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1.
The baculovirus P 10 protein has always represented a mystery in the feld of insect virology. Like the baculovirus polyhedrin protein it is expressed at high levels very late in infection. Homologues of the Autographa californica nucleopolyhedrovirus plO gene are conserved in all Alphabaculoviruses and in other viruses of lepidopteran hosts yet is completely dispensable for virus replication and transmission. P10 is a microtubule interacting protein whose expression has been associated with the formation of a variety of complex and extensive cytoplasmic and nuclear structures. P10 has been associated with a number of roles during infection ranging from the formation of virus occlusion bodies, to affecting the rate of cellular and/or nuclear lysis during the final stages of the virus replication cycle. In this article we review recent work aimed at understanding the role of this enigmatic protein, putting them into context with recent advances in understanding of protein structure and function. We look back at a number of historical studies and observations, reanalysing their conclusions based on recent data and our own observations. The role of the P 10 protein during baculovirus replication remains elusive, however, novel avenues of investigation have been identified that will, we are sure, eventually lead to an understanding of this protein. 相似文献
2.
Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli 总被引:2,自引:0,他引:2
Somodevilla-Torres MJ Morton H Zhang B Reid S Cavanagh AC 《Protein expression and purification》2003,32(2):276-287
Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E. coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding approximately 42 and 36mg EPF from 300ml bacterial and 1L Sf9 cultures, respectively. The preparations were highly purified (#10878;99% purity on SDS-PAGE for the bacterial products and #10878;97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immunosuppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. 相似文献
3.
The influence of the promoter and an N-terminal hexahistidine tag on human cardiac actin (ACTC) expression and function was investigated using four baculovirus constructs. It was found that both non-tagged ACTC and hisACTC expression from the p10 promoter was higher than from the polh promoter. Characterization showed that an N-terminal hexahistidine tag has a negative effect on ACTC. Recombinant ACTC inhibits DNase-I and binds myosin S1, indicative of proper folding. Our data support the hypothesis that the actin protein down-regulates the polh promoter. 相似文献
4.
不同氮磷比条件下浮游藻类群落变化 总被引:30,自引:1,他引:30
选取天津市小型人工湖湖水样品,调整氮磷起始质量比(分别为0.5∶1、7.2∶1、25∶1、50∶1),在水族试验箱内进行生态模拟试验,探讨城市湖水浮游藻类群落对不同氮磷比例的响应.与对照组相比,高氮磷比组绿藻种类减少,蓝藻种类变化不大;低氮磷比组蓝、绿藻种类数均减少.高氮磷比的中氮和高氮箱内,藻类生物量、细胞密度与叶绿素a都远高于对照和高磷箱;高氮箱中叶绿素a均值最高,为69.7 μg·L-1,中氮箱次之,为54.3 μg·L-1,对照与高磷箱分别为30.3和29.7 μg·L-1.试验中后期,高磷箱绿藻门美丽胶网藻(Dictyosphaerium pulchellum)一直占据优势地位;而中氮箱和高氮箱主要优势藻种为皮状席藻(Phormidium corium)、细胶鞘藻(Phormidium tenue)、湖泊鞘丝藻(Lyngbya limnetica)和铜绿微囊藻(Microcystis aeruginosa)等蓝藻.对照箱内物种丰富度最高,中氮箱内生态优势度最高. 相似文献
5.
YUUJI TSUKII TERUE HARUMOTO KAZUMORI YAZAKI 《The Journal of eukaryotic microbiology》1995,42(2):109-115
ABSTRACT. Some strains of P. caudatum contain macronuclear inclusion bodies that are morphologically distinct from bacteria. They vary in number as well as in size in each macronucleus. The inclusion bodies are basically divided into peripheral and inner areas. The peripheral area consists of fibrillar proteins of 22–24 nm in thickness, which are specifically stained with fast green in 45% acetic acid. On the other hand, chromatin-like granules are within the inner area of large inclusion bodies. The granules within the inner area changed their distribution depending upon the physiological state of their host cells. Transplantation experiments and crossbreeding analyses revealed that genetic factors responsible for the multiplication of the inclusion bodies can 'infect' other macronuclei (or cells) via the cytoplasm. These results suggest that the inclusion bodies are a non-bacterial macronuclear endosymbiont, possibly produced by a virus or a virus-like element. 相似文献
6.
