化,对于能否应用神经生长因子干预认知障碍疾病具有重要意义.目的:观察双侧穹窿海马伞切断大鼠脑内不同部位酪氨酸激酶A表达的变化,了解穹窿海马伞切断对神经生长因子受体系统的影响.设计:以动物为观察对象,完全随机对照实验.单位:青岛大学医学院附属医院脑血管病研究所.材料:实验于2003-03/12在青岛大学医学院附属医院脑血管病研究所完成.14只雌性Wistar大鼠随机分为对照组、模型组两组,每组7只.方法:模型组大鼠行双侧穹窿海马伞切断,对照组同样手术,但不切断穹窿海马伞.两组大鼠术后观察28 d后麻醉状态下处死,取脑,行免疫组化染色.主要观察指标:两组大鼠脑海马CA1区、皮质区、杏仁复合体区、基底前脑Meynert核区酪氨酸激酶A阳性细胞数量.结果:14只大鼠全部进入结果分析.模型组大鼠大脑皮质区、脑海马CA1区、杏仁复合体区、Meynert核区酪氨酸激酶A阳性细胞数显著少于对照组[(18.91±6.27),(15.17±5.23),(18.71±9.05),(8.03±2.33)个;(54.77±11.84),(59.69±10.40),(49.23±15.84),(21.49±15.54)个,t=4.17～10.00;P＜0.01].结论:穹窿海马伞切断大鼠脑内多部位酪氨酸激酶A表达减少,这种病理变化可能是认知障碍和老年性痴呆的原因之一.【总页数】3页(P141-143)【作者】郭宗君;金丽英;杜芳【作者单位】青岛大学医学院附属医院脑血管病研究所,山东省青岛市,266003;青岛大学医学院附属医院脑血管病研究所,山东省青岛市,266003;青岛大学医学院附属医院脑血管病研究所,山东省青岛市,266003【正文语种】中文【中图分类】R338【相关文献】1.穹窿海马伞切断和卵巢切除对大鼠不同脑区雌激素受体α与淀粉样β蛋白前体蛋白β位点裂解酶共存表达的影响 [J], 金丽英;郭宗君;崔红波2.去卵巢及穹窿海马伞切断复合模型大鼠不同脑区雌激素α受体mRNA的表达及倍美力的干预作用 [J], 赵珩;贾晓静;张昱;李晶3.穹窿海马伞切断大鼠不同脑区胆碱乙酰化转移酶表达的变化 [J], 郭宗君;金丽英;银建军4.穹窿海马伞切断大鼠不同脑区雌激素受体α的表达 [J], 金丽英;郭宗君;崔红波5.穹窿-海马伞切断与卵巢切除对大鼠不同脑区酪氨酸激酶A表达的影响 [J], 金丽英;郭宗君;辛萍;杨学伟因版权原因，仅展示原文概要，查看原文内容请购买。
姜黄素对荷瘤小鼠红细胞免疫功能的影响
姜黄素对荷瘤小鼠红细胞免疫功能的影响贾绍华;王巧玲;彭海生;赵瑛;曲中原;张秀娟【期刊名称】《哈尔滨商业大学学报（自然科学版）》【年(卷),期】2009(025)006【摘要】研究姜黄素对S180和H22荷瘤小鼠红细胞免疫功能的影响.观察姜黄素对荷瘤小鼠红细胞免疫黏附肿瘤细胞的能力;观察姜黄素对荷瘤小鼠红细胞免疫调节因子活性的影响;采用DPH荧光探针,用荧光偏振法测定荧光偏振度(P),并计算膜的微黏度(η)研究红细胞膜脂流动性(LFU).姜黄素能对两种荷瘤小鼠均具有非常显著的抑瘤作用;能够升高两种荷瘤小鼠的红细胞黏附肿瘤细胞的花环率;显著提高两种荷瘤小鼠红细胞C3b受体花环促进率,降低其抑制率;提高两种荷瘤小鼠的红细胞膜的流动性.姜黄素可能是通过提高两种荷瘤小鼠红细胞免疫调节因子活性及红细胞膜脂流动性.从而恢复荷瘤小鼠红细胞免疫功能进而达到抗肿瘤目的.【总页数】4页(P657-660)【作者】贾绍华;王巧玲;彭海生;赵瑛;曲中原;张秀娟【作者单位】哈尔滨商业大学药学院,哈尔滨150076;哈尔滨商业大学生命科学与环境科学研究中心,哈尔滨150076;哈尔滨医科大学大庆校区,黑龙江大庆163319;哈尔滨商业大学药学院,哈尔滨150076;哈尔滨商业大学药学院,哈尔滨150076;哈尔滨商业大学药学院,哈尔滨150076【正文语种】中文【中图分类】R285【相关文献】1.补虚化瘀方对化疗荷瘤小鼠红细胞免疫功能及血液流变学的影响 [J], 田卫卫;李偶;黄映红;邓道昌;王庆苗2.秦皮乙素对荷瘤小鼠红细胞免疫功能的影响 [J], 许冉达;邵天宇;贾绍华3.黑木耳多糖对荷瘤小鼠红细胞免疫功能的影响 [J], 张秀娟;耿丹;于慧茹;季宇彬4.糙叶败酱大孔吸附树脂提取物对荷瘤小鼠红细胞免疫功能的影响 [J], 王学习;赵健雄;程卫东;陈茹;白德成5.龙葵碱对荷瘤小鼠红细胞免疫功能的影响 [J], 季宇彬;万梅绪;高世勇;邹翔因版权原因，仅展示原文概要，查看原文内容请购买。
高尔基体概述
高尔基体概述高尔基体（Golgi apparatus）是由许多扁平的囊泡构成的以分泌为主要功能的细胞器。
又称高尔基器或高尔基复合体；在高等植物细胞中称分散高尔基体。
最早发现于1855年，1898年由意大利人卡米洛•高尔基（Camillo Golgi，1844-1926）在光学显微镜下研究银盐浸染的猫头鹰神经细胞内观察到了清晰的结构，因此定名为高尔基体。
因为这种细胞器的折射率与细胞质基质很相近，所以在活细胞中不易看到。
高尔基体从发现至今已有100多年的历史，其中一半以上的时间是进行关于高尔基体的形态甚至是它是否真实存在的争论。
细胞学家赋予它几十种不同的名称，也有很多人认为高尔基体是由于固定和染色而产生的人工假像。
直到20世纪50年代应用电子显微镜才清晰地看出它的亚显微结构。
它不仅存在于动植物细胞中，而且也存在于原生动物和真菌细胞内。
形态与组成高尔基体是由数个扁平囊泡堆在一起形成的高度有极性的细胞器。
常分布于内质网与细胞膜之间，呈弓形或半球形，凸出的一面对着内质网称为形成面（forming face）或顺面（cis face）。
凹进的一面对着质膜称为成熟面（mature face）或反面（trans face）。
顺面和反面都有一些或大或小的运输小泡，在具有极性的细胞中，高尔基体常大量分布于分泌端的细胞质中。
