Effects of Molybdenum on the Intermediates of Chlorophyll Biosynthesis in Winter Wheat Cultivars
牡蛎寡肽对免疫低下小鼠模型免疫功能的影响
牡蛎寡肽对免疫低下小鼠模型免疫功能的影响刘淑集;蔡水淋;刘智禹;许旻;苏永昌;王茵;潘南;乔琨;吴靖娜;苏捷;张伯超【期刊名称】《华南师范大学学报（自然科学版）》【年(卷),期】2018(050)002【摘要】The effect of oyster oligopeptide on regulation of immune function in cyclophosphamide （CTX)-induced immunosuppressive mice was evaluated.The indexes of immune organs such as liver and spleen,the transformation and propagation ratio of T lymphocyte,the activity of NK cell,the humoral immunity,macrophage phagocytosis,the level of cell cytokines and hemolysin were assessed.The experiment results revealed that compared with the model immunosuppressed group,oyster oligopeptide could significantly enhance the proliferation of spleen lymphocyte,NK cell activity,expurgation index k,devouring index a,ability of devouring neutral red,the level of TNF-α、IL-6 and hemolysin,and regulate the distribution of CD3+ 4T and CD3+ 8T spleen lymphocytesubsets.However,the influence of oyster oligopeptide on the immune organs was not significant.It was concluded that oyster oligopeptide can improve the cell immunity,humoral immunity and non-specific immune function of immunosuppressive mice induced by CTX,and it can regulate the immune function positively in mice.%采用腹腔注射环磷酰胺(Cyclophosvnamide,CTX)建立免疫低下小鼠模型,研究不同剂量牡蛎寡肽对小鼠免疫器官脏器指数、脾淋巴细胞转化增殖情况、自然杀伤细胞(Natural Killercell,NK)细胞活性、小鼠体液免疫、巨噬细胞吞噬能力、血清TNF-α、IL-6和溶血素水平的影响.结果显示:与免疫抑制模型组小鼠相比,牡蛎寡肽能够显著提高脾淋巴细胞增殖能力、NK细胞活性、廓清指数K、吞噬指数a、吞噬中性红能力、TNF-α、IL-6、溶血素水平和脾淋巴细胞CD3+ 4T淋巴细胞亚群及CD3+ 8T淋巴细胞亚群分布(P＜0.05),而对小鼠的肝、脾脏指数影响不显著,表明牡蛎寡肽能够提高由CTX引起的免疫低下模型小鼠的细胞免疫、体液免疫及非特异性免疫功能,对小鼠的免疫功能具有正面调控的作用.【总页数】7页(P70-76)【作者】刘淑集;蔡水淋;刘智禹;许旻;苏永昌;王茵;潘南;乔琨;吴靖娜;苏捷;张伯超【作者单位】福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建农林大学食品科学学院,福州350002;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013;福建省水产研究所∥福建省海洋生物增养殖与高值化利用重点实验室∥福建省海洋生物资源开发利用协同创新中心,厦门361013【正文语种】中文【中图分类】R151【相关文献】1.槲皮素对免疫低下小鼠免疫功能的影响 [J], 田瑞雪; 孙耀宗; 姚有昊; 张子健; 张继民; 宋维芳2.红景天当归不同配伍对免疫低下小鼠免疫功能的影响 [J], 史顶聪; 赵宏宇; 佟晓乐; 张凤清3.鹰嘴豆肽对免疫低下小鼠免疫功能的影响 [J], 李睿珺;秦勇;周雅琳;刘伟;李雍;于兰兰;陈宇涵;许雅君4.中药复方“益智灵”对臭氧所致衰老免疫低下小鼠模型免疫功能改善作用研究[J], 周勇;严宣佐;徐秋萍;吴金英5.绞股蓝总苷对免疫功能低下小鼠模型特异性免疫功能的影响 [J], 周俐;叶开和;任先达因版权原因，仅展示原文概要，查看原文内容请购买。
莫诺苷对局灶性脑缺血再灌注大鼠皮层IL-1β的影响
Ｋｅ ｏ ｄ ：ｍｏ ｒ ｎ ｓｄ ；ｃ ｒ ｂ ａ ｓ ｈ ｍｉ／ ｅ ｅ ｆ ｓｏ ；Ｉ 一 Ｂ ｎ ｉ ｆ ｍｍａ ｏ ｙ ｅ ｒ ｐ ｏ ｅ ｔ ｅ ｙｗ ｒ ｓ ｒ ｏ ｉｉ ｅ ｅ ｅ ｒ ｌｉ ｅ ａ ｒ ｐ ｒｕ ｉ ｎ Ｌ １ ；ａ ｔｉ ｌ ｃ ｎ ａ ｔ ｒ ；ｎ ｕ ０ ｒ ｔ ｃ ｉ ｖ
Ｈｏｐｉ ｌｏ ａ ｔ ｌ ｅ ｉａ ｎ ｖ ｒｉ ｓ ｔ ｆ Ｃ ｐｉ ｄ ｃ ｌＵ ｉｅｓｔ ａ ａ Ｍ ｙ，Ｂｅｊ ｎ ０ ０ ３ ｈ ｎ ｉｉｇ １ ０ ５ ，Ｃ ｉａ Ａｂｔａ ｔ ｓｒ ｃ：Ｏｂｅ ｔ ｅ Ｔｏｉｖ ｓｉａｅｔ ｅｅｆｃｓｏ ｒｏ ｉｉｅｏ Ｌ １ ｎ ｒｔｃ ｒｅ ｔ ｏ ａ ｃ ｒｂ ａ ｓ ｈ ｍｉ／ ｅ ｅ ｆｓｏ ． ｊｃｉ ｎ ｅ ｔ ｔ ｈ ｆｅｔ ｆｍｏ ｒｎｓｄ ｎ Ｉ 一ｐ ｉ ａ ｏ ｔｘ ｗｉ ｆｃ ｌ ｅｅ ｒ ｌｉｃ ｅ ａ ｒｐ ｒｕ ｉｎ ｖ ｇ ｈ
艾厚 喜 汪 莹 ， 栋 明。 王 文 ， 东 明 ， 丽 ， 林 ， 许 ， 高 张 李
［ 要］ 目 的 研 究 山茱 萸有 效 成 分 莫 诺 苷对 局 灶 性 脑 缺血 再 灌 注 大 鼠皮 层 Ｉ 一８的影 响 。方 法 用 线 栓法 制 备 大 鼠局 灶性 脑 摘 Ｉｌ
缺 血 再 灌 注模 型 ， 缺血 ３ ｎ， 灌 注 ７ ０ ｍｉ 再 ２ｈ。 以秋 水 仙 碱 为 阳 性 对 照 药 ， 用 酶 联 免 疫 吸 附 法 （ ＩＩ Ａ） 测 大 鼠脑 皮 层 炎 症 因子 采 Ｅ ｓ 检 Ｉ ｔ Ｄｌ ３的变 化 。结果 奠 诺 苷 可 剂 量依 赖 性 地 明 显 降低 由脑 缺 血 再灌 引起 的 Ｉ Ｂ含 量 增 加 （ Ｌ１ Ｐ＜ ｏ ０ １ 。结 论 莫 诺 苷 能 通 过 增 强 ．０ ） 大 鼠皮层 抗 炎 能 力 发挥 神 经 保 护 作 用 。
基于网络药理学探讨白藜芦醇治疗肺癌的生物分子机制
基于网络药理学探讨白藜芦醇治疗肺癌的生物分子机制 张丽慧，耿其顺，朱子家，王文斌，沈志博，李砺锋，薛文华，赵杰郑州大学第一附属医院，河南郑州 450052摘要：目的 基于网络药理学，运用在线数据库研究白藜芦醇治疗肺癌的潜在作用机制。
方法 通过中药系统药理学数据库与分析平台（TCMSP）、PubChem、SwissTargetPrediction、GeneCards数据库分别获取白藜芦醇和肺癌的相关基因，取二者交集基因，使用String数据库获得交集基因的蛋白相互作用（PPI）网络图，使用DA VID6.8对交集基因进行GO和KEGG富集分析。
采用Cytoscape3.7.2软件构建化合物-靶点-通路-疾病网络模型。
使用AutoDock4.2.6软件对白藜芦醇和重要靶点进行分子对接。
Western blot检测白藜芦醇对人肺癌H1975细胞p-Akt蛋白表达的影响。
结果 获得白藜芦醇和肺癌交集基因78个，PPI网络图表明交集基因关系密切。
富集分析得到生物过程55项、分子功能26项、细胞组分18项，以及86条相关通路，其中以PI3K-Akt 通路富集靶点较多。
分子对接结果显示，白藜芦醇与PIK3CB、PIK3CA这2个重要靶点均能稳定结合。
Western blot检测结果显示，白藜芦醇能够显著降低人肺癌H1975细胞p-Akt蛋白表达。
结论 白藜芦醇可能主要通过作用于PIK3CB、PIK3CA靶点，介导PI3K-Akt信号通路发挥治疗肺癌作用。
