豌豆叶绿体偶联因子腺三磷酶的分离与纯化

豌豆叶绿体经焦磷酸钠盐溶液洗涤,并以加蔗糖的Tris—Tricine缓冲液作分离介质,其抽提液通过 DEAE—Sephadex A_59柱层析,可得较高纯度的腺三磷酶制剂。经免疫沉淀反应、需镁腺三磷酶活和 SDS—聚丙烯酸胺凝胶电泳证明,这种豌豆叶绿体偶联因子腺三磷酶具有五种蛋白带,与菠菜叶绿体偶联因子腺三磷酶的五种亚单位(α,β,γ,δ和ε亚单位)具有相近的分子量,但两者的α和β亚单位大小有异。     

大鼠皮质星形胶质细胞尼古丁型乙酰胆碱受体的表达

该文研究了尼古丁型乙酰胆碱受体(nicotinic acetylcholine receptors,n ACh Rs)不同亚单位在大鼠大脑星形胶质细胞的表达情况。分离新生大鼠大脑皮质培养原代星形胶质细胞并纯化、传代鉴定后,运用RT-PCR方法、蛋白质印迹法检测正常大鼠大脑皮质星形胶质细胞n ACh Rs不同亚单位的表达情况。结果显示,α2、α3、α4、α7、β2等n ACh Rs亚单位在星形胶质细胞均有表达,而β3 n ACh Rs亚单位在星形胶质细胞没有表达,为深入研究星形胶质细胞的功能提供了可能。     

G蛋白βγ亚单位介导的信号转导途径

跨膜信息传递有关的Ｇ蛋白由α、β和γ亚单位所组成,受体激动后,引起ＧＴＰ与α亚单位结合,导致Ｇα与Ｇβγ分离。近年来发现Ｇα、受体本射和许多效应分子如Ｋ＾＋通道、Ｇａ＾２＋通道、磷脂酶Ｃ－β、腺苷酸环化酶、酷氨酸、ＭＡＰＫ和受体激酶等都受Ｇβγ的调节,Ｇβγ同Ｇα一样均可引起效应蛋白的激活,在细胞信号转导中起同样重要作用,共同介导一系列的生物学效应。     

胰岛素受体的研究进展

胰岛素受体是靶细胞膜表面固有的糖蛋白、由两个α亚单位和两个β亚单位组成、亚单位间由二硫键连结的大分子物质。其中α亚单位为结合亚单位,β亚单位为效应/调质亚单位。胰岛素与受体结合后引起β亚单位磷酸化,可能是胰岛素发挥一系列生理功能的早期现象。受体与胰岛素结合后一起内移,在细胞内分别以不同途径代谢。激素-受体复合物的内移及受体降解使细胞表面受体数减少,这是胰岛素对受体数进行减数调节的原因。     

重组逆转录病毒载体pLXPXSN—TCRα12-2-IRES-Vβ7．1的构建及其杀伤肝癌细胞作用的研究

目的：构建双表达逆转录病毒载体pLXPXSN—TCRα12—2-IRES—Vβ7．1,包装成病毒颗粒后有效地感染PBMC。方法：以实验室保存舍TCRVβ7．1基因和TCRα12．2基因的质粒为模板,分别扩增得到两个基因,亚克隆入载体pLXPXSN,得到重组质粒pLXPXSN—TCRα12—2．IRES—Vβ7．1。重组体质粒经酶切鉴定后,将鉴定好的阳性重组质粒用脂质体介导转染PA317细胞,包装成完整的病毒后测定滴度,感染PBMC,用流式细胞仪和提取基因组DNA检测目的蛋白的表达。最后病毒感染PBMC,用流式细胞仪检测目的蛋白的表达。然后用流式细胞术细胞凋亡率,MTT比色法检测pLXPXSN—TCRα12—2-IRES-Vβ7．1感染的PBMC对肝癌细胞BEL-7402和HEPG2的杀伤作用。结果：从重组病毒基因组中扩增出目的基因TCRα12．2和TCRVβ7．1,流式细胞仪检测表明目的基因可以在PBMC中有效的表达。pLXPXSN—TCRα12—2-IRES—Vβ7．1感染PBMC组对肿瘤细胞的杀伤率明显高于PBMC组和空载体感染组。结论：TCRα12．2和TCRVβ7．1能够整合进宿主PBMC的基因组中,并能得到有效地表达。pLXPXSN—TCRα12—2-IRES-Vβ7．1感染PBMC后可提高其对肝癌细胞的杀伤活性。     