顺面和反面都有一些或大或小的运输小泡（图6-24），在具有极性的细胞中，高尔基体常大量分布于分泌端的细胞质中（图6-25）。
图6-24高尔基体各部分的名称图6-25培养的上皮细胞中高尔基体的分布（高尔基体为红色，核为绿色）引自/因其看上极像滑面内质网，因此有科学家认为它是由滑面内质网进化而来的。
扁平囊的直径为1μm，由单层膜构成，膜厚6~7nm，中间形成囊腔，周缘多呈泡状，4~8个扁平囊在一起，某些藻类可达一二十个，构成高尔基体的主体，称为高尔基堆（Golgi stack）。
高尔基体膜含有大约60%的蛋白和40%的脂类，具有一些和ER共同的蛋白成分。
利用网络药理学和分子对接技术分析“猫爪草-_冬凌草”对肝癌的治疗作用机制
[基金项目]贵州省科技计划项目（黔科合支撑[2020]4Y156号）。
△杨青斌，李进 贵州中医药大学2021级中西医结合临床专业在读硕士研究生△司维群，刘静 贵州中医药大学2022级中西医结合临床专业在读硕士研究生△吴佳龙 贵州中医药大学2023级中西医结合临床专业在读硕士研究生▲通讯作者利用网络药理学和分子对接技术分析“猫爪草-冬凌草”对肝癌的治疗作用机制杨青斌1△ 徐 静2▲ 李 进1△ 司维群1△ 刘 静1△ 吴佳龙1△1.贵州中医药大学，贵州贵阳 550000；2.贵州中医药大学第二附属医院肿瘤科，贵州贵阳 550000[摘要]目的 利用网络药理学和分子对接技术对猫爪草与冬凌草进行分析，探讨其治疗原发性肝癌的作用机制。
方法 基于对中药系统药理学数据库和分析平台（TCMSP）与传统中药百科全书（ETCM）数据库的检索，掌握有关药物的有效成分与其作用靶点，借助有机小分子生物活性数据库（PubChem）以及小分子药物预测作用靶点平台（Swiss Target Prediction）的支持下，收集一系列肝癌的靶点。
参照蛋白互作数据库（STRING）进行药物与肝癌的蛋白互作（PPI）网络的构建。
在将网络系统应用于探索药物的抗癌过程和作用机理的同时，结合Cytoscope3.9.1软件成功构建“成分-靶点-通路”体系，并借助Metascape 功能实现对共同靶点和京都基因与基因组百科全书（KEGG）通路的富集处理。
此外，利用分子模拟软件AutoDock 展开活性成分与关键靶点间分子对接情况的探究，获取所需的系列研究成果。
结果 筛选出冬凌草与猫爪草的有效活性成分分别有23、9个；两味药的药物靶点总共1020个，共同交集靶点299个；药物和肿瘤疾病共有26个常见靶点。
KEGG 通路的富集显示，药物靶点涉及的通路主要有癌症通路、脂质与动脉粥样硬化通路、PI3K-Akt 信号通路等。
结论 猫爪草与冬凌草相须配伍，具有多成分、多靶点、多通路的作用特点，能协同发挥抗肝癌作用，为后续机制研究提供了切实可行的参考依据。
雷公藤内酯醇对阿尔茨海默病模型大鼠海马突触素表达及突触超微结构的影响
（ ． １ 土 ０ ０ ７８ ｏ Ｏ ３ ８Ｏ ． ２ ） ｆｉ ｍｍｕ ｏ ｅｃｉｅｐ ｏ ｕ ｔｏ ｙ ａ ｔｐ ｙ ｉ ｎ ｒ ａｅ ｎ ｔ ｅｔｅ ｔ ｎ ｒ ｕ ；ｔｅｖ ｌｍｅ（ １ ． １ ｎ ｒａ ｔ ｒ ｄ ｃ ｆｓ ｎ ｐ ｏ ｈ ｓｎ ｉｃｅ ｓｄ ｉ ｈ ｒａｍｅ ｔｇ ｏ ｐ ｈ ｏｕ ｖ １９ ７ ７ ±
（ 南昌大学医学院解剖 学教研室 ，南 昌 ３０ ０ ） ３ ０ ６
摘要 目的： 探讨雷公藤 内酯醇对 阿尔茨海默病模型大 鼠海马突触素表达及 突触超微 结构 的影 响。方法 ： 鼠随机 分成对 大
照组、 模型组 、 治疗组 。模 型组给予 双侧海 马各一次性注射凝 聚态 Ａ 。 ０ ， １ ｇ 治疗 组在海 马注射凝 聚态 Ａ 。 ， 日腹 后 每
腔注射雷公藤 内酯醇 ０ ４ｒｇ ｋ ，１ ． ／ ｇ ５ ａ ｄ后用免疫组织化学方法和蛋白免疫印迹技 术检 测海 马突触 素表 达情况 ， 透射 电镜观 察 突触结构 的变化 。结果 ： 与模 型组 相 比， 治疗 组海 马 区突触 素免疫 反应 阳性 产物 数量 （ ５ ． ０ ５ ７ ） １ ２８ 士１ ．６ 及平 均光 密度 （ ． １ ±０ ０７８ 均增加 ； Ｏ３８ Ｏ ． ２ ） 突触素表达总量 （ １ ． １ －１ ０ ） １ ７ ７ ４４ ． ２及密度 比值 （． ７ ． ３ 亦增加 ； ９ Ｏ ８ ±０ ０ ） 突触结构较清 晰 ， 界面增 长， 突触后 电子致 密物增厚 。结论 ： 雷公藤 内酯醇 可以增加 阿尔 茨海默病 模型大 鼠海马突触 素 的表 达 ， 轻阿尔茨 海默病 减 模 型大 鼠海马突触损伤程度 。 关键词 雷公藤 内酯醇 ；阿尔茨海 默病 ； 突触素 ； 突触 ； 马 ； 鼠 海 大
《基于RNA-Seq技术分析藻蓝蛋白对自然衰老小鼠卵巢功能恢复的机理研究》范文
《基于RNA-Seq技术分析藻蓝蛋白对自然衰老小鼠卵巢功能恢复的机理研究》篇一一、引言随着人口老龄化问题日益突出，研究如何延缓卵巢功能衰退，提高老年女性生活质量已成为科研领域的重要课题。
藻蓝蛋白作为一种天然的生物活性物质，其具有抗氧化、抗衰老等生物活性，被认为对卵巢功能恢复具有潜在的作用。
本研究利用RNA-Seq技术，深入探讨藻蓝蛋白对自然衰老小鼠卵巢功能恢复的机理。
二、材料与方法1. 材料（1）实验动物：选用自然衰老小鼠作为研究对象。
（2）藻蓝蛋白：购买自正规生物制品公司，纯度较高。
（3）RNA-Seq技术：用于检测小鼠卵巢组织中基因表达的变化。
2. 方法（1）分组与处理：将小鼠分为对照组和实验组，实验组小鼠给予藻蓝蛋白处理，对照组则不作处理。
（2）卵巢组织收集：处理一段时间后，收集小鼠卵巢组织。
（3）RNA提取与测序：提取卵巢组织中的RNA，进行RNA-Seq测序。
