两种亨廷顿蛋白相关蛋白1异构体—HAP1A和HAP1B在大鼠脊髓灰质内的分布特征

目的明确2种亨廷顿蛋白相关蛋白1(huntingtin-associated protein 1,HAP1)异构体—HAP1A和HAP1B在大鼠脊髓灰质内的分布特征。方法提取重组表达的谷胱甘肽S-转移酶(GST)-HAP1AC末端融合蛋白和GST-HAP1BC末端融合蛋白,免疫兔和豚鼠制备分别抗HAP1A和HAP1B特异性多克隆抗体,并采用免疫印迹技术对其特异性和效价进行鉴定和检测;用免疫组织化学技术检测HAP1A和HAP1B在大鼠脊髓灰质内的分布和定位特征。结果成功制备了分别特异性识别HAP1A和HAP1B的高效价多克隆抗体,应用这些抗体进行的免疫组织化学ABC法检测显示,HAP1A与HAP1B在大鼠脊髓灰质内的分布区域相似,二者均在大鼠脊髓灰质RexedⅠ-Ⅹ层表达,在RexedⅠ、Ⅱ层表达最强,中央管周围灰质(RexedX层)其次,在RexedⅢ-Ⅵ层表达水平较低,在脊髓前角(RexedⅦ-Ⅸ层)只有极微弱的或无阳性表达。HAP1A免疫反应产物主要定位在Stigmoid小体,在神经元胞质和近端突起内只有极少的弥散反应产物;HAP1B免疫反应性较强,其免疫反应产物弥散、均匀分布在神经元核周质和近端突起内,也定位在Stigmoid小体上。免疫荧光双重标记显示,约有80%的Stigmoid小体既表达HAP1A也表达HAP1B,其余的Stigmoid小体仅表达HAP1A。结论 HAP1A和HAP1B蛋白在大鼠脊髓灰质内具有相同的区域分布模式,但在神经元内的定位具有明显区别,提示二者可能具有不同的功能。     

慢性复合应激对大鼠胰腺HAP1表达的影响

目的探讨慢性复合应激大鼠胰腺内胰岛内分泌细胞HAP1表达的变化及其意义。方法36只大鼠随机分为两组:慢性复合应激组和正常对照组。应激组动物无规律交替暴露于垂直旋转、睡眠剥夺、捆绑(6h/d)和夜间光照等慢性复合性应激,共6周;实验结束后,采用免疫组织化学和Western-blot等方法检测两组大鼠胰腺胰岛内分泌细胞内HAP1蛋白表达的变化。结果HAP1在大鼠胰腺内选择性表达于胰岛内分泌细胞中。与对照组相比,慢性复合应激组大鼠胰腺HAP1的表达明显增强(P<0.05)。结论6周慢性复合性应激可使大鼠胰腺中胰岛内分泌细胞的HAP1表达加强,提示HAP-1在慢性复合应激促进胰腺内分泌功能中可能发挥一定作用。     

Cytoplasmic Inheritance of Sweet Potato: with Respect to the Study of Plastids and Mitochondria and the Existence of Their DNA in Sperm Cells

The mature pollen of sweet potato ( Ipomoea batatas lam. ) was bicellular. After pollination generative cell divided into a pair of sperm cells before its germination. The pair of sperm cells remained in the hydrated pollen was similar in their shape and volume with enriched cytoplasmic plastids and mitochondria. The specific fluorescence of cytoplasm DNA indicated that the sperm cells and the generative cell contained numerous organelle nucleoids. The pair of sperm cells had no significant difference in their numbers of organelle nucleoids. Two kinds of organelle nucleoids existed in the pair of sperm cells. Tile ones as big and strong fluorescent dots appeared to be the plastid nucleoids and the others as tile small and weak fluorescent dots could be the mitochondrial nucleoid. Few of the angiosperms were of biparental or paternal plastid inheritance. The result of this study has provided the cytological evidence for another genus, Ipomoea, which is of biparental or paternal plastid inheritance besides Pharbitis and Calystegia in Convolvulaceae.     