钝顶螺旋藻藻胆蛋白的分离,纯化及其理化特性

钝顶螺旋藻(Spirulina Platensis var.nanjingensis)一变异株的水溶性色素精提物,经固体硫酸铵沉淀,羟基磷灰石(HA)和Sephadex G-100柱层析后可分离、纯化出藻蓝蛋白(C-PC)和别藻蛋白(APC)。它们的纯度可分别达到AS 620/A_(277)=4.71;A_(650)/A_(270)=5.62。纯化后的C—PC和APC在聚丙烯酰胺凝胶电泳(PAGE)中仅见一条色带,其最大吸收峰分别在620nm和050nm。经12％的十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS—PAGE),以及高效液相色谱(HPLC)分离,C—PC和APC均可分为α和β两个亚单位。两者的亚单位分子量分别为:C—PC—α,15000;C—PC—β,14500;APC—α,15000;APC—β,13500。依此推算,该藻的C—PC和APC的最小分子量应为29.5kD和28.5kD。经等电电泳法测定,其C—PC和APC的等电点分别在4.8和4.9。氨基酸组成和含量分析结果表明,除色氨酸(Try)未测外,c—PC含有14种氨基酸,APC含有15种氨基酸,两者都缺乏组氨酸(His)和脯氨酸(Pro),C—PC还缺少蛋氨酸(Met)。     

哇巴因对人血管内皮细胞死亡和钠泵α1、β1,亚单位表达的影响

目的：研究钠泵抑制剂哇巴因（ouabain）对人血管内皮细胞死亡的影响及其作用机制。方法：以脐静脉内皮细胞系ECV304为靶细胞,应用MTT实验检测哇巴因对细胞生长的作用 采用Hoechst33342/PI双荧光染色、透射电镜和DNA琼脂糖凝胶电泳等分析细胞死亡特征,半定量RT-PCR法检测钠泵α1和β1亚单位mRNA的表达。结果：哇巴因以浓度和作用时间依赖的方式抑制ECV304细胞生长。10μmol/L哇巴因作用24 h,引起细胞坏死 0.1μmol/L哇巴因作用24～48 h,细胞明显脱落,细胞间连接丧失,细胞出现染色质凝集、分布于核膜内缘、DNA裂解等凋亡特征。哇巴因能明显上调ECV304细胞钠泵α1亚单位mRNA的表达,下调β1亚单位mRNA表达,且两者均呈时间依赖性。结论：哇巴因能诱导人血管内皮细胞ECV304死亡,其上调钠泵α1亚单位表达、下调β1亚单位表达,可能与亚单位介导信号传递、降低细胞黏附有关。     

建议采用抑素及其相关物的新定义与新命名

1987年5月21～22口在日本东京召开的题为《卵泡刺激素分泌的抑素-非甾体激素调节》的学术讨论会上,建议采用抑素及其相关物的新定义与新命名。抑素(inhibin)为一种由两个彼此不同的亚单位(α和β)组成(借二硫键连接),抑制垂体促性腺激素(尤其是 FSH),产生或分泌的一种糖蛋白激素。α亚单位是指已从猪、牛、羊和人体中提纯及克隆的抑素(31～23KD)的20KD 亚单位。高分子量抑素的相应亚单位,如分子量为55～65KD 的抑素,应根据其分子量命名,例如,α-44。这一命名可清楚地区分从性腺分离的抑素与     

血小板整合蛋白α2β1分子遗传变异与血栓性疾病相关性研究进展

血小板整合蛋白α2β1是血小板胶原受体,其α亚单位遗传变异影响α2β1表达水平并进一步影响血小板的功能。α2β1遗传多态性与卒中,心肌梗塞,糖尿病视网膜病和糖尿病肾病等血栓性疾病具有相关性。     

整合素的构象变化与亲和力调控

整合素(integrin)是由α、β两个亚单位通过非共价键连接而组成的异源二聚体。每种α、β亚单位都是含有多种结构域的大分子量Ⅰ型穿膜糖蛋白。它在细胞与细胞间、细胞与基质间相互作用的过程中发挥着十分关键的作用。整合素多种结构域的空间排列决定了其构象特征,而整合素的不同构象状态与其亲和力呈高度相关。对αVβ133整合素晶体结构的解析使我们对整合素的结构与功能有了更进一步的理解。     

Partial reduction of disulfide bonds in the hormone-specific subunits of TSH and LH.