（4）数据分析：对测序结果进行生物信息学分析，比较两组间基因表达差异。
三、结果与分析1. 基因表达差异分析通过RNA-Seq技术，我们发现在实验组小鼠卵巢组织中，有大量基因的表达发生了改变。
其中，与抗氧化、抗炎、细胞增殖和凋亡等相关的基因表达明显上调，而与衰老相关的基因表达则明显下调。
这表明藻蓝蛋白可能通过调节这些基因的表达，从而发挥其对卵巢功能的恢复作用。
2. 信号通路分析进一步分析发现，藻蓝蛋白处理的实验组小鼠卵巢组织中，与PI3K/Akt、MAPK等信号通路相关的基因表达发生了明显变化。
这些信号通路在细胞生长、增殖、存活和凋亡等过程中发挥重要作用，可能与藻蓝蛋白对卵巢功能的恢复有关。
3. 生物学功能验证为了进一步验证RNA-Seq结果的可靠性，我们通过Western blot、PCR等方法检测了部分关键基因和蛋白的表达情况。
结果表明，RNA-Seq结果与生物学功能验证结果基本一致，进一步证实了藻蓝蛋白对自然衰老小鼠卵巢功能恢复的作用。
羊驼皮肤中核糖体蛋白L41的表达
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羊 驼 皮 肤 中核 糖 体 蛋 白 Ｌ １的表 达 ４
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核糖 体 蛋 白 （ｉｏｏ ｌ ｒｔｉ， Ｐ） 一 种 细 ｒ ｓ ｍａ ｐ ｏｅ Ｒ 是 ｂ ｎ 胞 蛋 白合成 的重 要组 成部分 。 目前 为止 ， ８ 约 ０种 核 糖体 蛋 白已经确 定 。核糖 体在 蛋 白质合 成 中起 关 键 作用 ， 如参 与 Ｄ ＮＡ 修 复 、 胞 凋 亡 、 录调 控 和 翻 细 转 译 调 控口 ］ 。核 糖 体 蛋 白 ＲＰ ４ （ｉｏ ｏ ｌ ｒｔｉ Ｌ １ ｒ ｓ ｍａ ｐ ｏｅｎ ｂ
本 的精氨 酸和 赖 氨 酸组 成 的小 肽 ， 蛋 白功 能 包 括 其 Ｎ 端 缺 失 的 蛋 氨 酸 ， 酰 化 、甲 基 化 或 羟 基 化 ， 一 乙 Ｒ Ｌ １ 因 在 细 胞 凋 亡 过 程 中 发 挥 很 重 要 的 作 Ｐ４基
用＿ 。羊驼 毛 色 和 毛 品 质 受 诸 多 基 因 调 控 。Ｂ ｎ ５ ］ ｅ—
经分 析发 现 羊 驼 Ｒ Ｌ １基 因序 列 中存在 Ｃ ＧＧ 位 点 ， 测 羊驼 ＲＰ ４ Ｐ ４ Ｃ 推 Ｌ １基 因在 此位 点 发 生 甲基 化 。表 明 ＲＰ ４ 基 因可能参 与皮肤 细胞 凋 亡及毛 生 长周期 的调 节 。 Ｌ１
跨物种扩增筛选获得蓝马鸡Crossoptilon auritum微卫星多态分子标记英文
Chinese Birds 2012,3(2):103–107SHORT COMMUNICA TIONReceived 04March 2012;accepted 10May 2012Author for correspondence (Zhengwang Zhang)E-mail:zzw@*Y ang L iuPresent address:State Key L aboratory of Biocontrol and School of LifeSciences,Sun Y at-Sen University,Guangzhou 510275,ChinaA panel of polymorphic microsatellites in the Blue Eared P heasant (Crossoptilon auritum)developed by cross-species amplificationLangyu GU 1,Y ang LIU 2,*,N ngA NG 1,Zhengwang ZHA NG 1,1MOE K ey Laboratory for Biodiv ersity Sciences and Ecological Engineering,College of L ife Sciences,Beijing Normal University ,Beijing 100875,China2Computational and Molecular Population Genetics,Institute of Ecology and Ev olution,Univ ersity of Bern,Baltzerstrasse 6,3012,Bern,SwitzerlandAbstr act Polymorphic microsatellites are among the versatile genetic markers in molecular ecology studies.In contrast to de no vo isolation of microsatellites from target species,cross-species ampli ca-tion is a cost-effective approach for a fast development of microsatellite markers from closely related taxa.In our study ,we cross-ampli ed a panel of poly morphic microsatellite markers for the Blue Eared Pheasant (Crosso ptilon auritum),a species endemic to China of considerable conservation con-cern.We obtained 11polymorphic microsatellite markers selected from 