关键词：白藜芦醇；肺癌；网络药理学；作用机制；分子对接中图分类号：R273.42；R285 文献标识码：A 文章编号：1005-5304(2021)06-0046-06DOI：10.19879/ki.1005-5304.202004113 开放科学（资源服务）标识码（OSID）：Biomolecular Mechanism of Resveratrol Against Lung Cancer Based on Network Pharmacology ZHANG Lihui, GENG Qishun, ZHU Zijia, WANG Wenbin, SHEN Zhibo, LI Lifeng, XUE Wenhua, ZHAO Jie First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China Abstract:Objective To use the online databases to study the potential mechanism of resveratrol in treating lung cancer based on network pharmacology. Methods The genes related to resveratrol and lung cancer were obtained from TCMSP, PubChem, SwissTargetPrediction and GeneCards database, and the related intersection genes of resveratrol and lung cancer were screened out. The String database was used to draw the PPI network diagram of the intersection genes, and the DA VID 6.8 was used to perform GO and KEGG enrichment analysis. The compound-target-signal pathway-disease network model was constructed by using Cytoscape 3.7.2 software. AutoDock 4.2.6 software was used to perform molecular docking of resveratrol and important targets. Western blot was used to detect the effect of resveratrol on the expression of p-Akt protein in human lung cancer H1975 cell line. Results Totally 78 intersection genes of resveratrol and lung cancer were obtained, and the PPI network diagram showed that there was a close relationship between them. The enrichment analysis mainly obtained 55 biological processes, 26 molecular functions, 18 cellular components, and 86 related pathways, among which the PI3K-Akt pathway enriched the most targets. Molecular docking results showed that resveratrol could stably bind to two important targets, PIK3CB and PIK3CA. Western blot test results showed that resveratrol could significantly reduce the expression of p-Akt protein in human lung cancer H1975 cells. Conclusion Resveratrol may mainly act on the targets of PIK3CB and PIK3CA, and mediates the PI3K-Akt signaling pathway to exert anti-lung cancer action.Keywords: resveratrol; lung cancer; network pharmacology; action mechanism; molecular docking研究表明，肿瘤的发生发展与基因突变及多条信号通路改变有关[1-2]。
白念珠菌群体感应分子法尼醇对巨噬细胞Ana-1和RAW264.7增殖、凋亡和迁移能力的影响
doi：10.3969/j.issn.1000-484X.2021.09.004白念珠菌群体感应分子法尼醇对巨噬细胞Ana-1和RAW264.7增殖、凋亡和迁移能力的影响①胡万超王丹朱俊峰周子阳唐建国（复旦大学附属上海市第五人民医院急危重症医学科，上海200240）中图分类号R392.5文献标志码A文章编号1000-484X（2021）09-1041-06［摘要］目的：探讨白念珠菌群体感应分子法尼醇对巨噬细胞Ana-1和RAW264.7增殖、凋亡和迁移能力的影响。
方法：实验分为Ana-1细胞对照组、RAW264.7细胞对照组、不同浓度法尼醇+Ana-1细胞组和不同浓度法尼醇+RAW264.7细胞组。
对照组给予无血清培养基及1‰二甲基亚砜（DMSO），法尼醇+Ana-1细胞组和法尼醇+RAW264.7细胞组给予无血清培养基配制的各半数抑制浓度（IC50）及不同浓度法尼醇。
CCK-8法检测各组巨噬细胞增殖抑制率，Annexin V-FITc法检测各组巨噬细胞凋亡率，Western blot检测凋亡相关蛋白表达，Transwell小室检测巨噬细胞迁移能力。
结果：与对照组相比，法尼醇处理组对巨噬细胞Ana-1和RAW264.7增殖的抑制作用显著增强，且随浓度增加，抑制效果越明显（P<0.05）；法尼醇处理巨噬细胞组细胞凋亡率显著升高，且呈浓度依赖性（P<0.05），法尼醇可能通过影响磷酸化AKT、Bcl-2、Cleaved Caspase-3、Cleaved Caspase-9及Cleaved PARP表达促进RAW264.7和Ana-1巨噬细胞凋亡；Transwell结果显示随法尼醇浓度增加，巨噬细胞迁移数增加。
结论：白念珠菌群体感应分子法尼醇可有效抑制巨噬细胞Ana-1和RAW264.7增殖、促进其凋亡和迁移，其机制可能与降低磷酸化AKT、Bcl-2、PARP，上调Cleaved Caspase-3，Cleaved Caspase-9和Cleaved PARP表达有关。
新教材同步备课2024春高中生物第3章基因的本质3.3DNA的复制课件新人教版必修2
(2)注意碱基的单位是“对”还是“个”。 (3)切记在DNA复制过程中，无论复制了几次，含有亲代脱氧 核苷酸单链的DNA分子都只有两个。 (4)看清试题中问的是“DNA分子数”还是“链数”，“含” 还是“只含”等关键词，以免掉进陷阱。
二、DNA分子的复制
例1.某DNA分子中含有1 000个碱基对(被32P标记)，其中有胸腺 嘧啶400个。若将该DNA分子放在只含被31P标记的脱氧核苷酸的 培养液中让其复制两次，子代DNA分子相对分子质量平均比原来 减少 1 500 。
F2:
提出DNA离心
高密度带 低密度带 高密度带
低密度带 高密度带
一、DNA复制的推测—— 假说-演绎法
1.提出问题 2.提出假说
（1）演绎推理 ③分散复制
15N 15N
提出DNA离心
P:
3.验证假说
15N 14N
F1:
细胞分 裂一次
转移到含 14NH4Cl的培养 液中
提出DNA离心
细胞再 分裂一次
二、DNA分子的复制
例3.若亲代DNA分子经过诱变，某位点上一个正常碱基变成了5-溴 尿嘧啶（BU），诱变后的DNA分子连续进行2次复制，得到4个子 代DNA分子如图所示，则BU替换的碱基可能是（ C ）
A.腺嘌呤 C.胞嘧啶
B.胸腺嘧啶或腺嘌呤 D.鸟嘌呤或胞嘧啶
二、DNA分子的复制
例4. 5-BrU(5-溴尿嘧啶)既可以与A配对，又可以与C配对。将一 个正常的具有分裂能力的细胞，接种到含有A、G、C、T、5-BrU 五种核苷酸的适宜培养基上，至少需要经过几次复制后，才能实现 细胞中某DNA分子某位点上碱基对从T—A到G—C的替换（ B ）
去甲基化和去乙酰化
Repression of induced apoptosis in the 2-cell bovine embryo involves DNA methylation and histone deacetylationSilvia F.Carambula,Lilian J.Oliveira,Peter J.Hansen *Department of Animal Sciences,University of Florida,P.O.Box 110910,Gainesville,FL 32611-0910,USAa r t i c l e i n f o Article history:Received 2August 2009Available online 8August 2009Keywords:ApoptosisPreimplantation embryo DNA methylation Histone acetylation 5-Aza-20-deoxycytidine Trichostatin Aa b s t r a c tApoptosis in the bovine embryo cannot be induced by activators of the extrinsic apoptosis pathway until the 8–16-cell stage.Depolarization of mitochondria with the decoupling agent carbonyl cyanide 3-chlo-rophenylhydrazone (CCCP)can activate caspase-3in 