Cytoplasmic illuminations: In planta targeting of fluorescent proteins to cellular organelles

Summary Use of the jellyfish green-fluorescent protein as an in vivo reporter is in the process of revolutionising plant cell biology. By fusing the protein to specific targeting peptides or to sequences of complete proteins, it is now possible to observe the location, structure, and dynamics of a number of intracellular organelles over extended periods of time. In this review we discuss the most recent developments and unexpected results originating from the targeting of this unique protein and its derivatives to elements of the cytoskeleton and to membrane-bounded organelles in a range of plant cell types.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Evolution of the Cytoplasmic and Mitochondrial Phosphagen Kinases Unique to Annelid Groups

Creatine kinase (CK) is a member of a group of phosphoryl transfer enzymes called phosphagen kinases that play a key role in cellular energy transactions in animals. Three CK isoform gene families are known—cytoplasmic CK (CK), flagellar CK (fCK), and mitochondrial CK (MiCK). Each of the isoforms has a unique gene structure (intron/exon organization). A broad array of other phosphagen kinases is present in animals. Some of these enzymes are found only in annelids and closely related groups including glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK), and a unique arginine kinase (AK) restricted to annelids. Phylogenetic analyses of these annelid phosphagen kinases indicate that they appear to have evolved from a CK-like ancestor. To gain a greater understanding of the relationship of the CK isoforms to the annelid enzymes, we have determined the intron/exon organization of the genes for the following phosphagen kinases: Eisenia LK, Sabellastarte AK, and Arenicola mitochondrial TK (MiTK). Analysis of genomic database for the polychaete Capitella sp. yielded two putative LK genes [cytoplasmic LK and mitochondrial LK (MiLK)]. The intron/exon organization of these genes was compared with available data for cytoplasmic and mitochondrial CKs, and an annelid GK. Surprisingly, these annelid genes, irrespective of whether they are cytoplasmic (LK, AK, and GK) or mitochondrial (MiTK and MiLK), had the same 8-intron/9-exon organization and were strikingly similar to MiCK genes sharing seven of eight splice junctions. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids.     

Precursor of brain-derived neurotrophic factor (proBDNF) forms a complex with Huntingtin-associated protein-1 (HAP1) and sortilin that modulates proBDNF trafficking, degradation, and processing

proBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is anterogradely transported and released from nerve terminals, but the mechanism underlying this process remains unclear. In this study, we report that proBDNF forms a complex with Huntingtin associated protein-1 (HAP1) and sortilin, which plays an important role in proBDNF intracellular trafficking and stabilization. The interaction of proBDNF with both HAP1A and sortilin in co-transfected HEK293 cells is confirmed by both fluorescence resonance energy transfer and co-immunoprecipitation. The frequent co-localization (>90%) of endogenous HAP1, sortilin, and proBDNF is also found in cultured cortical neurons. Mapping studies using GST pulldown and competition assays has defined the interacting region of HAP1 with proBDNF within amino acids 371-445 and the binding sequences of proBDNF to HAP1 between amino acids 65 and 90. Fluorescence recovery after photobleaching confirms the defective movement of proBDNF-containing vesicles in neurites of HAP1(-/-) neurons, which can be partially restored by reintroducing HAP1 cDNA into the neurons. However, the effect is significantly increased by simultaneously reintroducing both HAP1 and sortilin. proBDNF and HAP1 are highly co-localized with organelle markers for the Golgi network, microtubules, molecular motor, or endosomes in normal neurons, but this co-localization is reduced in HAP1(-/-) neurons. Co-immunoprecipitation and Western blot showed that sortilin stabilizes the proBDNF·HAP1 complex in co-transfected HEK293 cells, helping to prevent proBDNF degradation. Furthermore, the complex facilitates furin cleavage to release mature BDNF.     

文山松毛虫质型多角体病毒形态结构及理化性质的研究

对文山松毛虫质型多角体病毒的形态结构及理化特性进行了研究,多角体大部分为六边形,少数为四边形及近园形,其大小在0.47～2.45μ之间,平均为1.1μ。病毒粒子呈球形,无囊膜,致密的核芯区由一层外壳包裹,直径为60nm。病毒粒子表面有12个刺突,放大图象可见其亚单位排列。多角体蛋白的主要成分为一种,分子量为26200道尔顿,多角体蛋白氨基酸组成中不含半胱氨酸;其碱性氨基酸与酸性氨基酸之比为1:2.16。病毒粒子结构蛋白含五条多肽组分。用SDS-热酚法提取所得核酸,其热变性紫外吸收OD_(260)值增加51.6％。抗核酸酶S_1。Tm值为86℃。在1％琼脂糖凝胶电泳中可分为9个片段,而在5％PAGE中,则可分为10个片段。各片段大小在0.66×10~6～2.85×10~6道尔顿之间,总分子量为15.35×10~6。电镜分析研究显示了CPV RNA在0.4μ、0.8μ和1.2μ处有三个分布峰。     