112candidate loci,originally isolated from other Galliforme species.This panel of makers has shown moderate to high lev els of polymorphism and include a Z-chromosomal linkage locus.We carried out preliminary analy ses of parentage among captive individuals with a known pedigree using this new panel of microsatellites.Our results suggest that the high utility of these markers may be powerful tools for studies in conser-vation genetics of eared-pheasants and other endang ered Galliforme species.Keywords Crossoptilon auritum,microsatellites,cross-species ampli cation,Z-chromosomal linkag eInt roductionEndemic species have long been a key focus in conser-vation efforts (Myers et al.,2000),given that the levelof endemics might be positiv ely correlated with species richness (Lamoreux et al.,2006).Besides,endemic spe-cies with limited dispersal capacity might be sensitive to changes in local climate,or vulnerable to invasive spe-cies (Ohlem üller et al.,2008).A good understanding of ev olutionary processes such as population subdivisions,changes of effective population size and genetic connec-tivity of endemic species would shed light on evolution-ary processes as well as on conservation manag ement.The Blue Eared Pheasant (Crossoptilon auritum),belonging to Phasianidae,Galliformes,is a rare and en-demic pheasant species in western China (L ei and L u,2006).Its wild populations are found at Helan Moun-Chinese Birds2012,3(2):103–107 104tain,as well as along the eastern edge of the Qinghai-Tibetan Plateau(QTP),cov ering Qing hai,Gansu and Sichuan provinces(L ei and L u,2006).A lthough previ-ous studies have been carried out on the biology and ecology of C.auritum(Sun et al.,2005;Li et al.,2009; Wu and L iu,2010),a thorough assessment of genetic diversity is urg ently needed to assess the population vi-ability of this species.Furthermore,apart from C.auri-tum,the genus Crosso ptilon includes three other species, i.e.,C.mantchuricum,C.harmani and C.cro ssoptilo n. These species are endemic to China(Zheng,2011)and all are listed on the IUCN Red L ist of Threatened Spe-cies(IUCN2011)because of a rapid decline in the size of their population,caused by habitat fragmentation and hunting(Lei and Lu,2006).Giv en these concerns, obtaining molecular markers is a prerequisite in un-derstanding the genetic background of C.auritum and might be useful for population genetic studies in Cros-so ptilon species.Microsatellites are powerful