2-cell embryos but DNA fragmentation does not occur.Here we hypothesized that the repression of apoptosis is caused by methylation of DNA andTUNEL was affected by a treatment ÂCCCP interaction (P <0.0001).CCCP did not cause a large increase in the percent of cells positive for TUNEL in embryos treated with vehicle but did increase the percent of cells that were TUNEL positive if embryos were pretreated with AZA or TSA.Immunostaining using an antibody against 5-methyl-cytosine antibody revealed that AZA and TSA reduced DNA methylation.In conclusion,disruption of DNA methylation and histone deacetylation removes the block to apoptosis in bovine 2-cell embryos.Ó2009Elsevier Inc.All rights reserved.IntroductionDuring preimplantation development,the mammalian embryo goes through a period where it is resistant to proapoptotic signals.In the best studied example,the bovine,this period lasts from the 2-cell stage through the 8–16-cell stage [1–5].Inhibition of the extrinsic pathway for apoptosis at the 2-cell stage is caused in part by resistance of the mitochondria to depolarization [4,5].In addi-tion,a second block exists that is revealed when the mitochondrial membrane is artificially depolarized by carbonyl cyanide 3-chloro-phenylhydrazone (CCCP).In this case,caspase-9and caspase-3activation takes place but DNA fragmentation does not occur [4].Thus,DNA is resistant to caspase-3mediated events such as activa-tion of caspase-activated DNase (CAD).One possible explanation for DNA resistance to CAD may reside with the structure of DNA in the early preimplantation embryo.At the 2-cell stage,little transcription takes place [6–7]and DNA is highly methylated [8].DNA demethylation occurs over the next several cleavage divisions [9].Thus,the stage of development at which susceptibility to apoptosis is acquired (the 8–16-cell stage)is also a time of when DNA methylation is reduced [9]and tran-scription is activated [6].DNA methylation can reduce the accessibility of DNases to DNA as shown for DNase I [10].Here we hypothesize that the repression of apoptosis responses in response to mitochondrial depolarization in the 2-cell embryo is caused by DNA methylation that makes internucleosomal DNA inaccessible to activated CAD.Moreover,we hypothesize that repression requires deacetylated histones.Materials and methodsReagents.Materials for in vitro maturation of oocytes,in vitro fer-tilization,and embryo culture were obtained as described previously [11].Carbonyl cyanide 3-chlorophenylhydrazone (CCCP)was pur-chased from Sigma (St.Louis,MO)and was maintained in 100mM stocks in dimethyl sulfoxide (DMSO)at À20°C in the dark.The CCCP stock solution was diluted in embryo culture medium (called KSOM-BE2,see Ref.[12]for recipe)to 100l M in 0.1%DMSO on the day of use.5-Aza-20-deoxycytidine (AZA)and trichostatin-A (TSA)were ob-tained from Sigma and used at a final concentration of 100l M and 100nM,respectively.The In Situ Cell Death Detection Kit (TMR red)was from Roche Diagnostics Corporation (Indianapolis,IN),Hoescht 33342was from Sigma,polyvinylpyrrolidone (PVP)was from Eastman Kodak (Rochester,NY).Anti-5-methylcytosine (mouse IgG1;clone 16233D3)was purchased from Calbiochem (San Diego,CA).The Zenon Alexa Fluor 488mouse IgG1labeling kit 488and Prolong ÒAntifade Kit were obtained from Invitrogen0006-291X/$-see front matter Ó2009Elsevier Inc.All rights reserved.doi:10.1016/j.bbrc.2009.08.029*Corresponding author.Fax:+13523925595.E-mail address:Hansen@animal.ufl.edu (P.J.Hansen).Biochemical and Biophysical Research Communications 388(2009)418–421Contents lists available at ScienceDirectBiochemical and Biophysical Research Communicationsjournal homepage:www.else v i e r.c o m /l o ca t e /y b b r cMolecular Probes(Eugene,OR).All other reagents were purchased from Sigma or Fisher Scientific(Pittsburgh,PA).Experiment1—Effects of cytosine demethylation and inhibition of histone deacetylation on induction of apoptosis by CCCP in2-cell em-bryos.Procedures for production of embryos in vitro were per-formed as previously described[12].After fertilization of matured