双抗体免疫沉淀法纯化牛生长激素poly(A)~+RNA的研究

我们用双抗体免疫沉淀法,从牛垂体多聚核糖体中分离出牛生长激素特异多聚核糖体,由此多聚核糖体纯化的牛生长激素Poly(A)~+RNA,可以在麦胚体外翻译系统中和兔网织红细胞体外翻译系统中促进~(14)C-亮氨酸的参入。合成的含~(14)-亮氨酸的翻译产物中有91％可以被牛生长激素抗体沉淀。用SDS-11％PAGE对翻译产物进行鉴定表明,翻译产物在25KD处呈一条放射自显影带,与报导的牛生长激素前体分子量相吻合。     

保守GTP酶EF-G在蛋白质合成中的功能研究北大核心CSCD

延伸因子G(elongation factor G,EF-G)是一种保守的GTP水解酶,它是蛋白质翻译过程中一个重要的调控因子。同源模拟发现EF-G与核糖体保护蛋白Tet(O)具有相似的空间结构且都包含5个结构域,序列比对发现E.coliEF-G与Campylobacter jejuniTet(O)结构域Ⅳ保守的两个环状区不同。通过分子克隆构建EF-G嵌合体,表达纯化后的蛋白突变体通过核糖体依赖的GTP水解酶(GTPase)活性检测、多聚尿嘧啶(polyU)为mRNA合成苯丙氨酸多肽链、多聚核糖体的解聚检测及相关的体内实验,检测EF-G在肽链合成中的作用,结果发现EF-G嵌合体能够影响肽链生成过程中tRNA-mRNA复合物的移位,但不影响核糖体的再循环过程。     

小鼠大脑抑制性神经元特异性多聚核糖体结合型mRNA的提取

目的:建立能够高效、快速地从小鼠大脑提取抑制性神经元特异性多聚核糖体结合的mRNA的方法,为进行小鼠大脑抑制性神经元特异性翻译表达谱分析提供材料。方法:依据Cre-loxp系统,将RiboHA标签小鼠与抑制性神经元特异性VGAT-Cre/PV-Cre小鼠杂交,启动抑制性神经元中核糖体上HA标签的表达。后代小鼠进行基因型鉴定,获得同时表达Cre和HA的小鼠。利用免疫荧光染色检测HA的表达。通过免疫共沉淀从目标细胞群体中获得HA标记的多聚核糖体。提取多聚核糖体结合的mRNA。荧光定量PCR法检测所得mRNA的细胞类型特异性。利用琼脂糖凝胶电泳及bioanalyzer 2100检测所得mRNA及cDNA的质量。结果:HA标记的多聚核糖体在目标细胞群体中能够被高效启动表达。抑制性神经元特异性多聚核糖体结合的mRNA能够被特异性地富集提取。所获细胞类型特异性的多聚核糖体结合的mRNA及cDNA的质量足以用来进行高通量测序及翻译表达谱的分析。结论:利用Cre-loxp遗传系统标记,结合蛋白免疫共沉淀实验,建立了从小鼠大脑抑制性神经元中高效特异地获得多聚核糖体结合的mRNA的方法,为进一步进行小鼠大脑抑制性神经元特异性翻译表达谱的分析奠定了基础。     

真核翻译起始因子5A在蛋白质合成中的功能及机制

在蛋白质合成过程中,除核糖体、氨酰 tRNA和mRNA外,还有多种翻译因子参与其中。真核翻译起始因子5A（eukaryotic translation initiation factor 5A, eIF5A）是维持细胞活性必不可少的翻译因子,在进化上高度保守。eIF5A是真核细胞中唯一含有羟腐胺赖氨酸（hypusine）的蛋白质,该翻译后修饰对eIF5A的活性至关重要。1978年,人们首次鉴定出eIF5A,认为它在翻译起始阶段促进第1个肽键的形成。直到2013年才证实它主要在翻译延伸阶段调控含多聚脯氨酸基序蛋白质的翻译。在经过四十多年研究后,人们对eIF5A的功能有了新的认识。近期基于核糖体图谱数据的分析表明,eIF5A能够缓解翻译延伸过程中核糖体在多种基序处的停滞,并不局限于多聚脯氨酸基序,并且它还能够通过促进肽链的释放增强翻译终止。此外,eIF5A还可以通过调控某些蛋白质的翻译,间接影响细胞内的各种生命活动。本文综述了eIF5A的多种翻译后修饰、在蛋白质合成和细胞自噬过程中的调控作用以及与人类疾病的关系,并与细菌及古细菌中的同源蛋白质进行了比较,探讨了该因子在进化中的保守性,以期为相关领域的研究提供一定的理论基础。     