Partial reduction at pH 7.0 of the hormone specific (β) subunit of either bovine thyrotropin or luteinizing hormone with dithioerythritol results primarily in the opening of a single disulfide bridge. The partially reduced subunits were alkylated with [1-¹⁴C] iodoacetic acid, followed by complete reduction and alkylation with non-radioactive iodoacetic acid. Isolation and degradation of the radioactive tryptic peptides shows that the bond primarily reduced in each β subunit links analogous half-cystine residues in the two sequences (88–95 in TSH-β and 93–100 in LH-β). These results are the first direct evidence of similar disulfide structures in hormone specific subunits of glycoprotein hormones.     

Promoter hypermethylation and Up-regulation of thyroid-stimulating-hormone-alpha (TSH-α) in thyroid cancer

Thyroid-stimulating-hormone-alpha (TSH-α) is the common subunit of the heterodimeric hormone TSH and also of other glycoprotein hormones. Although both expression and promoter-methylation profiles of the gene have been observed in the pituitary gland and placenta, no observation has been reported in the thyroid gland. We examined TSH-α expression in normal and cancer thyroid tissues. Real-time RT-PCR and immunohistochemistry indicated that TSH-a was repressed in normal tissues while activated in cancer tissues. To identify the epigenetic mechanism of upregulation of TSH-α, the methylation status of the seven CpG sites in the TSH-a promoter was examined in sixty thyroid cancer tissues. Two CpG sites showed remarkably higher levels of methylation in cancer (46 and 45%) than in normal tissues (24 and 23%) (p=0.010 and 0.003). These findings indicate that TSH-α is expressed in the thyroid cancer tissue per se and that its expression can be affected by promoter methylation.     

Mono-ADP-ribosylation of the G protein betagamma dimer is modulated by hormones and inhibited by Arf6

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the β subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the βγ dimer is inactive toward its effector enzymes. To better understand the physiological relevance of this post-translational modification, we have studied its hormonal regulation. Here, we report that Gβ subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces Gβ subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits Gβ subunit mono-ADP-ribosylation. We also provide the first demonstration that activation of the GnRHR can activate the ADP-ribosylation factor Arf6, which in turn inhibits Gβ subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of Gβ subunit mono-ADP-ribosylation, which is fully restored by re-addition of purified, myristoylated Arf6. We show that Arf6 acts as a competitive inhibitor of the endogenous ADP-ribosyltransferase and is itself modified by this enzyme. These data provide further understanding of the mechanisms that regulate endogenous ADP-ribosylation of the Gβ subunit, and they demonstrate a novel role for Arf6 in hormone regulation of Gβ subunit mono-ADP-ribosylation.     

G protein β subunit is closely associated with microtubules

Previously, we have identified the association of G protein β subunit (Gβ) with mitotic spindles in various mammalian cells. Since microtubules are the main component of mitotic spindles, here we have isolated bovine brain microtubules and purified Gβ subunit to identify the close association of Gβ subunit with purified brain microtubules and have shown the direct incorporation of Gβ subunit into the microtubules both in vitro and in vivo. It was found that: (1) microtubular fraction isolated from bovine brain contained Gβ subunit, (2) coimmunoprecipitation demonstrated that Gβ subunit could be coprecipitated with tubulin, (3) addition of purified Gβ subunit into cytosolic extract for microtubule assembly caused direct incorporation of Gβ subunit into assembled microtubules and increased the association of microtubule-associated proteins with microtubules, and (4) incubation of exogenous Gβ subunit with detergent-permeabilized cells resulted in direct incorporation of Gβ subunit into microtubule fibers and depolymerized tubulin molecules. We conclude that G protein β subunit is closely associated with microtubules and may play an important role in the regulation of microtubule formation in addition to its regulatory role in cellular signal transduction. J. Cell. Biochem. 70:553–562, 1998. © 1998 Wiley-Liss, Inc.     

The recombinant equine LHβ subunit combines divergent intracellular traits of human LHβ and CGβ subunits

The pituitary LHβ and placental CGβ subunits are products of different genes in primates. The major structural difference between the two subunits is in the carboxy-terminal region, where the short carboxyl sequence of hLHβ is replaced by a longer O-glycosylated carboxy-terminal peptide in hCGβ. In association with this structural deviation, there are marked differences in the secretion kinetics and polarized routing of the two subunits. In equids, however, the CGβ and LHβ subunits are products of the same gene expressed in the placenta and pituitary (LHβ), and both contain a carboxy-terminal peptide. This unusual expression pattern intrigued us and led to our study of eLHβ subunit secretion by transfected Chinese hamster ovary and Madin–Darby canine kidney cells. In continuous labeling and pulse-chase experiments, the secretion of the eLHβ subunit from the transfected Chinese hamster ovary cells was inefficient (medium recovery of 16%–25%) and slow (t_1/2 > 6.5 hours). This indicated that, the secretion of the eLHβ subunit resembles that of hLHβ rather than hCGβ. In Madin–Darby canine kidney cells grown on Transwell filters, the eLHβ subunit was preferentially secreted from the apical side, similar to the hCGβ subunit secretory route (∼65% of the total protein secreted). Taken together, these data suggested that secretion of the eLHβ subunit integrates features of both hLHβ and hCGβ subunits. We propose that the evolution of this intracellular behavior may fulfill the physiological demands for biosynthesis of the LH and CG β-subunits in the pituitary and placenta, respectively.     