Lattice Structure of Cytoplasmic Microtubules in a Cultured Mammalian Cell

Tubulin can polymerize in two distinct arrangements: “B-lattices,” in which the α-tubulins of one protofilament lie next to α-tubulins in the neighboring protofilaments, or the “A” configuration, where α-tubulins lie beside β-tubulins. Microtubules (MTs) in flagellar axonemes and those assembled from pure tubulin in vitro display only B-lattices, but recent work shows that A-lattices are found when tubulin co-polymerizes in vitro with an allele of end-binding protein 1 that lacks C-terminal sequences. This observation suggests that cytoplasmic MTs, which form in the presence of this “tip-associating protein,” may have A-lattices. To test this hypothesis, we have decorated interphase MTs in 3T3 cells with monomeric motor domains from the kinesin-like protein Eg5. These MTs show only B-lattices, as confirmed by visual inspection of electron cryo-tomograms and power spectra of single projection views, imaged at higher electron dose. This result is significant because 13 protofilament MTs with B-lattices must include a “seam,” one lateral domain where adjacent dimers are in the A-configuration. It follows that cytoplasmic MTs are not cylindrically symmetric; they have two distinct faces, which may influence the binding patterns of functionally significant MT-interacting proteins.     

胃溃疡对大鼠胃中HAP1表达的影响

建立冰乙酸性大鼠胃溃疡模型,采用ABC法进行免疫组织化学染色,观察并探讨胃溃疡对胃壁中HAP1(huntingtin as-soc iated protein 1,HAP1)表达的影响。结果溃疡组大鼠胃壁中HAP1阳性细胞数目及光密度与正常进食的大鼠相比显著增多。     

脑胶质瘤组织中胞质多聚腺苷酸化成分结合蛋白1、细胞周期蛋白B2的表达及临床意义

目的:检测脑胶质瘤组织中胞质多聚腺苷酸化成分结合蛋白1(CPEB1)、细胞周期蛋白B2(CCNB2)的表达,分析CPEB1、CCNB2表达与脑胶质瘤患者临床病理特征以及预后的关系。方法:选取2016年1月至2018年1月东莞松山湖中心医院神经外科收治的经手术切除的55例脑胶质瘤患者瘤组织标本(脑胶质瘤组)和50例颅脑损伤患者额叶或颞叶组织标本(对照组)。检测CPEB1、CCNB2表达,分析CPEB1、CCNB2表达与脑胶质瘤患者临床病理特征的关系。结合随访资料,采用Kaplan-Meier生存分析CPEB1、CCNB2阳性/阴性表达脑胶质瘤患者的预后差异及采用Cox比例风险回归分析其预后的影响因素。结果:脑胶质瘤组CPEB1、CCNB2阳性表达率均高于对照组(P<0.05)。肿瘤直径>2 cm、WHO分级Ⅲ级及远处转移的患者CCNB2阳性表达率高于肿瘤直径≤2 cm、WHO分级Ⅰ~Ⅱ级及无远处转移的患者(P<0.05);WHO分级Ⅲ级、远处转移患者的CPEB1阳性表达率高于WHO分级Ⅰ~Ⅱ级、无远处转移的患者(P<0.05)。CPEB1、CCNB2阳性表达患者3年生存率低于CPEB1、CCNB2阴性表达患者(P<0.05)。WHO分级Ⅲ级、CPEB1及CCNB2阳性表达是脑胶质瘤患者术后3年死亡的危险因素(P<0.05)。结论:脑胶质瘤组织中CPEB1、CCNB2的阳性表达率均升高,其与脑胶质瘤恶性生物学行为以及不良预后有关。     

家蚕3-羟酰辅酶A脱氢酶基因全长cDNA克隆及其在质型多角体病毒感染家蚕的差异表达

家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus,BmCPV)是家蚕的重要病毒病原之一,往往给养蚕业生产造成极大危害。我们以前的研究运用基因芯片技术在感染质型多角体病毒的家蚕中肠中鉴定出一个差异表达的3-羟酰辅酶A脱氢酶蛋白基因(Bombyx mori3-hydroxyacyl-CoA dehyrogenase protein gene-Bm3HAD)。本研究利用cDNA末端快速扩增技术(RACE)克隆了该基因,其全长cDNA序列为1168bp,包含一个83bp5’端非翻译区序列(5’-UTR)、一个930bp的开放阅读框(ORF)和一个155bp的3’端非翻译区序列(3’-UTR);基因结构分析发现该基因由5个外显子和4个内含子组成。RT-PCR结果显示该基因在家蚕中肠、脂肪体、血液、丝腺及生殖体中均有表达。荧光定量PCR结果表明该基因在BmCPV感染初期为上调表达,随着病毒感染的进展,该基因的表达水平逐渐降低,并转变为下调表达。研究结果为进一步研究BmCPV对家蚕致病的分子机制提供了有益的信息。     