oocytes for8h at38.5°C in an atmosphere of5%(v/v) CO2in humidified air,putative zygotes were cultured in groups of30in50-l l microdrops of KSOM-BE2overlaid with mineral oil at38.5°C in a humidified atmosphere of5%(v/v)CO2and5%(v/ v)O2with the balance N2.At18h post insemination(hpi),embryos were harvested and placed in groups of30in fresh50-l l micro-drops of KSOM-BE2containing either0.1%DMSO(vehicle), 100l M AZA or100nM TSA.At28–30hpi,2-cell embryos were harvested and placed in groups of10–20in50-l l microdrops of KSOM-BE2containing the same treatment as previously(vehicle, AZA or TSA)and either vehicle(0.1%DMSO,v/v)or100l M CCCP. Embryos were cultured for24h,harvested and then analyzed for TUNEL labeling.Procedures for TUNEL were performed as described previously [13].Slides were examined using a Zeiss Axioplan2epifluores-cence microscope(Zeiss,Gottingen,Germany)with Zeissfilter sets 02(DAPIfilter)and15(rhodaminefilter).Digital images for epi-fluorescence and for light microscopy using differential interfer-ence contrast were acquired using AxioVision software(Zeiss) and a high-resolution black and white Zeiss AxioCam MRm digital camera.Images were merged for presentation.The Hoescht stain-ing was digitally converted to green before merger.The experiment was replicated six times using a total of458 embryos.Experiment2—Effects of cytosine demethylation and inhibition of histone deacetylation on DNA methylation.The experiment was con-ducted as for Experiment1except embryos were examined for DNA methylation at the end of the experiment using immunocyto-chemistry with an antibody against5-methylcytosine.Unless otherwise stated,reactions were at room temperature and re-agents were diluted in phosphate-buffered saline(PBS;10mM KPO4,pH7.4containing0.9%(w/v)NaCl)containing1mg/ml pol-yvinylpyrrolidone(PVP).Embryos were washed in PBS–PVP,fixed in4%(w/v)paraformaldehyde,washed in PBS–PVP,permeabilized with0.3%(v/v)Triton X-100for30min,washed extensively in 0.05%Tween20and treated with3M HCl for30min at37°C.After neutralization with100mM Tris–HCl,pH8.5containing1mg/ml PVP,embryos were washed in0.05%(v/v)Tween20and non-spe-cific binding sites blocked by incubation in a blocking buffer con-sisting of PBS–PVP containing2%(w/v)bovine serum albumin overnight at4°C.The anti-5-methylcytosine antibody used for visualization of DNA methylation was labeled with Fab fragments against mouse IgG conjugated to Alexa Flour488(ZenonÒMouse Labeling IgG kits,Invitrogen Molecular Probes)as per manufacturer’s instruc-tions.An irrelevant mouse IgG1was similarly labeled as an isotype control.The labeled complex was diluted in blocking buffer at afi-nal concentration of5l g/ml primary antibody and embryos were incubated for1h at room temperature in the dark.After several washes in0.05%(v/v)Tween20in PBS-PVP,embryos were placed on slides and coverslips mounted using ProlongÒAntifade reagent (Invitrogen).Embryos were examined using a Zeiss Axioplan2epi-fluorescence microscope with Zeissfilter sets02(DAPIfilter)and 03(FITC).Intensity of methylation was subjectively scored for each embryo on a scale of0(no methylation)to3.A total of61embryos in two replicates were analyzed.Statistical analysis.Data were analyzed by least-squares analysis of variance using the General Linear Models procedure of the Sta-tistical Analysis System(SAS for Windows,version9.2,SAS Insti-tute,Inc.,Cary NC).Dependent variables for Experiment1,calculated on an embryo basis,were total cell number and percent of cells that were apoptotic(i.e.,TUNEL positive).Independent variables included pretreatments(vehicle,AZA or TSA),CCCP(yes vs.no)and replicate.The mathematical model included main ef-fects and all interactions.Replicate was considered random and other main effects were consideredfixed.F tests were calculated using error terms calculated from expected means squares.Differ-ences between individual means were determined using the pdiff procedure of SAS.The dependent variable for Experiment3was methylation score and the independent variable was treatment.ResultsExperiment1—Effects of cytosine