人巨细胞病毒感染过程中宿主翻译因子在宿主和病毒蛋白合成中的不同作用

<正>宿主eIF4F翻译起始复合物在带帽mRNA的翻译中起关键作用。虽然人巨细胞病毒(HCMV)感染增加宿主eIF4F复合物的丰度和活性,但HCMV复制和mRNA翻译中eIF4F元件的作用尚不明确。该研究发现,降低eIF4F的丰度或活性在感染起始抑制HCMV的复制。然而随着感染进展,病毒mRNA的翻译和复制对eIF4F抑制越来越有抵抗性。在感染晚期,典型的即刻早期、早期和晚期mRNA与多聚核糖体的     

反式翻译模型

反式翻译是细菌体内一种修复翻译水平上受阻的遗传信息表达过程的机制。tmRNA是反式翻译的核心分子,它兼具tRNA和mRNA的特点,在SmpB蛋白的帮助下特异性识别携带mRNA缺失体的核糖体,在核糖体蛋白S1的传递作用下结合在A位点上,一方面延续被中断的mRNA上的遗传信息,一方面终止蛋白质的合成,释放被束缚的核糖体和tRNA进入新的翻译过程。本文对近年来关于反式翻译模型的研究进行综述。     

核仁含磷蛋白B_(23)mRNA的部分提纯

用蔗糖梯度离心法对Novikoff肝癌细胞的多聚(A)~+mRNA进行了链长分部。沉降在13S和15S的组分6和组分7富集了B_(23)mRNA,其体外转译产物可特异地被抗蛋白B_(23)的抗体免疫吸附,被吸附的蛋白在SDS-PAGE中迁移到大约37,000道尔顿的区带。另外,用多聚核糖体免疫吸附技术提纯了少量B_(23)mRNA,它在体外转译系统中也指导合成了分子量约为37,000道尔顿的蛋白质。     

Sindbis病毒的蛋白质合成与细胞骨架的关系

我们以Sindbis病毒感染BHK-21细胞为模式,研究了病毒的感染与细胞骨架的关系。结果显示:在病毒感染早期,细胞的蛋白质合成迅速被抑制,细胞的多聚核糖体(polysome)和mRNA从骨架上脱落,而病毒的RNA结合到骨架上。我们的结果还进一步表明,病毒的RNA是通过其3′-尾端与骨架结合的。另一方面在对Sindbis病毒非结构蛋白在体内与体外合成与加工的比较中,我们发现病毒蛋白在体外翻译加工的速度远低于体内,并且出现很多未成熟蛋白(premature protein),这种区别可能在某种程度上反应细胞骨架在蛋白质合成与加工中的作用。此外,在用秋水仙素和细胞松驰素B破坏微管和微丝后,病毒非结构蛋白的合成与加工没有明显变化,而结构蛋白的合成则受到明显的抑制。这表明病毒的两类蛋白的合成所依赖的细胞骨架成分可能有所不同,在结构蛋白合成过程中,微丝和微管起了重要作用,在非结构蛋白合成过程中,中间丝很可能起了重要作用。     

蛋白质合成活动与卵母细胞成熟

蛋白质合成率的增加是卵母细胞成熟过程中的重要现象之一;合成率的增加是 mRNA与核糖体机构两者协调活动的结果。S6蛋白磷酸化和 pHi 上升似乎与蛋白质合成增加有关,但有证据表明这种联系是表面现象,非必然的。翻译机构中的蛋白质因子 cIF-4 A 可能参与成熟中蛋白质合成活动的调控。本文还分析了蛋白质合成的总体变化特点,蛋白质合成、磷酸化活动与 MPF 活性和 GVBD 现象之间的关系,这是研究 MPF 活性以及卵母细胞成熟机理的一个重要方面。     