tools for conservation genetic studies such as population genetics,mating systems and inv estigations into kinship(Primmer et al., 2005;Karl et al.,2011).Compared with isolated novel microsatellite markers,cross-species microsatellite am-pli cation from closely related species is cost-effective (Zane et al.,2002).More importantly,it has been sug-gested that this method has successfully worked among species belonging to the same genera,different genera and even different families(Barbaráet al.,2007;Huang and L iao,2010).Given that numerous microsatellite markers have been dev eloped for various Phasianidae species(Cheng et al.,1995;Wang et al.,2009;Zhou and Zhang,2009),we attempted to establish microsatellite markers for C.auritum through cross-species ampli-cation from a large number of marker candidates.In order to test the effectiveness of these markers,we also carried out preliminar y parentage analysis among cap-tive individuals of known pedigree.Mate ria ls and methodsA total of20C.auritum blood samples from brachial veins were collected to develop microsatellite loci by cross-species ampli cation,nine of which were from the Linxia Zoo in Gansu Province and the other11 from Huzhu in Qinghai Province,China.Additionally, nine individual birds from two families of the Beijing Wildlife Park in Daxing and Beijing Zoo were used to conduct parentage analy sis.We have detailed the known pedig ree of these two families of birds.Genomic DNA was extracted using DNA extraction kits(Tian Gen Biotech,Beijing,China).The cross-spe-cies microsatellite markers(Table1)came from various Galliforme species,including16loci from Meleag risgal-lopavo(Burt et al.,2003),30from T rag opan temminck ii (Zhou and Zhang,2009),20from Syrmatic us reevesii (Wang et al.,2009),41from Gallus gallus(Cheng et al., 1995;Dawson et al.,2010),four from C.mantchuric um (Zhao et al.,Beijing Normal Univ ersity,unpublished results)and one from Syrmaticus mikado(S.H.L i,T ai-wan Normal Univ ersity,unpublished results).After ltering out those loci with poor cross-ampli cation, the polymorphism of the remaining pairs were tested with either6-FA M or HEX uorescent dyelabeled on 5′of a single forward primer.Polymerase chain reac-tion(PCR)was carried out in a10L reaction system containing100ng DNA,0.25L of each primer,1L of a10×PCR buffer,1.5mM Mg Cl2,0.2mM dNTP mix and0.75U T aq polymerase(Takara,Japan).The reac-tion was denatured at94°C for5min,followed by40 cy