The human IKKbeta subunit kinase domain displays CK2-like phosphorylation specificity

NF-κB activation in response to pro-inflammatory stimuli relies upon phosphorylation of IκBα at serines 32 and 36 by the β subunit of the IκB kinase complex (IKK). In this study, we build upon the observation that highly purified human IKKβ subunit preparations retain this specificity in vitro. We show that IKKβ constructs that lack their carboxy-terminus beginning at the leucine zipper motif fail to phosphorylate IκBα at Ser-32 and Ser-36. Rather, these constructs, which contain the entire IKKβ subunit kinase domain, phosphorylate serine and threonine residues contained within the IκBα carboxy-terminal PEST region. Furthermore, removal of the leucine zipper and helix-loop-helix regions converts IKKβ to monomer. We propose that the helix-loop-helix of the human IKKβ subunit is necessary for restricting substrate specificity toward Ser-32 and Ser-36 in IκBα and that in the absence of its carboxy-terminal protein structural motifs the human IKKβ subunit kinase domain exhibits a CK2-like phosphorylation specificity.     

Identification and partial characterization of Rhizopus nigricans Gβ proteins and their expression in the presence of progesterone

The mammalian steroid hormone progesterone actuates a signalling pathway in the zygomycete Rhizopus nigricans which includes heterotrimeric G proteins. To investigate the possibility that the Gβ subunit of these proteins is involved in the signalling, a cDNA library from R. nigricans exposed to progesterone was prepared and a sequence coding for a Gβ subunit was searched for. Using degenerate primers, two sequences, RnGPB1 and RnGPB2, were identified that exhibited a high degree of identity with those for Gβ from other filamentous fungi, but not from yeast. The presence of more than one Gβ subunit is very rare among the fungi, and it has been to date reported only for Rhizopus oryzae. We have shown that progesterone increases the expression of RnGPB1, but has no influence on the expression of RnGPB2. Therefore, our studies imply the involvement of Gβ subunit 1 in the response of R. nigricans to progesterone. Moreover, the Gβ subunit is subjected to endogenous ADP-ribosylation in the presence of NAD, which could be important in some, as yet unknown, cell process. Article from a special issue on steroids and microorganisms.     

Amyloid-β Peptide Binds to Cytochrome C Oxidase Subunit 1

Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aβ 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aβ 1-42 in ELISA as well as to Aβ aggregates present in AD brain. Aβ 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aβ 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aβ 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.     

Functional morphology of thyrotropic and corticotropic cells during reproductive cycle in ewe

A histological examination has been carried on the pars distalis of the pituitaries of twelve, healthy sexually mature balady ewes which were classified into three groups Resting, follicular and luteal phases according to the different phases of reproduction. The histological features of the TSH- ACTH-cells during resting phase has been used to establish a standard with which the subsequent changes during the follicular and luteal phases were compared. Using PAS/OG, AB/OG and PFA/AB/PAS/OG stains the TSH- and ACTH-cells were differentiated and identified from the other forms of the pars distalis cells. The topographical study of the TSH- and ACTH-cells in various regions and levels in the pars distalis of balady ewe as well as their frequencies during the different phase of reproductive cycle were discussed. Statistical analysis of the different cell count during the different phases of reproductive cycle was conducted. TSH- and ACTH-cells exhibited signs of increased cellular activities and significantly increased in number during follicular phase and in the other phases of the reproductive cycle.     

FoF1-ATPase activity regulated by external links on β subunits

F_oF₁-ATPase activity is regulated by external links on β subunits with different molecular weight. It is inhibited when anti-β subunit antibody, streptavidin and H9 antibody link on the β subunits successively, but is activated when virus was binded. Western blotting indicated that the employed anti-β antibody target was on the non-catalytic site of the β subunit. Furthermore, an ESR study of spin-labeled ATP (SL-ATP) showed that the affinity of ATP to the holoenzyme increases with increasing external links on the β subunits. This simple regulation method may have great potential in the design of rapid, free labeled, sensitive and selective biosensors.