一种金针菇核糖体失活蛋白的分离纯化研究

获得一种为研究其他菌类核糖体失活蛋白的对照品,并论述一种金针菇核糖体失活蛋白的分离纯化及其活性的研究结果。实验中采用了DEAE和CM-离子交换纤维素与Bio-Gel 100柱层析方法。从1 000 g新鲜金针菇中得到5.58 mg的核糖体失活蛋白-Velutin,并证明其具有明显的抑制蛋白质的翻译作用,同时简要介绍了它的应用及展望。分离纯化到具有活性的Velutin,分子量为13.8 ku。     

Binding of globin mRNA, beta-globin mRNA segments and RNA homopolymers by immobilized protein of polysomal globin messenger ribonucleoprotein

The binding of rabbit globin mRNA, in-vitro-generated beta-globin mRNA segments, and RNA homopolymers by proteins of rabbit reticulocyte polysomal messenger ribonucleoproteins (mRNP) after SDS gel electrophoresis and electroblotting was examined. The polysomal mRNP proteins have a higher affinity for mRNA than for rRNA and tRNA while having a higher affinity for polypurine than polypyrimidine homopolymers. Binding experiments with synthetic poly(A) and with segments of beta-globin mRNA transcribed from a cDNA in vitro revealed a set of polysomal mRNP proteins which preferentially bind the poly(A)-free beta-globin mRNA. A protein of Mr 90,000 binds specifically the 3'-nontranslated trailer of the poly(A)-free beta-globin mRNA and not the poly(A)-containing globin mRNA. Another set of proteins preferentially binds poly(A). The latter group of proteins contains a prominent species of Mr 72,000, which is most likely the rabbit poly(A)-binding protein. Three polysomal mRNP proteins which bound rabbit globin mRNA did not bind preferentially any of the other RNA probes used.     

Ovine osteopontin: I. Cloning and expression of messenger ribonucleic acid in the uterus during the periimplantation period.

Trophoblast-derived interferon tau (IFNtau) acts on the endometrium to increase secretion of several proteins during the pregnancy recognition period in ruminants. One of these is a 70-kDa acidic protein that has not been identified. Our hypothesis was that the 70-kDa acidic protein is osteopontin (OPN). OPN is an acidic glycoprotein that fragments upon freezing and thawing or treatment with proteases including thrombin. OPN contains a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins to promote cell-cell attachment and cell spreading. Using antisera to recombinant human OPN, both 70-kDa and 45-kDa proteins were identified in uterine flushings from pregnant ewes by Western blotting. A clone containing the entire ovine OPN cDNA coding sequence was isolated by screening a Day 15 pregnant ovine endometrial cDNA library with a partial ovine OPN cDNA. In pregnant ewes, steady-state levels of OPN endometrial mRNA increased (P < 0. 01) after Day 17. In both cyclic and pregnant ewes, in situ hybridization analysis showed that OPN mRNA was localized on unidentified immune cells within the stratum compactum of the endometrium. In pregnant ewes, OPN mRNA was also expressed by the glandular epithelium. Results suggest that progesterone and/or IFNtau induce expression and secretion of OPN by uterine glands during the periimplantation period and that OPN may induce adhesion between luminal epithelium and trophectoderm to facilitate superficial implantation.     