cles at94°C for30s,a touch-down annealing process from58–47°C,reducing in steps of0.5°C per cycle and another20cycles annealing at47°C and then72°C for 50s,with a nal extension at72°C for5min.Fragment analy sis was conducted on an ABI PRISM3100Genetic Analyzer using the GeneMapper software(Applied Biosystems)with ROX-500as the standard for size.We conducted PCR ampli cation and fragment analysis at least twice to ensure the accuracy of individual geno-types.Sequencing of selected homozygotes was also conducted in both directions with ampli cation prim-ers(BGI Bio T ech,Beijing,China)to ensure that the products were genuine microsatellites.We blast the acquired microsatellite sequences from C.auritum on the chicken genome in GenBank to nd their locations and to inv estigate the potential linkages among the loci.If some loci were mapped in sex chro-mosome,we carried out sex ing identi cation with ourLangyu Gu et al.Microsatellite DNA loci in Blue Eared Pheasant105sample set by using primer sex1/sex2(Wang and Zhang,2009)to cross-validate the sex-linkage loci.We tested observed heterozygosity(HO),expectedheterozygosity(HE),the number of allele per locus, the Hardy-Weinberg equilibrium(HWE)and linkage-disequilibrium in each population using A rlequinv.3.11 (Excof er et al.,2005).W e calculated the frequencies of null alleles by using FreeNA(Chapuis and Estoup, 2007).High polymorphic markers with HWE were used for paternity tests in CERVUS3.0with100000 times simulations to estimate their resolving power. Signi cant levels were recorded after application of the sequential Bonferroni correction(Rice,1989;Ex cof er et al.,2005).ResultsSixty-two(55.4%)of the112cross-species markers could be ef ciently ampli ed in C.auritum,while11 (17.7%)of the62markers had a moderate to high level of polymorphism(3–11loci),with the expected het-erozyg osities ranging from0.42to0.89(T able1).Locus 4C12was found to be homozyg ous in heterogametic fe-males and heterozy gous in56.2%of the males(n=28) and thus most likely to link with the Z chromosome. Three loci(1H4,2420and4F8)were not targ eted on the chicken genome.A ll loci were at the HWE;neither linkage disequilibrium among pairs of loci nor null al-leles was found.The parentage analysis results showedTable1Characterization of11microsatellite lociL ocus Primer sequences(5′–3′)A ccessionNo.Repeatmotifsn NASize range(bp)HOHESpecies origin Chromosome2580F:TTAACCTA TCAGGTCGTTGCG AL592580(CA)n208191–213 