demethylation and inhibition of histone deacetylation on induction of apoptosis by CCCP in2-cell embryosIn thefirst experiment,embryos were treated with either AZA or TSA at the zygote stage to block cytosine methylation or histone deacetylation and then treated with CCCP at the2-cell stage.Rep-resentative images of TUNEL labeling are shown in Fig.1A–F,least-squares means±SEM for total cell number are in Fig.1G and least-squares means±SEM for the percent of cells that were TUNEL-po-sitive are in Fig.1H.Embryo growth,as determined by total cell number at the end of the experiment,was reduced by AZA,and to a lesser extent,TSA (P<0.05)(Fig.1G).Regardless of pretreatment,CCCP induced cell-cycle arrest as determined by a reduction in cell number (P<0.001)(Fig.1G).As shown in Fig.1H,the percent of blastomeres positive for TUN-EL was affected by a treatmentÂCCCP interaction(P<0.0001). CCCP did not cause a large increase in the percent of cells positive for TUNEL in embryos treated with vehicle(2.0±3.4%vs.7.7±5.5%;compare Fig.1A with D)but did cause a large increase in the percent of cells that were positive for TUNEL for embryos pre-treated with AZA(5.4±2.9%vs.42.3±3.2%;compare Fig.1B,E)or TSA(17.1±2.8%vs.24.9±4.2%;compare Fig.1C,F).The magnitude of the TUNEL labeling after CCCP depolarization was less for TSA than AZA(P<0.01)(Fig.1H).The degree of TUNEL labeling in the absence of CCCP was great-er for embryos treated with TSA than for control embryos or em-bryos treated with AZA(P<0.01).A total of32%of TSA-treated embryos were P8cells,a stage when apoptosis is possible.In this subset of TSA-treated embryos,the proportion of cells that were TUNEL-positive was25.7±4.1%.In control embryos P8cells,only 1.3±4.6%of cells were TUNEL positive.Thus,some of the TSA-trea-ted embryos underwent apoptosis when developing past the8-cell stage.None of the AZA-treated embryos were>8cells.Further analysis of the effect of CCCP on TSA-treated embryos focused on the subset of embryos that were<8cells(i.e.,those that are ordi-narily not susceptible to apoptosis).In this subset,which repre-sents68%of the TSA-treated embryos,there was an increase in the percent of blastomeres that were TUNEL positive after CCCP treatment(10.0±4.2%vs.24.4±4.5%,P<0.025).Experiment2—Effects of cytosine demethylation and inhibition of histone deacetylation on DNA methylationAs determined by reactivity with an antibody to5-methylcyto-sine,treatment of putative zygotes with AZA or TSA reduced DNA methylation at52–54hpi(Fig.2).In control embryos treated with vehicle,nuclei reacted strongly with antibody against5-methyl-cytosine(Fig.2A).Immunoreaction product was greatly reduced in embryos treated with AZA(Fig.2B)and reduced to a lesser ex-tent for embryos treated with TSA(Fig.2C).The subjective score for degree of DNA methylation was greatest in control embryosS.F.Carambula et al./Biochemical and Biophysical Research Communications388(2009)418–421419(2.5±0.1),least in AZA-treated embryos (1.0±0.1),and intermedi-ate in TSA-treated embryos (1.9±0.1).Differences between each mean were significant (P <0.0001).DiscussionThe bovine preimplantation embryo undergoes a period from the 2-cell stage to 8–16-cell stage when it is resistant to activators of the extrinsic pathway for induction of apoptosis [1–5].The block to apoptosis involves resistance of the mitochondria to depolariza-tion and failure of caspase-3activation to lead to DNA fragmenta-tion [4,5].Here we show that the resistance of DNA to caspase-3mediated events is the result of inaccessibility of the DNA caused by a chromatin structure dependent upon DNA methylation and histone acetylation.5-Aza-20-deoxycytidine inhibits DNA methylation by incor-poration into DNA during replication and subsequent inhibition of DNA methyltransferases (DNMT)[14].Treatment of embryos with AZA reversed the block to apoptosis so that CCCP treat-ment caused DNA fragmentation.Experiments with AZA indi-cate that inhibition of apoptosis caused by mitochondrial depolarization involves DNA methylation preventing accessibil-ity of CAD to DNA.One can visualize two