Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals

Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca²⁺ in the presence of ATP. The specific activity of ATP-dependent uptake is ~20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca²⁺ stimulation of ATP hydrolysis was not discernible above the high background of Ca²⁺-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca²⁺ concentration, suggesting that Ca²⁺ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca²⁺ at half maximal velocity of uptake (S_0.5) was 12.5 micromolar and the Hill coefficient (n_H) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca²⁺, although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca²⁺ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca²⁺ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca²⁺ into microsomal vesicles declined by ~70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S_0.5 or n_H, but V_max for ATP-dependent Ca²⁺ uptake decreased by ~40%. A similar decline in ATP-dependent uptake of Ca²⁺ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene.     

Protein Subcellular Localization in Bacteria

Like their eukaryotic counterparts, bacterial cells have a highly organized internal architecture. Here, we address the question of how proteins localize to particular sites in the cell and how they do so in a dynamic manner. We consider the underlying mechanisms that govern the positioning of proteins and protein complexes in the examples of the divisome, polar assemblies, cytoplasmic clusters, cytoskeletal elements, and organelles. We argue that geometric cues, self-assembly, and restricted sites of assembly are all exploited by the cell to specifically localize particular proteins that we refer to as anchor proteins. These anchor proteins in turn govern the localization of a whole host of additional proteins. Looking ahead, we speculate on the existence of additional mechanisms that contribute to the organization of bacterial cells, such as the nucleoid, membrane microdomains enriched in specific lipids, and RNAs with positional information.Our view of the organization of the bacterial cell has changed radically over the past two decades. Once seen as an amorphous vessel harboring a homogeneous solution of proteins, these primitive organisms are now known to have an intricate subcellular architecture in which individual proteins localize to particular sites in the cell, often in a dynamic manner. Of course, bacteria frequently show conspicuous morphological features, such as division septa, flagella, pili, and stalks, which implied a nonuniform, underlying distribution of proteins. But it was not until the early 1990s that it became clear that proteins can, and often do, have distinctive subcellular addresses. Among the earliest discoveries were: (1) the formation of a ringlike structure at the mid cell position by the cytokinetic protein FtsZ (Bi and Lutkenhaus 1991), (2) the clustering of chemotaxis proteins at the poles of cells (Alley et al. 1992), (3) the compartment-specific production of sporulation proteins and their assembly into shell-like structures (Driks and Losick 1991), and (4) the asymmetric distribution of proteins involved in actin polymerization along the cell surface (Goldberg et al. 1993; Kocks et al. 1993). These discoveries were initially made by immunoelectron and immunofluorescence microscopy with fixed cells, but the discovery of green fluorescent protein (GFP) and the demonstration that proteins could retain their proper subcellular localization as GFP fusions opened the way to visualizing proteins and their dynamic behavior in living cells, including, importantly, in bacteria (Arigoni et al. 1995).Knowing where proteins are in the cell is often critical to understanding their function. Thus, the position of the aforementioned FtsZ ring (the Z-ring) dictates where cytokinesis will take place (Margolin 2005). The clustering of chemotaxis proteins plays an important role in the extraordinary gain in the responsiveness of chemotatic behavior to small changes in attractants (Ames and Parkinson 2006). Where sporulation proteins are produced and the way in which they assemble governs spore morphogenesis (Stragier and Losick 1996; Errington 2003). The asymmetric distribution of actin-polymerization proteins on the cell surface explains how certain pathogens harness host cytoskeletal proteins for their own motility (Smith et al. 1995). From these and other examples emerge a view of the bacterial cell as a dynamic, three-dimensional system in which protein localization and changes in protein localization over time orchestrate growth, the cell cycle, behavior, and differentiation.Here, after an initial discussion of general principles governing the positioning of proteins within the cell, we consider five broad categories of subcellular localization: the divisome, polar assemblies, cytoplasmic clusters, cytoskeletal elements, and organelles. We end by looking ahead to exciting new aspects of bacterial cytology just emerging from current research. Our goal is not to be comprehensive but rather to focus on examples that are illustrative of general principles of protein localization. Comprehensive treatment of individual topics can be found in other articles on this topic.     