小麦胞质突变型雄性不育系85EA与其保持系线粒体DNA的比较研究

８５ＥＡ是通过电子束辐照获得的胞质突变型小麦不育要用ＲＦＬＰ和ＲＡＰＤ技术对８５ＥＡ及春保持系的线粒体ＤＮＡ进行了比较研究。ＲＦＬＰ分析表明８５ＥＡ线粒体基因组中ｃｏｘⅡ基因的位置结构与保持系发生了变化；ＲＡＰＤ分析中引物ＯＰＤ－１５扩增产物在不育系和保持系间有明显差异，不育系的扩增产物比保持系多１条分子量为０．６ｋｂ的特展览 要带，用Ｔ－ｅａｓｙ ｖｅｃｔｏｒ克隆该不育系特异条带并命名为ＯＰＤ－     

家蚕CPV感染离体细胞的电镜观察

本文报道了BmCPV感染家蚕细胞系后的电镜观察。病毒感染早期,细胞质内形成电子致密的病毒发生基质,由病毒发生基质形成BmCPV球状病毒粒子;病毒感染48小时后,多角体在病毒发生基质周围形成,大量的病毒粒子随机包埋在多角体内;病毒接种后96小时,多角体数目增多,其形状有三角,四角,五角及六角形,细胞质内充盈多角体致使细胞核被挤向细胞一侧并伴有形态的改变,受染细胞约为40％。     

木毒蛾质型多角体病毒形态结构及理化特性的研究

木毒蛾质型多角体病毒呈不规则多面体,大小差异较大,大多数在０．８－２．１μｍ之间;病毒粒子在多角体内随机分布,病毒粒子为正二十面体结构,有１２个顶体,每个顶上均有一个球状结构,平均直径约为６０ｎｍ。经ＳＤＳ－ＰＡＧＥ分析,多角体蛋白由一条分子量为３１ｋＤ之间。多角体蛋白富含天冬氨酸,谷氨酸及酪氨酸,而半胱氨酸和甲硫氨酸含量较少,其碱性氨基酸与酸性氨基酸之比为１．４３。病毒核酸对ＲＮａｓｅ和ＤＮａｓ     

Isolation and Sequence of Partial cDNA Clones of Human L1: Homology of Human and Rodent L1 in the Cytoplasmic Region

We have isolated cDNA clones coding for the human homologue of the neuronal cell adhesion molecule L1. The nucleotide sequence of the cDNA clones and the deduced primary amino acid sequence of the carboxy terminal portion of the human L1 are homologous to the corresponding sequences of mouse L1 and rat NILE glycoprotein, with an especially high sequences identity in the cytoplasmic regions of the proteins. There is also protein sequence homology with the cytoplasmic region of the Drosophila cell adhesion molecule, neuroglian. The conservation of the cytoplasmic domain argues for an important functional role for this portion of the molecule.     

红菜薹新型细胞质雄性不育系的转育研究

以新型甘蓝型油菜（Brassica napus L.）细胞质雄性不育系（Eru CMS）为不育源,通过远缘杂交结合回交的方法,并运用组织培养、单株选择、异地加代种植的手段成功地将该不育基因转育到了红菜薹上。经检测所得的红菜薹（Brassica campestris L.ssp.chinensis L.var.utilis Tsen et Lee.）不育系苗期遇低温叶片不黄化,不育性稳定,蜜腺正常,不育度和不育株率均为100%。     

Cytoplasmic streaming in internodal cells ofNitella under centrifugal acceleration: a study done with a newly constructed centrifuge microscope