mechanisms by which methylated cytosines could prevent enzymatic cleavage of DNA.Methylated cytosines can repel certain proteins,for example transcription factors [15],and may also repel CAD.Alternatively,methylated cytosines can attract other proteins,such as the Sin3A histone deacetylase complex and a methyl-CpG binding protein called MeCP2that binds tightly to chro-mosomes [15].Fig.1.DNA demethylation and histone acetylation allows DNA fragmentation in 2-cell embryos after apoptosis triggered by mitochondria depolarization.Putative zygotes were treated with vehicle (DMSO),5-aza-20-deoxycytidine (AZA)or trichostatin-A (TSA);2-cell embryos were collected at 28–30h post insemination and exposed to 100l M CCCP.Total cell number and the percent of cells positive for the TUNEL reaction were determined 24h later.Representative images of embryos following the TUNEL procedure are shown in panels A–F.Nuclei were labeled with Hoechst 33342(digitally colored as green)and those that are TUNEL-positive nuclei are additionally labeled with TMR red (red).(G)Total cell number and (H)the percent of blastomeres that are TUNEL positive.Data are least-squares means ±SEM for embryos cultured in the absence (black bars)and presence (white bars)of CCCP.Cell number was affected by pretreatment (P <0.025),CCCP (P <0.001)and the interaction (P <0.001).Percent of blastomeres positive for TUNEL was affected by CCCP (P <0.025)and the interaction between pretreatment and CCCP (P <0.001).Bars with different superscripts differ (P <0.05orless).Fig.2.Reduction in DNA methylation caused by treatment of putative zygotes with 5-aza-20-deoxycytidine (AZA)and trichostatin-A (TSA).DNA methylation was observed by the immunolocalization of 5-methylcytosine in bovine embryos at 52–54h after insemination using an anti-5-methycytosine tagged with Alexa Fluor 488(green).(For interpretation of color mentioned in this figure the reader is referred to the web version of the article.)420S.F.Carambula et al./Biochemical and Biophysical Research Communications 388(2009)418–421The fact that embryos treated with TSA were also capable of undergoing DNA fragmentation in response to CCCP suggests that repression of apoptosis involves histone interactions with DNA controlled by histone deacetylation.Results with TSA were more complex to interpret than for AZA because more TSA-treated em-bryos not exposed to CCCP experienced TUNEL labeling than for AZA-treated embryos.This effect is due to an increase in TUNEL labeling among TSA-treated embryos that were8cells or greater. Unlike for AZA,which caused a large reduction in cell number, TSA reduced developmental competence only slightly and many TSA embryos reached the8–16-cell stage.In these more advanced embryos,TSA caused apoptosis in the absence of CCCP.Given the role of DNA methylation and histone deacetylation in repressing apoptosis in early stages of development,it is proposed that the acquisition of the capacity for apoptosis at the8–16-cell stage is dependent upon loss of DNA methylation or changes in his-tone acetylation.There are large species differences in the pattern of DNA methylation during early development with some species like the mouse experiencing a continual reduction in DNA methyl-ation until the blastocyst stage while other species like the pig and rabbit do not experience large scale demethylation during early development[16].In the cow,DNA methylation is reduced from the2-cell to8-cell stage and then increases by the16-cell stage [9,17].There are also changes in histone acetylation that occur dur-ing development with Histone H4K5and K12becoming deacety-lated at the one and2-cell stages,followed by reacetylation that reaches a maximum at the8-cell stage[17].Given the importance of DNA methylation and histone deacet-ylation for repressing apoptosis in early cleavage-stage embryos, it is possible that some types of embryonic death result from inad-equate DNA methylation or histone deacetylation.Patterns of DNA methylation during early development are clearly important for embryonic development because AZA caused a large