During the last decade, microRNAs (miRNAs) have emerged as fine tuners of gene expression in various biological processes including host–pathogen interactions. Apart from the role of host encoded miRNAs in host–virus interactions, recent studies have also indicated the key role of virus-encoded miRNAs in the regulation of host defense responses. In the present study, we show that bmnpv-miR-3, a Bombyx mori nucleopolyhedrovirus (BmNPV) encoded miRNA, regulates the expression of DNA binding protein (P6.9) and other late genes, vital for the late stage of viral infection in the host, Bombyx mori. We have performed both cell culture and in vivo experiments to establish the role of bmnpv-miR-3 in the infection cycle of BmNPV. Our findings showed that bmnpv-miR-3 expresses during early stage of infection, and negatively regulates the expression of P6.9. There was an upregulation in P6.9 expression upon blocking of bmnpv-miR-3 by Locked Nucleic Acid (LNA), whereas overexpression of bmnpv-miR-3 resulted in a decreased expression of P6.9. Besides, a remarkable enhancement and reduction in the viral loads were observed upon blocking and overexpression of bmnpv-miR-3, respectively. Furthermore, we have also assessed the host immune response using one of the Lepidoptera-specific antimicrobial proteins, Gloverin-1 upon blocking and overexpression of bmnpv-miR-3, which correlated viral load with the host immune response. All these results together; clearly imply that bmnpv-miR-3-mediated controlled regulation of BmNPV late genes in the early stage of infection helps BmNPV to escape the early immune response from the host. 相似文献
7.
To obtain an overall three-dimensional picture of the interaction between microtubules and the motor proteins of the kinesin family it will be necessary to take account of both atomic resolution structures obtained by X-ray crystallography and medium resolution reconstructions obtained by electron cryomicroscopy. We examine the problems associated with obtaining the required structural information from electron micrographs of vitreous ice-embedded microtubules decorated with motor domains. We find that the minus-end directed motor, ncd, decorates microtubules with an 80 Å periodicity as for kinesin. Our theoretical analysis and experiments with ncd illustrate the difficulty in determining unambiguously the surface lattice organization by diffraction analysis of micrographs. 3D reconstructions of decorated microtubules are required to accurately locate the motor domains. Helical diffraction theory is not usually applicable because microtubules are cylindrical structures that rarely have complete helical symmetry. We propose using a back-projection method based on the long pitch helices formed by individual protofilaments. Model reconstructions show that this approach is feasible. © 1995 Wiley-Liss, Inc. 相似文献
8.
The structural proteins of Amsacta moorei, Euxoa auxiliaris, and Melanoplus sanguinipes entomopoxviruses (EPVs) were separated by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels. More than 35 structural proteins were detected in each virus. Based on the distribution and the variation in the molecular weights of the virus structural proteins little homology was detected between the EPVs and vaccinia virus. The molecular weight of Amsacta EPV occlusion body matrix protein (110,000) was determined by SDS-acrylamide gel electrophoresis. The occlusion body matrix protein of Amsacta EPV occluded virus isolated from infected E. acrea larvae was rapidly degraded at pH 10.6 to peptides of approximately 94,000 and 60,000 daltons. After 2 hr incubation at alkaline pH, Amsacta EPV occlusion body protein was degraded to approximately 56,000 daltons. Proteolysis of occlusion body protein was inhibited by SDS. No proteolytic degradation was detected in occlusion body matrix protein isolated from Amsacta EPV infected BTI-EAA cells. Amino acid analysis indicates that entomopoxvirus occlusion body matrix protein consists of approximately 20% acidic amino acids and 9% of the sulfur-containing amino acids cysteine and methionine. 相似文献
9.