Diversity of Halogenated Secondary Metabolites in Okinawan Aplysia argus Including 12-Hydroxypinnaterpene C and Their Feeding Targets

A new irieane-type diterpene, 12-hydroxypinnaterpene C ( 1 ), and 21 known compounds, angasiol acetate ( 2 ), angasiol ( 3 ), 11-deacetylpinnaterpene C ( 4 ), palisadin A ( 5 ), 12-acetoxypalisadin B ( 6 ), 12-hydroxypalisadin B ( 7 ), aplysistatin ( 8 ), luzodiol ( 9 ), 5-acetoxy-2-bromo-3-chloro-chamigra-7(14),9-dien-8-one ( 10 ), neoirietriol ( 11 ), neoirietetraol ( 12 ), (3Z)-laurenyne ( 13 ), cupalaurenol ( 14 ), cupalaurenol acetate ( 15 ), (3Z)-venustinene ( 16 ), 10-hydroxykahukuene B ( 17 ), aplysiol B ( 18 ), (3Z)-13-epipinnatifidenyne ( 19 ), 3Z,6R,7R,12S,13S-obtusenyne ( 20 ), (3Z,9Z)-7-chloro-6-hydroxy-12-oxo-pentadeca-3,9-dien-1-yne ( 21 ), and cholest-7-en-3,5,7-triol ( 22 ) were isolated from the digestive diverticula of Aplysia argus from the Ikei Island in Okinawa, Japan. The structures of these compounds were determined using spectroscopic methods such as NMR and HR-ESI-MS. These compounds were tested for their antibacterial activity against the phytopathogen Ralstonia solanacearum. Compounds 11 and 21 exhibited antibacterial activity at 30 μg/disc. In this study, we also discuss the types of red algae that A. argus feeds on in the shallow waters of Okinawa Prefecture.     

Age-Dependent Discrimination between Stereoisomers of 1-Amino-2-Ethylcyclopropane-1-Carboxylic Acid in Carnation Petals

The ability of carnation petals (Dianthus caryophyllus L. cv White Sim) of different ages to convert the cis and trans isomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) to 1-butene was studied. Young petals, which produce ethylene at a low rate, convert both cis- and trans-AEC to 1-butene with low efficiency and at equal rates. In senescing petals, the rate of conversion of cis-AEC remains low, but there is a marked increase in the rate of trans-AEC conversion. Thus there is a clear evidence of stereodiscrimination between the isomers. Stimulating the rate of senescence by treatment with either 1-aminocyclopropane-1-carboxylic acid or ethylene further increases the rate of trans-AEC conversion. Delaying of petal senescence by silver thiosulphate or aminooxyacetic acid inhibits the rise in trans-AEC conversion.     

Endoglycanase-Catalyzed Degradation of Hemicelluloses during Development of Carnation (Dianthus caryophyllus L.) Petals

Large molecular-size hemicelluloses, including xyloglucan, decreased in quantity during development of carnation (Dianthus caryophyllus L. cv White Sim) petals, along with a relative increase in polymers with an average size of 10 kilodaltons. An enzyme extract from senescing petal tissue depolymerized the large molecular-size hemicelluloses in a pattern similar to that occurring in vivo during petal development. The products generated in vitro were composed of polymeric and monomeric components, the latter consisting primarily of xylose, galactose, and glucose. The 10 kilodalton hemicelluloses were resistant to in vitro enzymic hydrolysis. Glycosyl-linkage composition of the large molecular-size polymers provided evidence for the presence of xyloglucan with smaller amounts of arabinoxylan and arabinan. The 10 kilodalton polymers were enriched in mannosyl and 4-linked glucosyl residues, presumably derived from glucomannan. During petal development or enzymic hydrolysis, no change was observed in the relative glycosyl-linkage composition of the large molecular-size hemicelluloses. The in vitro activity of carnation petal enzymes active toward native hemicelluloses increased threefold at the onset of senescence and declined slightly thereafter. Gel chromatography revealed 23 and 12 kilodalton proteins with hemicellulase activity. The enzymes hydrolyzed the large molecular-size hemicelluloses extensively and without formation of monomers. Endoxylanase activity was detected in the partially purified enzyme preparation. Xyloglucan was depolymerized in the absence of cellulase activity, suggesting the presence of a xyloglucan-specific glucanase. These data indicate that the hemicellulose molecular-size changes observed during development of carnation petals are due, in part, to the enzymic depolymerization of large molecular-size hemicelluloses.     