reduction in embryo cell number.In conclusion,repression of apoptosis in the2-cell embryo in-volves inaccessibility of caspase-activated DNases to the DNA med-iated by a chromatin structure determined by DNA methylation and histone deacetylation.Future work should focus on the particular interactions between methylated cytosines and histones responsi-ble for this inaccessibility as well as the importance of aberrant chro-matin structure and premature apoptosis in embryonic death.AcknowledgmentsFunding was provided by the National Research Initiative Com-petitive Grants Program Grant No.2007-35203-18070from the U.S.Department of Agriculture Cooperative State Research,Educa-tion and Extension Service.Lilian Oliveira was supported by a CAPES/Fulbright Fellowship.The authors thank William Rembert for collecting ovaries;Marshall,Adam,and Alex Chernin and employees of Central Beef Packing Co.(Center Hill,FL)for provid-ing ovaries;and Scott A.Randell of Southeastern Semen Services (Wellborn,FL)for donating semen.None of the authors have a con-flict of interest.References[1]F.F.Paula-Lopes,P.J.Hansen,Heat-shock induced apoptosis in preimplantationbovine embryos is a developmentally-regulated phenomenon,Biol.Reprod.66 (2002)1169–1177.[2]C.E.Krininger III,S.H.Stephens,P.J.Hansen,Developmental changes ininhibitory effects of arsenic and heat shock on growth of preimplantation bovine embryos,Mol.Reprod.Dev.63(2002)335–340.[3]P.Soto,R.P.Natzke,P.J.Hansen,Actions of tumor necrosis factor-a on oocytematuration and embryonic development in cattle,Am.J.Reprod.Immunol.50 (2003)380–388.[4]A.M.Brad,K.E.M.Hendricks,P.J.Hansen,The block to apoptosis in bovine two-cell embryos involves inhibition of caspase-9activation and caspase-mediated DNA damage,Reproduction134(2007)789–797.[5]L.A.de Castro e Paula,P.J.Hansen,Ceramide inhibits development andcytokinesis and induces apoptosis in preimplantation bovine embryos,Mol.Reprod.Dev.75(2008)1063–1070.[6]R.E.Frei,G.A.Schultz,R.B.Church,Qualitative and quantitative changes inprotein synthesis occur at the8–16-cell stage of embryogenesis in the cow,J.Reprod.Fertil.86(1989)637–641.[7]D.R.Natale,G.M.Kidder,M.E.Westhusin, A.J.Watson,Assessment bydifferential display-RT-PCR of mRNA transcript transitions and alpha-amanitin sensitivity during bovine preattachment development,Mol.Reprod.Dev.55(2000)152–163.[8]J.S.Park,Y.S.Jeong,S.T.Shin,K.K.Lee,Y.K.Kang,Dynamic DNA methylationreprogramming:active demethylation and immediate remethylation in the male pronucleus of bovine zygotes,Dev.Dyn.236(2007)2523–2533.[9]W.Dean,F.Santos,M.Stojkovic,V.Zakhartchenko,J.Walter,E.Wolf,W.Reik,Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos,A98 (2001)13734–13738.[10]S.Kochanek, D.Renz,W.Doerfler,Differences in the accessibility ofmethylated and unmethylated DNA to DNase I,Nucleic Acids Res.21(1993) 5843–5845.[11]K.E.Hendricks,L.Martins,P.J.Hansen,Consequences for the bovine embryo ofbeing derived from a spermatozoon subjected to post-ejaculatory aging and heat shock:development to the blastocyst stage and sex ratio,J.Reprod.Dev.55(2009)69–74.[12]P.Soto,R.P.Natzke,P.J.Hansen,Identification of possible mediators ofembryonic mortality caused by mastitis:actions of lipopolysaccharide, prostaglandin F2a,and the nitric oxide generator,sodium nitroprusside dihydrate,on oocyte maturation and embryonic development in cattle,Am.J.Reprod.Immunol.50(2003)263–272.[13]Z.Roth,P.J.Hansen,Involvement of apoptosis in disruption of developmentalcompetence of bovine oocytes by heat shock during maturation,Biol.Reprod.71(2004)1898–1906.[14]W.G.Zhu,G.A.Otterson,The interaction of histone deacetylase inhibitors andDNA methyltransferases inhibitors in the treatment of human cancer cells, Curr.Med.Chem.Anticancer Agents3(2003)187–199.[15]A.Bird,DNA methylation patterns and epigenetic memory,Genes Dev.16(2002)6–21.[16]H.Fulka,J.C.St.John,J.Fulka,P.Hozák,Chromatin in early mammalianembryos:achieving the pluripotent state,Differentiation76(2008)3–14. [17]W.E.Maalouf,R.Alberio,K.H.Campbell,Differential acetylation of histone H4lysine during development of in vitro fertilized,cloned and parthenogenetically activated bovine embryos,Epigenetics3(2008)199–209.S.F.Carambula et al./Biochemical and Biophysical Research Communications388(2009)418–421421。