以5和6种限制性内切酶分别消化昆虫杆状病毒SeNPV和LsNPV DNA,求出它们的基因组平均长133kb和164kb.为了定位这两种杆状病毒的p10基因,构建了含有AcNPVp10基因序列的两个探针载体pAcHP106和pAcEP102.从探针载体回收0.18kb和0.42kb的AcNPV p10基因序列,制备探针,与SeNPV和LsNPV的酶切片段杂交,得到清晰的杂交图谱,准确的定位了这两种病毒的p10基因. 相似文献
10.
对虾杆状病毒感染螃蟹组织的电镜观察 总被引:3,自引:0,他引:3
应用电子显微技术对人工养殖对虾池内的感病螃蟹作了组织切片的电镜观察,发现肝脏、胃、肠组织细胞核内和肌纤维间质中有大量杆状病毒,电镜下的病毒粒子的形态、大小与在中国对虾体内观察到的无病毒包涵体杆状病毒一致。 相似文献
11.
王宗舜 《Entomologia Sinica》1994,(1)
应用细胞整装制备和穿透电子显微镜技术,在家蚕4龄幼虫睾丸育精囊的初级精母幼胞内检查出四个基体—轴丝复合体结构.呈两对,每对结构是由连结纤维将基体牢固地联结并使其相互成直角排列.生长的复合体呈现出丰满的远心端膨大.在此阶段,轴丝是由9个具有马达蛋白臂的双微管组成,缺乏辐射轴和两个中央单微管.在基体的三联体微管和轴丝的双微管的接合点,观察到连结节. 相似文献
12.
Zongshun Wang 《Insect Science》1994,1(1):94-99
Abstract Using cell whole mount preparation, tetrad basal boby-axoneme complexes in the primary spermatocyte from testicular cysts of fourth instar larvae of Bombyx mori are examined by transmission electron microscopy. There exist two paired basal body-axoneme complexes and the two orthogonally oriented basal bodies are linked together with distal and proximal linking fibers. The growing complex displays voluminous distal swelling. At this stage, the axoneme consists of nine microtubular doublets. A connecting nodule is found at the juncture of basal body's triplet and axoneme's doublet, and the A and B tubules of the former continue through the nodule to become the ones of the latter. 相似文献
13.
The human parasite Trypanosoma brucei contains a motile flagellum that determines the plane of cell division, controls cell morphology, and mediates cell–cell communication. During the cell cycle, inheritance of the newly formed flagellum requires its correct positioning toward the posterior of the cell, which depends on the faithful segregation of multiple flagellum-associated cytoskeletal structures including the basal body, the flagellar pocket collar, the flagellum attachment zone, and the hook complex. A specialized group of four microtubules termed the microtubule quartet (MtQ) originates from the basal body and runs through the flagellar pocket collar and the hook complex to extend, along the flagellum attachment zone, toward the anterior of the cell. However, the physiological function of the MtQ is poorly understood, and few MtQ-associated proteins have been identified and functionally characterized. We report here that an MtQ-localized protein named NHL1 interacts with the microtubule-binding protein TbSpef1 and depends on TbSpef1 for its localization to the MtQ. We show that RNAi-mediated knockdown of NHL1 impairs the segregation of flagellum-associated cytoskeletal structures, resulting in mispositioning of the new flagellum. Furthermore, knockdown of NHL1 also causes misplacement of the cell division plane in dividing trypanosome cells, halts cleavage furrow ingression, and inhibits completion of cytokinesis. These findings uncover a crucial role for the MtQ-associated protein NHL1 in regulating basal body segregation to promote flagellar inheritance in T. brucei. 相似文献
14.
The aim of this study is to examine changes in prevalence of overweight and obesity, using International Obesity Task Force criteria, in three cohorts of children and youth living in Cracow, Poland, in 1971, 1983 and 2000. Rates of overweight and obesity doubled among boys and girls, from 7.5% and 6.5% in 1971, to 15.2% and 11.8% in the year 2000. The greatest increases in prevalence occurred in the youngest age groups (7-12 years for boys and 7-10 years for girls), increases being less extensive among adolescents, and lowest of all in the oldest age groups (16-18 years in boys and 14-18 years in girls). The absence of a positive secular trend in BMI among adolescent females relative to males may be due to sociocultural pressures associated with transition to a free market economy in Poland. The extent to which girls attempt to achieve the ideal body, as portrayed by media and society more generally, increases across adolescence. 相似文献
15.