1.000.80M.gallopavo21 R:CA GTGCACA TGCA GGCA G3D2F:TCTCTGA CGTA TCGCA TCT FJ221373(GT)n196286–3040.470.58S.reevesii4 R:A CTTCCCCTGGTAAA CT1H4F:TGAACA AGTGA GGCGGAGC/(TG)n2010127–1610.650.81S.reevesii/ R:CTGCACA CAGCCCGAA GC2420F:CA TCA TCTGCCAA TGCA GAGG/(TTTA)n204118–1420.550.54S.mikado/ R:A AGCCCA TA TA TGCTTCCTGG4H1F:TA TGAAA CAGA CTTAA TCC FJ221388(GTTT)n204203–2110.850.67S.reevesii1 R:TGCAGCA TTTGA GT AAC5C9F:TA TGGGAA A TGTGTACCTTT A GQ184557(CA)n2010221–2590.950.89 C.mantchuricum10 R:TCCA GGCAA CACGTAA CATT06F:TGAGAGA TTTTGACCCA GQ181183(CA)n207225–2370.850.83T.temminckii6 R:CAA GACTTCA CCCTA CAGA TA4F8F:GTGGCA TGCCTAGTA GA TGTT/(A C)n2011186–2140.750.88 C.mantchuricum/ R:CCCTGTGGTA CGAACTGTCSR11F:A TCAA T A TGGACTGCTCCGT FJ221381(TG)n205210–2480.550.58S.reevesii17 R:TCCTTCA AGGCCAAGTG5H7F:CCAA GAGGGA GGCA CACGTTC U60782(TG)n203186–1940.550.42G.gallus8 R:A GCCA TAAA T AAGCAAA CGC4C12F:A T AGGCGGACA GAGGA T AGA FJ221385(CA)n204160–1700.300.59S.reevesii Z R:CCCCGCA TCGAGGTGNotes:n is the number of successfully genotyped individuals and NA the number of alleles.HOis observed heterozygosity and HEexpected heterozygosity.Chinese Birds2012,3(2):103–107 106that if neither parent was known,the successful assig n-ment rates were98%at a95%con dence lev el and 98%at an80%con dence level.Paternity test results are consistent with known pedigrees.DiscussionThe phylogenetic relationship between the original and target species seems to affect the success of cross-species ampli cation(Primmer et al.,1996).In our analyses, cross-species ampli cation from Crossoptilon was the most effective(2/4=50%),followed by those from Syrmaticus(6/21=28.6%),Meleag ris(1/16=6.25%), Trag opan(1/30= 3.0%)and Ga llus(1/41=2.4%). Phylogenetic analysis showed a successiv e relationship between Crossoptilon and the genera mentioned earlier (Kimball et al.,2011),indicating that evolutionary re-lationships may play an important role in cross-species ampli cation of microsatellite markers within Gallifor-mes.A lthough large numbers of microsatellite markers are found in autosomes,Z-linked microsatellites markers are still rarely av ailable,ev en in the well-characterized chicken genome(Groenen et al.,2000).One Z-linked marker TUT,originally isolated from the T etrao uro gal-lus(Seg elbacher et al.,2000,Wang et al.,2011),failed to be ampli ed in chickens and mig ht be speci c for T etr-aoninae grouse.In the present study,the Z-linked poly-morphic