曼氏无针乌贼墨汁黑色素对免疫低下模型小鼠的调节作用
曼氏无针乌贼墨汁黑色素对免疫低下模型小鼠的调节作用董慧;王力东;王春琳;宋微微;母昌考;李荣华【摘要】In this study, the immunomodulatory effects of MSMI ( melanin from Sepiella maindroni ink) on hypoimmune mice induced by Hydrocortisone( HYD) was investigated . ICR mice were selected as animal mode, which were randomly divided into six groups, in-cluding control groups, hypoimmune mice, positive control groups( Levamisole Hydrochloride) , and MSMI treated groups of low, me-dium and high dosage. Except for the control group, all the other groups were treated with Hydrocortisone by subcutaneous injection. After a trial period of 10 days, the liver and spleen indexes and phagocytic capacity of macrophages were analyzed, and which were found to be significantly enhanced in the treated groups(P<0. 01,P<0. 01),(P<0. 05,P<0. 01) as compared to that in the control group. Antioxidant enzyme activities were also measured and they significantly increased compared to the hypoimmune mice group. However, lipid peroxidation assayed as melondialdehyde ( MDA) reduced significantly in the treated groups compared to that in the control. It is suggested that MSMI can promote immune function of immune-depressed mice.%探究曼氏无针乌贼墨汁黑色素( Melanin from Sepiella Maindroni Ink,MSMI)对免疫低下小鼠的调节作用。
纳米材料在生物医学领域的应用英文文献综述
纳米材料在生物医学领域的应用英文文献综述There have been numerous studies on the applications of nanomaterials in the field of biomedical sciences. These materials have unique properties at the nanoscale, which make them attractive for use in various biomedical applications. In this review, we will summarize some of the recent studies on the applications of nanomaterials in the biomedical field.Nanoparticles have been widely investigated for drug delivery purposes. These particles can encapsulate drugs and provide controlled release, improving drug efficacy and reducing side effects. For example, liposomes and polymeric nanoparticles have been used to deliver anticancer drugs specifically to tumor cells, thereby reducing systemic toxicity. Additionally, gold nanoparticles have been utilized to enhance the delivery of therapeutic molecules, such assmall interfering RNA (siRNA), through their ability to penetrate cell membranes.In the field of tissue engineering, nanomaterials have been employed to develop scaffolds with enhanced properties for tissue regeneration. By modifying the surface properties of nanomaterials, researchers have been able to promote cell adhesion and proliferation. For instance, carbon nanotubes and nanofibers have been incorporated into scaffolds to enhance mechanical strength and electrical conductivity, which are essential for certain tissue engineering applications.Furthermore, nanomaterials have been used in the development of diagnostic tools, such as biosensors and imaging agents. Engineered nanoparticles can be functionalized with specific ligands or antibodies to selectively bind to disease markers, enabling the detection and monitoring of various diseases. In addition,superparamagnetic nanoparticles have been employed as contrast agents for magnetic resonance imaging (MRI),offering better visualization of anatomical structures and disease sites.Nanomaterials have also shown promise in the field of theranostics, which involves simultaneous diagnosis and therapy. By incorporating therapeutic agents and imaging agents into a single nanoplatform, researchers have been able to develop theranostic nanoparticles that can target specific cells or tissues, deliver therapy, and monitor treatment response in real-time.In conclusion, nanomaterials have demonstratedsignificant potential in the field of biomedical sciences. Their unique properties make them suitable for applicationsin drug delivery, tissue engineering, diagnostics, and theranostics. Continued research and development in thisfield will likely lead to further advancements and innovative applications of nanomaterials in medicine.。