Implantation-induced changes in uterine arylamidase localization in the rabbit, rat, hamster and guinea-pig

Localization of uterine arylamidase activity varied between species: arylamidase was found primarily in the apical aspect of uterine epithelial cells in the rabbit, hamster and non-pregnant rat; only moderate staining was observed in these animals in the endometrial stroma. By contrast, arylamidase localization was primarily stromal in the guinea-pig at all stages studied while the luminal epithelium was devoid of reactivity. In all species, uterine enzyme activity increased before implantation but decreased in the vicinity of the blastocyst once implantation had begun. A generalized increase over the entire length of the uterus was seen during the preimplantation phase in the uterine epithelium of the rabbit and in the endometrial stroma of the guinea-pig. Increase in stromal activity appeared to indicate predecidual transformations which were embryo-dependent (i.e. localized to the implantation site) in the rat, or embryo-independent (i.e. occurring throughout the uterus) in the guinea-pig. A subsequent decrease in enzyme activity occurred in the vicinity of the implanting embryo irrespective of the cell type involved (epithelium in the rabbit, stroma/decidua in the rat and guinea-pig). Since arylamidases of the type studied here are integrated membrane proteins, the uniformity of changes observed in different species may reflect profound changes in membrane properties of endometrial cells as an element of the implantation reaction.     

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.     

Expression of the interferon tau inducible ubiquitin cross-reactive protein in the ovine uterus.

Ubiquitin cross-reactive protein (UCRP) is a 17-kDa protein that shows cross-reactivity with ubiquitin antisera and retains the carboxyl-terminal Leu-Arg-Gly-Gly amino acid sequence of ubiquitin that ligates to, and directs degradation of, cytosolic proteins. It has been reported that bovine endometrial UCRP is synthesized and secreted in response to conceptus-derived interferon-tau (IFNtau). In the present studies, UCRP mRNA and protein were detected in ovine endometrium. Ovine UCRP mRNA was detectable on Day 13, peaked at Day 15, and remained high through Day 19 of pregnancy. The UCRP mRNA was localized to the luminal epithelium (LE), stromal cells (ST) immediately beneath the LE, and shallow glandular epithelium (GE) on Day 13, but it extended to the deep GE, deep ST, and myometrium of uterine tissues by Day 15 of pregnancy. Western blotting revealed induction of UCRP in the endometrial extracts from pregnant, but not cyclic, ewes. Ovine UCRP was also detected in uterine flushings from Days 15 and 17 of pregnancy and immunoprecipitated from Day 17 pregnant endometrial explant-conditioned medium. Treatment of immortalized ovine LE cells with recombinant ovine (ro) IFNtau induced cytosolic expression of UCRP, and intrauterine injection of roIFNtau into ovariectomized cyclic ewes induced endometrial expression of UCRP mRNA. These results are the first to describe temporal and spatial alterations in the cellular localization of UCRP in the ruminant uterus. Collectively, UCRP is synthesized and secreted by the ovine endometrium in response to IFNtau during early pregnancy. Because UCRP is present in the uterus and uterine flushings, it may regulate endometrial proteins associated with establishment and maintenance of early pregnancy in ruminants.     

Regulation of synthesis of uterine secretory proteins: evidence for differential induction of porcine uteroferrin and antileukoproteinase gene expression

Mechanisms regulating the expression of two pregnancy-associated proteins of the porcine uterus, namely the iron-transport protein uteroferrin (UF) and the lysosomal serine proteinase-inhibitor antileukoproteinase (ALP), were investigated by comparing the effects of estrogen (E), progesterone (P4), and conceptuses on the steady-state levels of their mRNAs. For UF, the expression of mRNA with production and secretion of the corresponding protein was also investigated. Progesterone increased the levels of endometrial UF mRNA and of secreted UF, but did not affect the levels of ALP mRNA in ovariectomized gilts that had received P4 treatment for 8 days. Estrogen inhibited the accumulation of endometrial UF mRNA, increased UF secretion in these gilts, but had no effect on levels of ALP mRNA. Administration of E to gilts on Day 11 of the cycle slightly diminished UF mRNA levels at 1 h post-E; had no effect at 6, 12, and 24 h post-E; and increased levels of secreted UF in uterine luminal fluids 24 h post-E. The presence of conceptuses increased levels of endometrial ALP mRNA and decreased UF protein in uterine luminal fluids, but did not affect levels of endometrial UF mRNA. Myometrium, endometrium, and placenta from Day 75 and Day 105 pregnant gilts were also evaluated for ALP and UF mRNA expression to determine regional expression of these steroid-regulated genes. Myometrium and endometrium expressed comparable levels of UF and ALP mRNAs within Days 75 or 105, but placenta did not express detectable levels of mRNA for either protein. Within the myometrium, UF protein is immunolocalized mostly to the inner circular and to a lesser extent to the outer longitudinal layer of smooth muscle. These results indicate that E, P4, and presence of conceptuses differentially affect endometrial expression of ALP and UF mRNAs and secretion of UF.     

