小鼠受精卵微注射Cdc25b mRNA可提高卵裂率并促进受精卵发育

为了观察Cdc25B蛋白及PKA/Cdc25B 信号途径在小鼠受精卵发育中的作用,将突变型和野生型Cdc25b转录成 mRNA,显微注射到小鼠受精卵中,放入含有或不含有dbcAMP的M16中,相差显微镜下观察受精卵卵裂情况;用蛋白激酶活性测定方法检测MPF的活性;利用Western 印迹检测Cdc2-Tyr15的磷酸化状态.结果显示,未加dbcAMP的Cdc25b- S321A mRNA注射组与Cdc25b-WT组相比,能够提前使受精卵发生G ₂/M期转变,导致卵裂,并明显提高卵裂率;MPF的活性测定和Cdc2-Tyr15磷酸化状态的检测结果也显示,Cdc25b-S321A组先于Cdc25b-WT组提前激活MPF.此外, Cdc25b-S321A mRNA注射组可以有效恢复由PKA引起的受精卵G ₂期阻滞,显著增加卵裂率;MPF的活性测定和Cdc2-Tyr15磷酸化状态的检测结果也显示,在PKA持续激活的情况下,对比于Cdc25b-WT组,Cdc25b-S321A组提前激活MPF.因此,在小鼠受精卵发育过程中PKA主要通过磷酸化Cdc25B的321位丝氨酸,从而调控MPF的激活与失活来控制有丝分裂进程.     

dbcAMP通过Cdc25B蛋白调控小鼠1-细胞期受精卵发育

为了研究PKA激活剂dbcAMP通过调控小鼠Cdc25B蛋白S149和S321位点磷酸化状态影响 小鼠1-细胞期受精卵的发育,将质粒pBSK-Cdc25B-WT、pBSK-Cdc25B-S149A、pBSK- Cdc25B-S321A和pBSK-Cdc25B-S149A/S321A体外转录成mRNA;显微注射入S期受精卵中 ,在2 mmol/L dbcAMP的M16培养基中培养,观察其对受精卵发育、MPF活性及CDC2- pTyr15磷酸化状态的影响. 结果显示,在有dbcAMP存在时,各组受精卵卵裂时间延迟 ,但Cdc25B-S/A mRNAs注射组受精卵卵裂率明显高于Cdc25B-WT mRNA注射组,MPF 活性提前达到高峰;CDC2-pTyr15磷酸化状态和MPF活性变化相一致. 因此,在小鼠1- 细胞期受精卵有丝分裂过程中,PKA对小鼠Cdc25B蛋白S149位点与S321位点的磷酸化 修饰是控制受精卵G2/M转换的重要方式.     

Cdc25B在小鼠受精卵的表达和定位

为阐明细胞分裂周期(Cdc)25B调控小鼠受精卵发育的机制,利用Western印迹检测小鼠受精卵各时期Cdc25B的表达及Cdc2-Tyr15的磷酸化状态。利用间接免疫荧光技术观察Cdc25B在小鼠受精卵的定位。构建pEGFP-Cdc25B融合表达载体并显微注射到受精卵中,观察Cdc25B在受精卵M期的定位变化。结果表明Cdc25B在G1和S期被磷酸化,在G2和M期去磷酸化。Cdc2-Tyr15在G1和S期处于磷酸化状态,G2期只检测到Cdc2-Tyr15轻微的磷酸化信号,M期未检测到任何Cdc2-Tyr15的磷酸化信号。Cdc25B在G1期定位于细胞质和细胞核中,S和G2期定位于细胞质的皮质部分,M期由细胞质转向核区。证明Cdc25B核输出后激活有丝分裂促进因子,从而启动小鼠受精卵的有丝分裂。     

Wee1B蛋白S15位点突变抑制小鼠1-细胞期受精卵的发育

为研究小鼠Wee1B蛋白S15位点磷酸化状态对小鼠1-细胞期受精卵发育的影响,构建pcDNA3.1/V5-His-TOPO-Wee1B-S15A（Ser突变成Ala）/D（Ser突变成Asp）突变体,体外转录成mRNAs. 对小鼠进行超排卵后当晚与雄鼠1∶1合笼,第2 d早取受精卵后培养至S期,显微注射Wee1B-WT(野生型)/KD（激酶失活型)-mRNAs和突变体Wee1B-S15A/D-mRNAs,观察其对受精卵发育、有丝分裂促进因子（MPF）活性及CDC2-pTyr15磷酸化状态的影响.结果表明,过表达Wee1B -WT和Wee1B-S15A/D可有效抑制受精卵有丝分裂进程,明显降低卵裂率. 过表达模拟磷酸化的突变明显抑制MPF的活性,CDC2-pTyr15磷酸化状态和MPF活性变化相一致. 因此,在小鼠1-细胞期受精卵有丝分裂过程中,PKA对小鼠Wee1B蛋白S15位点的磷酸化修饰是控制受精卵G2/M转换的重要方式.     