locus4C12seems to hav e general application in different Galliforme species and might yet provide a valuable tool in kinship and demographic analyses combined with other loci.Acknowledgements The study was supported by the National Key Project of the Scienti c and Technical Supporting Programs Funded by the Ministry of Science and T echnology of China (2012BA C01B06)and the National Natural Science Founda-tion of China(No.30570234).We thank Jiliang X u,Xinli Zhao, Jiang Chang and Ying Liu for the collection of samples,as well as administrations of the Y inchuan Zoo in Ningxia A utonomous Region,the Xining Zoo in Qinghai Province,the Linxia Dongjiao Zoo in Gansu Province and the Beijing Wildlife Park and Beijing Zoo for providing samples.We thank Xinli Zhao for provid-ing unpublished markers from Crossoptilon mantchuricum and Shou-Hsien Li for providing unpublished markers from Syrmati-cus mikado.We thank Lu Dong,Xiangjiang Zhan,Ning Wang and Y ingying Liu for guidance with data analysis.ReferencesBarbaráT,Palma-Silva C,Paggi GM,Bered F,Fay MF,L exer C.2007.Cross-species transfer of nuclear microsatellite markers: potential and limitations.Mol Ecol,16:3759–3767.Burt DW,Morrice DR,Sewalem A,Smith J,Paton IR,Smith EJ, Bentley J,Hocking PM.2003.Preliminary linkage map of the T urkey(Meleagris gallopavo)based on microsatellite markers.A nim Genet,34:399–409.Chapuis MP,Estoup A.2007.Microsatellite null alleles and esti-mation of population differentiation.Mol Biol Evol,24:621–631.Cheng HH,Levin I,V allejo RL,Khatib H,Dodgson JB,Critten-den LB,Hillel J.1995.Development of a genetic map of the chicken with markers of high utility.Poult Sci,74:1855–1874. 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改进的TROSY序列-BioNMRLabUSTC
PEP-HSQC
3. PFG-PEP-HSQC
通常称为”gradient-enhanced HSQC”， 梯度场脉冲可以用于相干传递途径的选择， 但经常导致一半信号的损失，因为梯度场脉 冲选择信号依据信号的绝对相干阶。但是梯 度场脉冲可以同PEP-HSQC结合起来，既保 留正交的两个分量，又没有一半传递途径被 抑制的问题。
750 MHz下的氘代蛋白质的理论线宽
蛋白质分子量 150 kDa 800 kDa
1H线宽（窄） 1H线宽（宽） 15N线宽（窄） 15N线宽（宽）
15 Hz 50 Hz
70 Hz 350 Hz
5 Hz 15 Hz
60 Hz 300 Hz
生物大分子波生物大分子波谱学原理 吴季辉
生物大分子波谱学原理 吴季辉
PEP-HSQC
生物大分子波谱学原理 吴季辉
PEP-HSQC
生物大分子波谱学原理 吴季辉
PEP-HSQC
生物大分子波谱学原理 吴季辉
PEP-HSQC
生物大分子波谱学原理 吴季辉
PEP-HSQC
生物大分子波谱学原理 吴季辉
PEP-HSQC
生物大分子波谱学原理 吴季辉
Problem set 1
1. 请查找IPAP-HSQC的相关文献，分析该 脉冲序列（包括乘积算符，相干阶途径， 相位循环，梯度，正交检波，FID处理 等）；
2. 请对这个脉冲序列提出进一步的改进思 路。 3. 对于大蛋白，如何测量N-H之间的耦合 常数？请提出自己的思路。 作业5.20日前email给我: kruan@,
液体NMR技术的发展： TROSY
（Transverse relaxation-optimized spectroscopy ）