Differences in the rabbit uterine response to progesterone as influenced by growth hormone or prolactin

The direct effect of growth hormone (GH) on the uterine response to progesterone was tested by using ovariectomized rabbits (at least 12 weeks) treated with GH; GH + progesterone; or progesterone alone. These results were compared with the effect of prolactin or prolactin + progesterone on the uterus. Prolactin treatment produced an increase (P less than 0.01) in the endometrial surface area and restored cytosolic oestrogen and progesterone receptor concentrations to oestrous control values. The sequential treatment of does with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on Day 5 of pregnancy. In contrast, GH treatment had no effect on endometrial surface area, produced an increase in the concentration of cytosolic oestrogen receptor but did not produce an increase in the concentration of progesterone receptor. The sequential treatment of does with GH + progesterone failed to stimulate uteroglobin secretion above control (progesterone alone) values. It is concluded that the action of prolactin in the rabbit uterus is no generally somatogenic; rather, prolactin increases the concentration of progesterone receptor and thereby enhances the uterine response to progesterone.     

The effect of beta-naphthoflavone on rabbit liver protein synthesis, and on the induction of cytochrome P-450 LM4 mRNA

A single injection of β-naphthoflavone dispersed in corn oil causes significant changes in rabbit liver polysome and polysomal poly(A+)mRNA driven $\text{in vitro}$ protein synthesis. The changes occur between 6–18 hr and 30–36 hr after the injection. Our data indicate that the first effect is due to the β-naphthoflavone and the second effect is due to the oil vehicle. $\text{In vitro}$ translation of rabbit liver polysomes obtained from treated rabbits followed by specific immunoprecipition and gel electrophoresis, showed that maximal levels of translatable cytochrome P-450 LM₄ occurred 18–24 hr after β-naphthoflavone treatment.     

Calcitonin down-regulates E-cadherin expression in rodent uterine epithelium during implantation

Previous studies indicated that calcitonin (CT), a peptide hormone involved in calcium homeostasis, is transiently expressed in the receptive rat and human endometrial epithelia within the window of implantation. Attenuation of uterine CT expression using antisense methods severely impaired implantation in the rat. The molecular pathway of CT in the pregnant uterus, however, remains unknown. In the present study, we investigated the cellular events following the binding of CT to its membrane receptors in human endometrial epithelial cell line Ishikawa. We observed that CT treatment triggers a transient rise in intracellular calcium in these cells. Most interestingly, CT treatment also led to the disappearance of E-cadherin, a critical cell adhesion molecule, from cell-cell contact sites. Blockade of intracellular calcium release by BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl) prevented the CT-induced disappearance of E-cadherin. Our studies further revealed that CT treatment markedly down-regulates the level of E-cadherin mRNA in Ishikawa cells. We also examined whether CT influences the expression of E-cadherin mRNA in intact rat uterine tissue during implantation. In pregnant rats, high levels of E-cadherin mRNA were expressed during the first 3 days of gestation when the CT mRNA in uterine epithelial cells is undetectable. Concomitant with a transient burst of CT expression during days 4-5 of pregnancy, the level of E-cadherin mRNA declined sharply. Furthermore, administration of exogenous CT to animals on day 2 of pregnancy led to a premature suppression of E-cadherin mRNA level on day 3, indicating a direct link between elevated levels of uterine CT and the down-regulation of E-cadherin expression in the surface epithelium. Collectively, our results are consistent with the hypothesis that CT-induced reduction in E-cadherin expression may remodel the adherens junctions between epithelial cells, and this change in epithelial cell phenotype might be a critical event during the implantation of the blastocyst.