蛋白质分子细胞定位的应用

蛋白质分子的功能与其在细胞内的定位密切相关,其细胞定位在新基因克隆、基因功能研究、多肽分子细胞内传送、临床药物设计方面发挥越来越重要的作用。     

copine V蛋白的亚细胞定位

目的：确定copine V蛋白的亚细胞定位,初步研究该蛋白的生物学功能.方法：将copine V编码区基因分别构建真核表达载体pEGFP-copine V（或pRED-copine V）,转染HEK293、HeLa细胞,在激光共聚焦荧光显微镜下与转染空载体pEGFP-N1（或pRED-N1）的细胞比较观察.结果：经限制性内切酶分析鉴定,构建的重组表达载体正确.通过激光共聚焦荧光显微镜观察,转染了重组载体pEGFP-copine V的细胞荧光信号集中分布于胞膜和内膜系统;进一步研究表明copine V定位于内质网而非线粒体,而空载体则在整个细胞中均匀分布.结论：copine V蛋白定位于细胞膜和内质网上,而不定位于线粒体.     

Subcellular Localization of IAA Oxidase in Peas

Indoleacetic acid (IAA) oxidase has been reported to be involved in plant growth because of its alleged role in the control of endogenous IAA levels. This purported role was reevaluated in terms of the properties and subcellular location of the enzyme in etiolated pea (Pisum sativum L. var. Alaska) epicotyls.     

蛋白质亚细胞定位的生物信息学研究

细胞中蛋白质合成后被转运到特定的细胞器中,只有转运到正确的部位才能参与细胞的各种生命活动,如果定位发生偏差,将会对细胞功能甚至生命产生重大影响.蛋白质的亚细胞定位是蛋白质功能研究的重要方面,也是生物信息学中的热点问题,数据库的构建和亚细胞定位分析及预测加速了蛋白质结构和功能的研究.     

放线菌次级代谢产物研究进展

随着耐药细菌和新型病毒的不断出现，癌症的发病率和死亡率持续上升，迫使人们不断寻找新的化合物来治疗疾病。放线菌次级代谢产物结构新颖，作用独特，具有抗菌、杀虫、抗肿瘤、免疫抑制等活性，广泛应用于医疗、农业、食品等领域，深入挖掘放线菌资源来开发新型抗生素潜力巨大。然而从自然界分离的放线菌生产目标化合物的能力较弱，这直接影响其工业应用，增加其生产成本，因此构建目标化合物高产菌株显得尤为重要。本文以此为出发点，从放线菌新药资源挖掘和放线菌产抗能力提高两个方面对近年来的研究情况进行概述，为放线菌资源开发提供参考。     

拟南芥血红蛋白3的亚细胞定位

拟南芥的血红蛋白3（AtGLB 3）属于截短的血红蛋白。与拟南芥血红蛋白1相比,拟南芥血红蛋白3具有不同的起源、不同的生化特性和结构;但其功能还不清楚。蛋白质的定位与蛋白质的功能息息相关。为深入研究该基因功能,构建了拟南芥血红蛋白3基因与绿色荧光蛋白融合的植物表达载体pUCGFP/AtGLB3。利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,通过检测融合蛋白在洋葱表皮细胞中的分布来确定拟南芥血红蛋白3在细胞中的定位。荧光显微镜检测结果表明,AtGLB3基因表达产物主要定位在细胞膜上。     

江西青霉的次生代谢产物研究

江西青霉是药用江西虫草的无性型,本文对江西青霉发酵菌丝体甲醇提取物的正丁醇萃取部位运用反复色谱层析进行了系统的分离纯化,得到了6个化合物。经波谱解析,并结合理化鉴定,确定这6个化合物结构为尿嘧啶(1)2、’-脱氧尿嘧啶核苷(2)、腺嘌呤(3)、腺苷(4)、L-焦谷氨酸甲酯(5)和2’-甲氧基腺苷(6)。其中化合物2、5和6为首次从虫草属中分离获得的化合物。     

拟南芥血红蛋白1的亚细胞定位

为研究拟南芥的血红蛋白1(AtGLB1)基因的亚细胞定位,该实验构建了拟南芥血红蛋白1基因与绿色荧光蛋白基因融合的植物表达载体pUCGFP/ AtGLB1.利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,通过检测融合蛋白在洋葱表皮细胞中的分布来确定拟南芥血红蛋白1在细胞中的定位.荧光显微镜检测结果表明,AtGLB1基因表达产物主要定位在细胞核中,少量定位在细胞质中.     