Summary We constructed a new centrifuge microscope of the stroboscopic type, with which the cytoplasmic streaming inNitella internodal cells under centrifugal acceleration was studied. Under moderate centrifugal acceleration (ca. 50–100×g), the direction of cytoplasmic streaming in an internodal cell ofNitella is parallel to the direction of the subcortical fibrils. The speed of endoplasm flowing contiguous to the subcortical fibrils is neither accelerated nor retarded by moderate centrifugal acceleration. The endoplasmic flow, however, stops suddenly following an electrical stimulus. The endoplasm contiguous to the subcortical fibrils is immobilized transiently at the time of streaming cessation induced by an electrical stimulus under centrifugal acceleration at 50–100×g, even at 900×g. It is suggested that transitory cross bridges between the immobilized endoplasm and the subcortical fibrils are formed at the time of streaming cessation. The bulk endoplasm flows as a whole in the direction parallel to that of the subcortical fibrils and stops promptly upon electrical stimulation. Soon after the stoppage the bulk endoplasm starts to flow passively in the direction parallel to that of the centrifugal acceleration as a result of the centrifugal force.Abbreviations APW artificial pond water - CMS centrifuge microscope     

泛素连接酶Smurf1不同亚型的功能研究

Smad泛素调节因子1(Smad ubiquitination regulatory factor 1,Smurf1)是一种HECT类的泛素连接酶,它参与许多生命活动的调节,如神经发育、细胞极性、骨的重塑和肿瘤形成等.虽然目前对Smurf1的了解较为深入,但在研究过程中并未对它的两种亚型Smurf1 L和Smurf1 S(两者在一级结构上仅相差26个氨基酸残基)进行详细区分,且偏重于对Smurf1 S的研究.因此本文对Smurf1上述两种亚型的功能(特别是Smurf1 L)进行了更加深入的探索.利用RT-PCR、免疫荧光和蛋白质印迹(Western blot)等实验技术,我们从组织表达、亚细胞定位和对底物的降解能力这三个方面深入研究了Smurf1 S和Smurf1 L的不同之处.实验结果表明:一方面,Smurf1 S的组织分布比Smurf1 L更加广泛,表达量更高;另一方面,两者的亚细胞定位也有所不同,Smurf1 L在有丝分裂期定位于纺锤体,而Smurf1 S则可能主要分布于胞质中.此外,Smurf1 S对底物的降解比Smurf1 L更彻底,且前者的降解效应有剂量依赖性.上述成果对今后更为精确地研究Smurf1的功能具有重要意义.     

The cytoplasmic domain of influenza M2 protein interacts with caveolin-1

The cytoplasmic domain of influenza M2 protein (M2c) consists of 54 amino acid (aa) residues from aa44 to aa97. In this paper, M2c and its deletion mutant M2c_Δ47-55 were expressed using prokaryotic expression system. First, glutaraldehyde crosslinking assay showed that M2c had multimerization potential mediated by aa47-55. Then, M2c, instead of M2c_Δ47-55, directed eGFP from the whole cell localization to a predominately perinuclear region in CHO cells, which indicated that aa47-55 of M2c mediated the localization. Moreover, M2c colocalized with caveolin-1 (Cav) when CHO cells were cotransfected with Cav. A caveolin-1 binding motif ΦxxxxΦxxΦ (Φ represents aromatic amino acid residues) in aa47-55 of M2c was found by sequence alignment and analysis. Further overlay ELISA result showed that M2c, but not M2c_Δ47-55, bound to prokaryotically expressed cholesterol-free Cav_2-101, which illustrated the interaction could be cholesterol-independent. That was the first report of cellular protein bound to M2c.     

Functional Study of Carboxylesterase 1 Protein Isoforms

Carboxylesterase 1 (CES1) is a primary human hepatic hydrolase involved in hydrolytic biotransformation of numerous medications. Considerable interindividual variability in CES1 expression and activity has been consistently reported. Four isoforms of the CES1 protein are produced by alternative splicing (AS). In the current study, the activity and expression of each CES1 isoform are examined using transfected cell lines, and CES1 isoform composition and its impact on CES1 activity in human livers are determined. In transfected cells, isoforms 3 and 4 show mRNA and protein expressions comparable to isoforms 1 and 2, but have significantly impaired activity when hydrolyzing enalapril and clopidogrel. In individual human liver samples, isoforms 1 and 2 are the major forms, contributing 73–90% of the total CES1 protein expression. In addition, the protein expression ratios of isoforms 1 and 2 to isoforms 3 and 4 are positively associated with CES1 activity in the liver, suggesting that CES1 isoform composition is a factor contributing to the variability in hepatic CES1 function. Further investigations of the regulation of CES1 AS would improve the understanding of CES1 variability and help develop a strategy to optimize the pharmacotherapy of many CES1 substrate medications.