CDC25B蛋白亚细胞定位调控小鼠1-细胞期受精卵发育

为探讨小鼠细胞分裂周期25B（CDC25B）蛋白149位丝氨酸磷酸化状态对小鼠1 细胞期受精卵中CDC25B的亚细胞定位和发育的影响,应用定制的CDC25B-pS149位的 磷酸化和非磷酸化抗体检测小鼠1-细胞期受精卵各细胞时期的磷酸化和非磷酸化状 态;应用免疫荧光观察各期受精卵中CDC25B蛋白的定位情况;将质粒pEGFP-CDC25B -WT、pEGFP-CDC25B-S149A和pEGFP-CDC25B-S149D融合质粒及空载体质粒显微注射入 G1期受精卵中,观察不同显微注射组小鼠1-细胞期受精卵中外源性CDC25B蛋白亚细 胞定位．结果显示,CDC25B-S149位丝氨酸在G1和S期被磷酸化,在G2和M期去磷酸化 ．1-细胞期受精卵从G2向M期的转换过程中,发生了CDC25B向细胞核区的移位,到2- 细胞初期,部分CDC25B蛋白又从细胞核回到细胞浆．实验结果提示,小鼠1-细胞期受精卵G2/M期转换过程中,CDC25B 的S149位点磷酸化修饰可能是对CDC25B细胞内定 位及其活性的精确调节方式．     

雷帕霉素靶在小鼠受精卵早期发育中的作用研究

哺乳动物雷帕霉素靶(mTOR)是细胞生长的中心调控因子,应用RT-PCR、免疫印迹、放射性同位素体外测定酶活性等方法,研究mTOR在小鼠受精卵第一次有丝分裂过程中在卵中的表达、活性变化以及对卵裂的影响.研究发现mTOR在小鼠卵母细胞和受精卵中都有表达,在mRNA水平,mTOR从G2期开始降解,在蛋白水平,则各期没有明显变化;mTOR的激酶活性在受精后明显升高,并且在整个1-细胞期保持较高活性;mTOR的特异性抑制剂雷帕霉素能抑制卵裂,并且能抑制成熟促进因子MPF的调节亚基cyclin B的表达,从而抑制了MPF的活性.结果表明mTOR可能通过促进MPF的激活而促进小鼠受精卵的分裂.     

过表达组蛋白H3K9me3去甲基化酶对猪克隆胚胎发育的影响

组蛋白异常修饰是克隆胚胎发育的重要制约因素，组蛋白H3K9me3去甲基化酶KDM4家族的过表达可以有效提高克隆胚胎的发育效率。为探究过表达H3K9me3去甲基化酶对猪克隆胚胎发育的影响，本研究在猪克隆胚胎1-细胞期和2-细胞期分别注射KDM4A mRNA和KDM4D mRNA检测胚胎的囊胚率；收集1-细胞期注射KDM4A mRNA和胚胎注射水(对照组)的2-细胞期克隆胚胎检测H3K9me3表达水平；此外，收集1-细胞期注射KDM4A mRNA和胚胎注射水的4-细胞期克隆胚胎进行单细胞转录组测序，并对测序数据进行GO与KEGG富集分析。结果显示：在1-细胞期注射KDM4A mRNA的猪克隆胚胎囊胚率显著高于对照组(25.32±0.74%vs14.78±0.87%)，注射KDM4D mRNA对猪克隆胚胎囊胚率无明显作用(16.27±0.77%vs 14.78±0.87%)；在2-细胞期注射KDM4A mRNA和KDM4D mRNA的克隆胚胎囊胚率与对照组相比均无显著差异(32.18±1.67%、30.04±0.91%vs 31.22±1.40%)。在1-细胞期注射KDM4A mRNA的克隆...     

褪黑素对小鼠2-细胞期胚胎发育的影响

目的：研究褪黑素对小鼠2-细胞期胚胎发育的影响。方法：在培养液中添加不同浓度褪黑素,在不同的作用时间点,观察并记录小鼠胚胎囊胚率、孵出率,研究其变化情况。结果：不同浓度褪黑素对小鼠2-细胞期胚胎存在双向作用,在10-13-10-5M之间促进细胞囊胚形成和孵出,在10-9M时促进效应达到最高,而在10-3M时则呈现出一定的毒性作用。结论：褪黑素对小鼠胚胎发育影响与褪黑素浓度密切相关。     

Ywhaz和ATPase6在小鼠植入前胚胎中阶段特异性表达的分析

Ywhaz基因与卵裂球之间的通讯以及细胞通讯系统的建立有关,而ATPase6基因对1-细胞向2-细胞转变是非常关键的并在氧化磷酸化系统的建立中起重要作用。本文报道小鼠2-细胞期胚胎特异表达的基因的表达分析。对mRNA差异显示技术（DDRT-PCR）进行改进,使之在小鼠植入前胚胎发育领域得以应用。通过对感兴趣的差异片段进行回收,亚克隆,序列分析和反向Northern杂交,结果表明：Ywhaz和ATPase6是2-细胞期胚胎特异表达的基因。     

小鼠受精卵早期发育过程中PKA在M/G1期进程中的作用

为研究小鼠体内l-细胞期受精卵蛋白激酶A(PKA)对M/G1期进程的影响,应用热稳定性抑制剂PKI显微注射入l-细胞期受精卵内,观察M期促进因子(MPF)及PKA活性变化以及MPF调节亚基Cyclin B含量情况。发现PKI显微注射后PKA活性低,而MPF活性在hCG后27．5h即达高峰,较对照组提前30分钟。PKI达一定浓度则MPF活性不下降,出现M/G1阻滞;与此同时Western blotting法显示PKI注射后Cyclin B含量在M末期相当于M中期水平。结果表明,PKI显微注射抑制PKA活性后MPF活性呈高峰值,高浓度PKI显微注射可引起M/Gl阻滞,其机制与PKI干扰了Cyclin B降解有关。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.