Subcellular Localization Studies Indicate That Lipoxygenases 1 to 6 Are Not Involved in Lipid Mobilization during Soybean Germination

Soybean (Glycine max) lipoxygenase (LOX) has been proposed to be involved in reserve lipid mobilization during germination. Here, subcellular fractionation studies show that LOX1, -2, -3, -4, -5, and -6 isozymes were associated with the soluble fraction but not with purified oil bodies. The purified oil bodies contained small amounts of LOX1 (<0.01% total activity), which apparently is an artifact of the purification process. Immunogold labeling indicated that, in cotyledon parenchyma cells of LOX wild-type seeds that had soaked and germinated for 4 d, the majority of LOX protein was present in the cytoplasm. In 4-d-germinated cotyledons of a LOX1/2/3 triple null mutant (L0), a small amount of label was found in the cytoplasm. In epidermal cells, LOX appeared in vacuoles of both wild-type and L0 germinated seeds. No LOXs cross-reacting with seed LOX antibodies were found to be associated with the cell wall, plasma membrane, oil bodies, or mitochondria. Lipid analysis showed that degradation rates of total lipids and triacylglycerols between the wild type and L0 were not significantly different. These results suggest that LOX1, -2, -3, -4, -5, and -6 are not directly involved in reserve lipid mobilization during soybean germination.     

Subcellular Localization of Isoleucine-Valine Biosynthetic Enzymes in Yeast

By using the method of stepwise homogenization of yeast spheroplast lysates employed previously with the leucine biosynthetic enzymes, it is shown that threonine deaminase, acetohydroxy acid synthase, Mg(2+)-dependent isomero-reductase, and dihydroxy acid dehydratase are particulate. Density gradient centrifugation and the behavior of marker enzymes suggest that all of the above enzymes of the isoleucine-valine biosynthetic pathway are associated with the mitochondria.     

Subcellular Localization of 5''-Nucleotidase in Rat Brain

The subcellular distribution of the ectoenzyme, 5'-nucleotidase, in cerebral cortex and cerebellum of the rat was studied both biochemically and cytochemically. The fractions were characterized biochemically by marker enzymes. The localization of 5'-nucleotidase activity was also investigated cytochemically in the myelin, synaptosomal, mitochondrial, and microsomal fractions. Biochemically 5'-nucleotidase was found to be enriched in the membrane-containing fractions, i.e., myelin, synaptosomal, and microsomal fractions. Cytochemistry showed the reaction product in the myelin fraction to be associated with myelin profiles. In the synaptosomal fraction reaction product could occasionally be seen at synaptosomal membranes, although it could not be attributed unequivocally to the synaptosome itself, since in positions with reaction product unidentifiable membrane structures could always be seen attached. Mitochondria were virtually without any reaction product. In the microsomal fraction 5'-nucleotidase activity was associated with unidentifiable membrane structures. It is concluded that 5'-nucleotidase is associated with myelin profiles and that the high activity found in the synaptosomal fraction is probably not associated with nerve ending plasma membranes.     

PF40 的亚细胞定位研究

pf40 基因是从谷子未成熟种子 cDNA 文库中获得的,与水稻、拟南芥的离子通道蛋白同源性很高 , 具有 8 个跨膜区 . 该基因转入烟草,可使烟草分枝增多,将其转入谷子,可使谷子分蘖增多 . 构建 pf40 与绿色荧光蛋白 gfp 的融合基因,转入烟草进行稳定表达,通过荧光显微镜和激光共聚焦显微镜观察研究其亚细胞定位,发现荧光主要集中在内质网 . 将 pf40 不同缺失区段的 4 个片段与 gfp 构建融合基因,转入烟草进行稳定表达,发现 PF40 N 端 93 个氨基酸残基就能够使得 PF40 定位在内质网, C 端缺失没有影响蛋白质的亚细胞定位 .     

蛋白质体积对其亚细胞定位的影响

蛋白质亚细胞定位信息对深入研究蛋白质的细胞生物学功能十分重要.通过Helix Systems在线计算程序和Vor计算程序两种方法讨论了蛋白质的体积对其亚细胞定位的影响,发现定位于细胞外的蛋白质体积显著小于定位于细胞核、细胞膜和细胞质的蛋白体积,证实了体积参数对区分蛋白质的亚细胞定位是有效的.     

p21亚细胞定位与肿瘤

p21是一种重要的周期蛋白依赖性激酶抑制因子(cyclin-dependent-kinase inhibitor, CKI),主要通过调控细胞周期维持细胞的生长和增殖。此外, p21还参与调控细胞凋亡、细胞衰老以及细胞运动等过程。近年来越来越多的研究表明, p21的功能具有双重性。当p21定位在细胞核时,其主要通过抑制周期蛋白依赖性激酶(cyclin-dependent kinases, CDKs)的活性介导细胞周期停滞,抑制细胞增殖;当定位在细胞质时, p21能够促进细胞增殖。本文主要对p21的生物学功能、亚细胞定位调控机制及其在肿瘤研究中的最新进展予以综述。