《Fly》2013,7(1):12-15
The ability of the microtubule cytoskeleton to rapidly and locally reorganize itself in response to intra- and extracellular signals is essential to its wide range of functions. A site of tightly regulated microtubule dynamics—and the major interface between the microtubule cytoskeleton and the extracellular environment—is the cell cortex, where the selective stabilization and destabilization of microtubule plus-ends is required for normal cell division, morphogenesis and migration. In a recent study, we found that the cortex of Drosophila S2 and D17 cells is coated with the microtubule severing enzyme and plus-end depolymerase, Kat-60, which actively suppresses microtubule growth and stability along the cell edge. We have proposed that cortical Kat-60 functions by uncapping plus-ends, thereby activating another microtubule depolymerase, KLP10A, preloaded onto the end. The localized destruction of microtubule plus-ends at a specific cortical could feed into larger regulatory pathways, such as those in control of the actin cytoskeleton, to influence cell polarization and motility. 相似文献
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18.
Shipley K Hekmat-Nejad M Turner J Moores C Anderson R Milligan R Sakowicz R Fletterick R 《The EMBO journal》2004,23(7):1422-1432
With their ability to depolymerize microtubules (MTs), KinI kinesins are the rogue members of the kinesin family. Here we present the 1.6 A crystal structure of a KinI motor core from Plasmodium falciparum, which is sufficient for depolymerization in vitro. Unlike all published kinesin structures to date, nucleotide is not present, and there are noticeable differences in loop regions L6 and L10 (the plus-end tip), L2 and L8 and in switch II (L11 and helix4); otherwise, the pKinI structure is very similar to previous kinesin structures. KinI-conserved amino acids were mutated to alanine, and studied for their effects on depolymerization and ATP hydrolysis. Notably, mutation of three residues in L2 appears to primarily affect depolymerization, rather than general MT binding or ATP hydrolysis. The results of this study confirm the suspected importance of loop 2 for KinI function, and provide evidence that KinI is specialized to hydrolyze ATP after initiating depolymerization. 相似文献
19.
Wang Yan Cai Qingyun Chen Jiannan Huang Zhihong Wu Wenbi Yuan Meijin Yang Kai 《中国病毒学》2019,34(6):712-721
Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus(AcMNPV) p48(ac103)gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions(ODVs). However,the exact role of p48 in the morphogenesis of ODVs remains unknown. In this study, we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles. To further understand its functional role in intranuclear microvesicle formation, we characterized the distribution of the P48 protein, which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs. In Ac MNPV-infected cells, P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs, with a limited but discernible distribution in the plasma membrane, nuclear envelope, intranuclear microvesicles, and ODV envelope. Furthermore,coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation, P48 associated with Ac93 in the absence of viral infection. 相似文献
20.
The soluble protein Ure2p from the yeast Saccharomyces cerevisiae assembles in vitro into straight and insoluble protein fibrils, through subtle changes of conformation. Whereas the structure of soluble Ure2p has been revealed by X-ray crystallography, further characterization of the structure of insoluble Ure2p fibrils is needed. We performed X-ray absorption near-edge spectroscopy (XANES) at the sulfur K-edge to probe the state of Cys221 in the fibrillar form of Ure2pC221 and provide structural information on the structure of Ure2p within fibrils. Although the Ure2p dimer dissociation into its constituent monomers has proven to be a prerequisite for assembly into fibrils, we showed the ability of every Ure2pC221 monomer to establish disulfide bonds upon incubation of the fibrils under oxidizing conditions. Our result indicates either that the constituent unit of the fibrillar form of the protein is a dimeric Ure2p or that the fibrils are made of protofilaments assembled in such a way that the residue C221 from a Ure2p molecule in one protofilament is located in the vicinity of a C221 residue from another molecule belonging to a neighbor protofilament. 相似文献